TtgV a key regulator in solvent tolerance
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S – SYMPOSIA
S01 Single Molecules
S01.1 Protein–Nucleic Acid Interactions
S01.1–1Unusual modes of RNA recognitions by RNArecognition motifsT. Afroz, A. Clery and F. Allain
Institute of Molecular Biology and Biophysics, Zurich, Switzerland
RRMs are the most common types of RNA recognition modules,
being present in about 1% of all human proteins. They are a typ-
ical bababfold although N- and C-terminal extensions of these
domains have been observed. We have recently characterized the
NMR structure of two RRM proteins bound to RNA, namely
SRSF1 (previously known as ASF/SF2) and CPEB (Cytoplasmic
polyadenylation element binding protein) which are an alterna-
tive-splicing factor and a regulator of translation, respectively.
The structure of both proteins bound to RNA present unusual
features. SRSF1 contains a so-called pseudo-RRM which medi-
ates sequence-specific recognition using almost exclusively its
a-helix 1 while the beta sheet surface of the RRM which is the
common RNA binding surface in RRM is not involved in RNA
recognition. In CPEB, the two RRMs from a V-shape surface in
the free form which is used to bind the RNA in its center. The
fold is unusual with several additional secondary structure ele-
ments. RRM1 binds the 5¢end of the RNA while RRM2 binds
only the 3¢-terminal nucleotide. This binding arrangement is
unprecedented among RRM-RNA structures.
These structural findings reinforce the idea that the mode the
RNA binding of RRM is still highly variable and unpredictable.
Functional data in support of these structural findings will be
presented.
S01.1–2RNA chaperones modulate RNA structuraldynamics through energy transferB. Furtig1, M. Doetsch1, S. Stampfl1, G. Kontaxis2 and
R. Schroeder1
1Max F. Perutz Laboratories, Vienna, Austria, 2Department for
Structural and Computational Biology, Max F. Perutz
Laboratories, Vienna, Austria
RNA molecules traverse rugged energy landscapes when folding
into functional structures. Thereby, they become easily trapped
in misfolded conformations. Proteins with RNA chaperone activ-
ity modulate RNA’s free-energy landscapes in order to accelerate
RNA folding. These proteins do not require any external energy
and the precise mechanism of how these proteins alter the ener-
getics of RNA folding landscapes was unknown. Here we show
that the C-terminal domain of the RNA chaperone StpA pro-
motes RNA folding by ‘transferring’ conformational energy to
the RNA molecule. We found that StpA presents a positively
charged surface of high plasticity for the interaction with the neg-
atively charged RNA backbone. Formation of the transient com-
plex renders the protein structurally less flexible and freezes out
micro- to millisecond timescale motions within the protein core.
Stabilisation of hydrophobic interactions between aromatic
amino acids in the core of CTD-StpA is the source of energy
transferred to the RNA molecule. Thereby, the RNA gains con-
formational freedom leading to a lower energy barrier for refold-
ing. Our results show how proteins contribute to the
fundamental role of RNA dynamics, an essential feature in all
steps of gene expression.
S01.1–3Structural studies on ribonucleoproteincomplexesA. Torres-Larios
Instituto de Fisiologıa Celular, Universidad Nacional Autonoma de
Mexico, Mexico D.F, Mexico
One of the hallmarks of life is the widespread use of certain
essential ribozymes. The ubiquitous ribonuclease P (RNase P) is
a ribonucleoprotein complex where a structured, noncoding
RNA acts in catalysis. Recent discoveries have elucidated the
three-dimensional structure of an ancestral complex and sug-
gested the possibility of a protein-only composition in organelles.
We will present data dealing with the use of the protein subunit
of RNase P to develop new inhibitors and our efforts leading to
the crystallization of an RNase P composed solely by protein.
S01.1–4DNA lesion recognition and mismatch repairW. Yang
Laboratory of Molecular Biology, NIDDK, NIH, Bethesda,
MD, USA
DNA base lesions consist of base adducts as well as normal bases
paired with wrong partners. Misincorporation or strand slippage
during replication results in mispaired or unpaired DNA bases,
which leads to mutations if not corrected. Exposure to environ-
mental and endogenous DNA damaging agents leads to modified
bases that potentially block replication and transcription. In
eukaryotes, two different MutS homlogs, MutSa and MutSb, areresponsible for mismatch recognition and initiation of mismatch
repair. MutSa, (a heterodimer of Msh2 and Msh6) is highly
homologous to bacterial homodimeric MutS and recognize a base
mispair or 1–2 unpaired bases. In contrast, MutSb, a heterodimer
of Msh2 and Msh3, recognizes insertion-deletion loops (IDL) of
2–15 nucleotides and DNA with a 3¢ single-stranded overhang.
Mismatched DNA bound by MutSa and MutSb is always bent,
but the bending angle and the disposition of the mispaired or
unpaired bases are dramatically different. All MutS homologs
are ATPases and the ATPase activity is modulated by DNA
binding and mismatch recognition. Based on genetic, biochemical
and structural data, we suggest that ATP hydrolysis enhances the
specificity of mismatch recognition. In the nucleotide-excision
repair, multiple ATPase activities are required and likely play a
similar role in enhancing lesion recognition. I will present crystal
structures of bacterial and human MutS proteins complexed with
their respective mismatch substrates and an ATP-dependent
kinetic profreading mechanism that enables specific recognition
of a large variety of mismatched DNA bases by MutS and
MutL. I will compare mismatch recognition with bulk DNA
lesion recognition in the nucleotide excision repair pathway.
6 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S01 Single Molecules
S01.2 Protein Interactions and Networks
S01.2–1A network medicine approach to humandiseaseP. Aloy
IRB Barcelona, Barcelona, Spain
High-throughput interaction discovery initiatives are providing
thousands of novel protein interactions which are unveiling many
unexpected links between apparently unrelated biological pro-
cesses. In particular, analyses of the first draft human interacto-
mes highlight a strong association between protein network
connectivity and disease. Indeed, recent exciting studies have
exploited the information contained within protein networks to
disclose some of the molecular mechanisms underlying complex
pathological processes. These findings suggest that both protein-
protein interactions and the networks themselves could emerge as
a new class of targetable entities, boosting the quest for novel
therapeutic strategies. In this talk, I will summarize our work
towards the characterization and modelling of the protein-inter-
action network underlying Alzheimer¢s disease, together with our
most recent attempts to decipher complex cell networks to the
point of being able to predict how the perturbation of a node
might affect the system as a whole.
S01.2–2Role of an adrenal ferredoxin in regulatingsteroid hormone biosynthesisR. Bernhardt
Universitaet des Saarlandes, Institut fur Biochemie, Saarbruecken,
Germany
Six different cytochromes P450 are involved in steroid hormone
biosynthesis. The role of redox partners in those P450-dependent
substrate conversions is often underestimated. Adrenodoxin
(Adx) plays a key role as electron transfer protein in mitochon-
drial P450 systems catalyzing the initial step of steroid hormone
biosynthesis, the side-chain cleavage of cholesterol to pregneno-
lone, as well as the formation of cortisol and and aldosterone.
While Adx can be replaced by Adx-like proteins from yeast
(Schizosaccharomyces pombe) and bacteria (Sorangium cellulosum
Soce 56), it was demonstrated that the Adx-like protein Fdx2
from mammalian mitochondria involved in iron-cluster biosyn-
thesis is not able to replace Adx in steroid hydroxylation. Investi-
gations of redox partner interactions have been performed to
understand this interesting observation. The interactions between
Adx and AdR as well as Adx and CYP11A1 are investigated in
great detail and described using protein mutants and various bio-
chemical and biophysical methods (surface plasmon resonance,
spectral studies, kinetic investigations, AFM). Moreover, we
demonstrate that the activity and selectivity of steroid hydroxyl-
ation can be affected by the amount and activity of adrenodoxin
and its variants. Furthermore, the dynamics of Adx and AdR
folding has been investigated using fluorescence methods und will
be discussed with respect to functional implications.
S01.2–3Network topology complements sequence asa source of biological informationN. Przulj
Department of Computing, Imperial College, London, UK
Sequence-based computational approaches have revolutionized
biological understanding. However, they can fail to explain some
biological phenomena. Since proteins aggregate to perform a
function, the connectivity of a protein-protein interaction (PPI)
network will provide additional insight into the inner working on
the cell. We argue that sequence and network topology give
insights into complementary slices of biological information,
which sometimes corroborate each other, but sometimes do not.
Hence, the advancement depends on the development of sophisti-
cated graph-theoretic methods for extracting biological knowl-
edge purely from network topology before being integrated with
other types of biological data (e.g. sequence). However, dealing
with large networks is non-trivial, since many graph-theoretic
problems are computationally intractable, so heuristic algorithms
are sought.
Analogous to sequence alignments, alignments of biological
networks will likely impact biomedical understanding. We intro-
duce a family of topology-based network alignment (NA) algo-
rithms, that we call GRAAL algorithms, which produces by far
the most complete alignments of biological networks to date.
Also, we demonstrate that topology around cancer and non-can-
cer genes is different and when integrated with functional genom-
ics data, it successfully predicts new cancer genes in
melanogenesis-related pathways. Finally, we find that aging, can-
cer, pathogen-interacting, drug-target and genes involved in sig-
naling pathways are topologically ‘central’ in the network,
occupying dense network regions and ‘dominating’ other genes in
the network. We conclude that network topology is a valuable
source of biological information that can suggest novel drug tar-
gets and impact therapeutics.
S01.2–4Molecular courtship: how proteins formtransient complexesM. Ubbink
Leiden University, Leiden, The Netherlands
Both functional and non-functional transient interactions between
proteins occur frequently. Functional protein complexes with low
affinity and fast turn-over are found in biological processes that
require a high flux, like redox chains. In photosynthesis, respira-
tion and other metabolic routes fast transport of electrons is
essential. Electron transfer protein complexes must compromise
between the specificity of the interaction –necessary for fast trans-
fer of the electron from one redox centre to the next – and rapid
turn-over of the complex [1]. In these complexes an encounter
state in which the proteins assume multiple orientations plays an
important role [2]. This state can be characterized using paramag-
netic NMR. For the complex of cytochrome c peroxidase and
cytochrome c the encounter state represents 30% of the complex,
whereas the specific complex accounts for 70% [3,4]. This delicate
balance can readily be shifted by mutations in the binding inter-
face [5]. Electrostatic interactions dominate this encounter com-
plex. Recent results on the encounter states of the complexes of
cytochrome f with plastocyanin [6] and cytochrome c6, two photo-
synthetic electron transfer complexes, show that in this case
hydrophobic interactions are involved and suggest that a specific
complex may be absent.
References
1. FEBS J. 2011; 278, 1391.
2. FEBS Lett. 2009; 583, 1060.
3. Proc. Natl Acad. Sci. USA 2006; 103, 18945.
4. J. Am. Chem. Soc. 2010; 132, 241.
5. J. Am. Chem. Soc. 2010; 132, 11487.
6. Scanu et al. Chem. Bio. Chem. 2012; 13, 1312.
S01 Single Molecules Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 7
S01.3 Membranes and Proteins
S01.3–1TRPducins: a novel paradigm to modulate ionchannel signallingP. Valente1, A. Fernandez-Carvajal1, I. Devesa1,
G. Fernandez-Ballester1, R. Planells-Cases2, A. Gomis3,
F. Viana3, J. M. Fernandez-Ros1, C. Belmonte3, W. Van Den
Nest4, C. Carreno4 and A. Ferrer1
1Instituto de Biologıa Molecular y Celular, Universidad Miguel
Hernandez, Alicante, Spain, 2Centro de Investigacion Prıncipe
Felipe, Valencia, Spain, 3Instituto de Neurociencias, UMH-CSIC,
Alicante, Spain, 4DiverDrugs, Gava, Spain
The transient receptor potential vanilloid 1 (TRPV1) channel is a
thermosensory receptor implicated in diverse physiological and
pathological processes. The TRP domain, a highly conserved
region in the C-terminus adjacent to the internal channel gate, is
critical for subunit tetramerization and channel gating. We found
that peptides patterned after this protein domain block TRPV1
activity by binding to the intracellular side of the receptor and,
presumably, interfering with protein-protein interactions at the
level of the TRP domain that are essential for the conformational
change that leads to gate opening. Palmitoylation of active pep-
tides reveals that they are moderate and selective TRPV1 antago-
nists both in vitro and in vivo, blocking receptor activity in intact
rat primary sensory neurons and their peripheral axons. The
most potent lipidated peptide, TRP-p5, blocked all modes of
TRPV1 gating with micromolar efficacy (IC50 £ 10 lM), without
significantly affecting other thermoTRP channels. In contrast, its
retrosequence or the corresponding sequences of other thermo-
TRP channels did not alter TRPV1 channel activity (IC50 > 100
lM). TRP-p5 display anit-hyperalgesic and anti-prurito activity
in a model of chronic hepatic failure. Therefore, these palmitoy-
lated peptides, that we coined TRPducins, are non-competitive,
voltage-independent, sequence-specific TRPV1 blockers with
in vivo activity. Our findings indicate that TRPducin-like peptides
may embody a novel molecular strategy that can be exploited to
generate a selective pharmacological arsenal for the TRP super-
family of ion channels, as well as other channel families.
Acknowledgements:
Funded by MICINN, CONSOLIDER-INGENIO, GVA-PRO-
METEO, and Diverdrugs.
S01.3–2Analysing the catalytic processes ofimmobilised redox enzymes by vibrationalspectroscopiesP. Hildebrandt
Technische Universitat Berlin, Institut fur Chemie, Berlin, Germany
Analysing the structure and function of redox enzymes attached
to electrodes is a central challenge in many fields of fundamental
and applied life science. Electrochemical techniques such as cyclic
voltammetry (CV) which are routinely employed do not provide
insight into the molecular structure and reaction mechanisms of
the immobilised proteins. Surface enhanced infrared absorption
(SEIRA) and surface enhanced resonance Raman (SERR) spec-
troscopy may fill this gap if nanostructured Au or Ag are used as
conductive support materials. In this account, we will first outline
the principles of the methodology including a description of the
most important strategies for biocompatible protein immobiliza-
tion. Subsequently, we will critically review SERR and SEIRA
spectroscopic approaches to characterise the protein and active
site structure of the immobilized enzymes. Special emphasis is
laid on the combination of surface enhanced vibrational spectros-
copies with electrochemical methods to analyse equilibria and
dynamics of the interfacial redox processes. Finally, we will assess
the potential of SERR and SEIRA spectroscopy for in-situ
investigations on the basis of the first promising studies on human
sulfite oxidase and hydrogenases under turnover conditions.
S01.3–3Anoctamin 1 is a Ca2+-activated chloridechannel and a heat sensor in nociceptorsU. Oh
WCU Dept Mol Med & Biopharmaceut Sci & College of
Pharmacy, Seoul National University, Seoul, Korea
A Ca2+-activated chloride channels (CaCCs) are activated by
intracellular Ca2+. CaCCs are known to mediate the apical
movement of Cl- in secretory epithelia in salivary glands, airways,
and kidneys. CaCCs are also known to control the excitability of
muscles and neurons. Moreover, CaCCs also regulate sensory
transduction in retina and other sensory organs. Many bioactive
ligands such as acetylcholine, ATP, endothelin-1, angiotensin II,
and histamine are known to activate CaCCs for initiating their
own physiological functions Previously, we cloned TMEM16A
and found that TMEM16A is a candidate for CaCCs.
TMEM16A retains the hall marks of characteristic pharmacolog-
ical and biophysical properties of endogenous CaCCs. Because it
has eight putative transmembrane domains and an anion chan-
nel, we changed its name as anoctamin 1 (ANO1). Anoctamin 1
has nine additional paralogs comprising superfamily and defines
a novel family of ionic channels because of its unique topology.
ANO1 is highly expressed in small sensory neurons, suggesting
a possible role in nociception. Surprisingly, ANO1 is activated by
heat over 44oC, a temperature for thermal pain. Application of
heat to dorsal root ganglion (DRG) neurons evokes inward cur-
rents and depolarization, thus capable of exciting sensory neu-
rons. Knock-out of ANO1 specifically from DRG neurons
induces hypoalagesic effects over heat. Thus, ANO1 play an
important role in mediating thermal pain in nociceptors.
S01.3–4The role of proton and sodium Ions in energytransduction by respiratory complex IA. P. Batista, B. C. Marreiros and M. Pereira
Instituto de Tecnologia Quımica e Biologica, Universidade Nova
de Lisboa, Oeiras, Portugal
Complex I is an energy transducing enzyme present in most bac-
teria and in all mitochondria. This enzyme catalyses the oxida-
tion of NADH and the reduction of quinone, coupled to ion
translocation across the membrane. The research on complex I
has gained a new enthusiasm by the recently obtained structural
data. Now, the investigation of the energy coupling mechanism(s)
has most dedication. The H+ has been identified as the coupling
ion, although in the some bacterial complexes I Na+ has been
proposed to have that role.
We have addressed the relation of complex I with Na+ by
developing an innovative methodology using 23Na-NMR spec-
troscopy, with the advantage of allowing a direct observation of
the sodium nuclei via its own resonance frequency. We used
membrane vesicles and concentration of sodium ions in the dif-
ferent compartments could be monitored and calibrated using
shift reagents [1].
We have shown that some bacterial complexes I are capable of
H+ and Na+ translocation, but to opposite directions, being the
H+ the coupling ion. A model for the functional mechanism of
Abstracts S01 Single Molecules
8 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
complex I was then proposed, suggesting the presence of two
different energy coupling sites, both operating by indirect
coupling mechanisms. One coupling site may work as a proton
pump and the other as a Na+/H+ antiporter [2,3]. We proposed
a correlation between the type of quinone used as substrate and
the presence of the antiporter activity [4]. We hypothesize that
complexes I that use menaquinone, a low reduction potential qui-
none, as substrates take advantage of the existent Na+ gradient
to reach the same stoichiometry of translocated H+ per electron
as the ubiquinone reducing complexes.
References
1. Batista, Marreiros, Louro & Pereira. Biochim Biophys Acta
2012.
2. Batista, Marreiros & Pereira. ACS Chem Biol 6, 2011, 477.
3. Batista A.S., Fernandes R.O., Louro J., Steuber M.M. &
Pereira. Biochim Biophys Acta 1797, 2010, 509.
4. Batista & Pereira. Biochim Biophys Acta 1807, 2011, 286.
S01.4 Intrinsically Disordered Proteins
S01.4–1Exploring the binding diversity of intrinsicallydisordered proteins involved in one-to-manysignalingW.-L. Hsu1, C. J. Oldfield1, B. Xue2, J. Meng1, P. Romero1,
V. N. Uversky2 and A. K. Dunker1
1Center for Computational Biology and Bioinformatics,
Department of Biochemistry and Molecular Biology, Indiana
University School of Medicine, Indianapolis, IN, USA,2Department of Molecular Medicine, University of South Florida,
Tampa, FL, USA
Molecular recognition features (MoRFs) are intrinsically disor-
dered protein regions that bind specifically to structured partners
via disorder-to-order transitions. Two MoRF-dependent multiple
partner binding processes have been observed: one-to-many sig-
naling, in which a single disordered MoRF binds to two or more
different partners, and many-to-one signaling, in which two or
more MoRFs bind to a single site on one partner. In this study,
we focus on one-to-many signaling with the goal of increasing
the number of examples that have been compared via their 3D
structures. After examining and clustering the existing crystallized
complex structures in Protein Data Bank (PDB), we found 23
MoRFs that were bound to between 2 and 9 partners, with all
pairs of partners binding to the same MoRF having <25%
sequence identity. Of these, eight MoRFs were bound to between
2 to 9 partners having completely different folds, while 15
MoRFs were bound to between 2 and 5 partners having basically
the same folds but with low sequence identity. For both types of
partner variation, the MoRFs exhibited both backbone and side
chain rotations in order to bring about large or small conforma-
tional changes as needed to fit onto the distinct partner surfaces.
Changes in MoRF secondary structure were observed for a few
examples. These data provide solid support for the often stated
concept that one advantage of intrinsically disordered protein
(IDP) for signaling is that an IDP’s flexibility allows the same
segment to adjust its shape to bind to more than one partner.
S01.4–2Role of intrinsic disorder in protein-proteinand protein-nucleic acid interactionsJ. Dyson
Department of Molecular Biology, The Scripps Research Institute,
La Jolla, CA, USA
While interactions between proteins may involve fully folded,
partly folded and disordered segments, the interactions between
proteins and nucleic acids more frequently use disordered seg-
ments, linkers, tails and other entities in complexes that must
form with high affinity and specificity but which must be capa-
ble of dissociating when no longer needed. Disorder is also
observed in free nucleic acids, particularly RNA, as well as in
the proteins that interact with them. The interactions of disor-
dered proteins with DNA most often manifest as molding of
the protein onto the B-form DNA structure, although remodel-
ing of the DNA structure occurs in some instances, and seems
to require that the interacting proteins be disordered to various
extents in the free state. Induced fit in RNA-protein interactions
has been recognized for many years, and provides evidence that
RNA and its interactions with proteins are highly dynamic, and
that the dynamic nature of RNA and its multiplicity of folded
and unfolded states is an integral part of its nature and
function.
S01.4–3Recent progress in NMR spectroscopy:towards the study of intrinsically disorderedproteins of increasing size and complexityI. C. Felli
CERM and Department of Chemistry ‘Ugo Schiff’, University of
Florence, Florence, Italy
Thanks to fast recent progress, NMR spectroscopy is now in a
strategic position to provide unique atomic resolution informa-
tion on a variety of different biological macromolecules in differ-
ent conditions (solution, solid state, in-cell). Among them,
intrinsically disordered proteins (IDPs) or intrinsically disordered
regions (IDRs) of proteins have attracted the attention of the sci-
entific community challenging well accepted ideas and concepts
stimulating us to expand our view of the structure function para-
digm. Recent developments in NMR spectroscopy that enable us
to focus on IDPs and IDRs of increasing size and complexity are
presented. The new methods are demonstrated on a paradigmatic
IDP, human a-synuclein.References:
1. Felli I. C. & Pierattelli R. IUBMB Life. 2012; 64: 473–81.
2. Bertini I., Felli I. C., Gonnelli L., Kumar M. V. V. & Pierat-
telli R. Angew Chem Int Ed Engl. 2011; 50: 2339–41.
3. Bertini I., Felli I. C., Gonnelli L., Kumar M. V. V. & Pierat-
telli R. ChemBioChem. 2011; 12: 2347–2352.
S01.4–4Protein interactions via intrinsically disorderedregions-specificity and fuzzinessM. Fuxreiter
Department of Biochemistry and Molecular Biology Medical and
Health Science Center, University of Debrecen, Debrecen,
Hungary
Proteins containing intrinsically disordered (ID) regions are
widespread in eukaryotic organisms and are mostly utilized in
regulatory processes. ID regions can mediate binary interactions
of proteins or promote organization of large assemblies. Why
S01 Single Molecules Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 9
Nature distinguished ID proteins in molecular recognition func-
tions? ID regions interact via a motif-based manner, which
enable preserving a large conformational freedom in bound
forms.
Thus residues far outside the binding region can critically influ-
ence selectivity or binding affinity via transient, dynamic interac-
tions, without adopting a well-defined structure in the complex.
This phenomenon is termed as fuzziness. The dynamic segments
can modulate conformational preferences or flexibility of the
interface, vary the spacing of the binding motif(s) or serve as a
competitive partner. Post-translational modifications, additional
interactions or alternative splicing of such structurally heteroge-
neous regions provide further means to regulate the activity of
the complex and expand the functional repertoire of the proteins
involved.
S01.5 Engineering and Design
S01.5–1Phototransformable fluorescent proteins:a mechanistic viewD. Bourgeois, C. Duan, M. Byrdin and V. Adam
Pixel Team, IBS, Institut de Biologie Structurale Jean-Pierre Ebel,
CEA, CNRS, Universite Joseph Fourier, Grenoble, France
Phototransformable fluorescent proteins (PTFPs) have received
considerable attention in recent years because they enable many
new exciting modalities in fluorescence microscopy and biotech-
nology. Upon illumination with proper actinic light, PTFPs are
amenable to long-lived transitions between various fluorescent or
nonfluorescent states, resulting in processes known as photoacti-
vation, photoconversion or photoswitching. These processes add
to photoblinking and photobleaching, which universally charac-
terize fluorescent molecules. The highly complex photophysical
behavior of PTFPs can be investigated at the molecular scale by
a combination of X-ray crystallography, in crystallo optical spec-
troscopy and simulation tools such as quantum-chemistry/mole-
cular-mechanics hybrid approaches. In this way, it is possible to
decipher the often subtle conformational dynamics driving photo-
transformations, eventually facilitating the rational engineering of
better performing markers for advanced nanoscopy or biotechno-
logical applications. We will detail how bi-photochromic fluores-
cent proteins such as IrisFP [1] or NijiFP [2] behave
mechanistically, opening the door to new exciting modalities for
their applications at the single molecule level.
References
1. Adam V., Lelimousin M., Boehme S., Desfonds G., Nienhaus
K. et al. (2008) Structural characterization of IrisFP, an optical
highlighter undergoing multiple photo-induced transforma-
tions. Proc. Natl. Acad. Sci. U. S. A. 105, 18343–18348.
2. Adam V., Moeyaert B., David C. C., Mizuno H., Lelimousin
M. et al. (2011) Rational design of photoconvertible and bi-
photochromic fluorescent proteins for advanced microscopy
applications. Chem. Biol. 18, 1241–1251.
S01.5–2Why protein engineering may not always be agood ideaJ. L. Martin
Institute for Molecular Bioscience, University of Queensland,
Brisbane, Qld, Australia
Engineered N- and C-terminal truncations are commonly used to
generate proteins with improved properties for biochemical stud-
ies – for example to optimise expression yields, to increase solu-
bility or to remove a membrane anchor. However, these
modifications can sometimes give rise to unintentional and often
undetected changes in the protein’s ability to interact with part-
ners.
We investigated two related single-span transmembrane pro-
teins, by generating these with and without N-terminal residues
and without the C-terminal membrane anchor. We used a range
of biochemical techniques to characterise interactions including:
pulldown assays, isothermal titration calorimetry, chemical
cross-linking, mass spectrometry, small angle X-ray scattering,
small angle neutron scattering, contrast variation and fluores-
cence spectroscopy. For these two proteins, we found that
removal of 20–30 N-terminal residues either changed the binding
mode for a partner protein or abolished binding altogether. We
also found that removal of the C-terminal membrane anchor
can alter protein interactions but that this may be offset to
some extent by replacing the anchor with a designed soluble
fusion protein.
References
1. Christie and Whitten et al. (2012) Proc Natl Acad Sci USA,
accepted May 3 2012.
S01.5–3Advances in DNA simulations, fromdodecamer to genomesM. Orozco
Institute for Research in Biomedicine (IRB Barcelona), Barcelona,
Spain
Atomistic simulation of DNA is reaching maturity, opening new
oportutinities for theoreticiens to impact into the mainstream of
research in biology. I will sumarize during my talk recent
advances on simulation techniques and how they can be used to
obtain information into the mechanism of chromatin compaction
and gene regulation.
S01.5–4AFM-based force spectroscopy probingof dengue virus capsid protein bindingto lipid droplets-towards a new drugtargetF. A. Carvalho1, F. A. Carneiro2, I. C. Martins1,
I. Assuncao-Miranda2, A. F. Faustino1, M. Castanho1,
R. Mohana-Borges2, A. T. Da Poian2 and N. C. Santos1
1Instituto de Medicina Molecular, Faculdade de Medicina da
Universidade de Lisboa, Lisboa, Portugal, 2Universidade Federal
do Rio de Janeiro, Rio de Janeiro, Brazil
Dengue virus (DENV) affects millions of people and causes more
than 20,000 deaths annually. No effective treatment is currently
available. We characterized the properties of the interaction
between DENV capsid (C) protein and lipid droplets (LD),
recently shown to be essential for the virus replication cycle.
Atomic force microscopy (AFM)-based force spectroscopy mea-
surements were performed with DENV C-functionalized AFM
tips, used to probe interactions in precise locations, at the single-
molecule level, by tapping at the surface of the sample until the
occurrence of a binding event between the protein at the tip and
a ligand on the LD, measuring afterwards the force necessary for
the unbinding. DENV C-LD interaction is dependent on the high
intracellular concentrations of potassium, not occurring in the
Abstracts S01 Single Molecules
10 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
presence of the same concentration of sodium. Limited proteoly-
sis of LD surface impaired the interaction. Force measurements
in the presence of specific antibodies indicate perilipin 3 (TIP47)
as the major DENV C ligand on LD. AFM studies were com-
plemented with zeta-potential measurements and cell biology
studies. Inhibition of Na+/K+-ATPase in DENV-infected cells
resulted in a 50-fold inhibition of virus production. The same
force spectroscopy approach was also used to demonstrate the
successful inhibition of the DENV C-LD binding by a peptide
corresponding to a conserved domain on several related capsid
proteins.
References
1. Carvalho et al. (2012) Dengue virus capsid protein binding to
hepatic lipid droplets (LD) is potassium ion dependent and is
mediated by LD surface proteins, J. Virol., 86, 2096–2108.
2. Martins et al. (2012) The disordered N-terminal region of den-
gue virus capsid protein contains a drug targetable lipid drop-
let-binding motif, Biochem. J., in press.
S01 Single Molecules Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 11
S02 Trends in Biochemistry
S02.1 Genome Dynamics
S02.1–1R-loops in recombination-mediated genomeinstabilityA. Aguilera, T. G. Muse, M. C. Pozo and S. Munoz-Galvan
CABIMER, Universidad de Sevilla, Seville, Spain
Specific cellular processes, such as mitochondrial DNA replica-
tion or class switching, but that may have deleterious conse-
quences. A number of data will be revised providing evidence
that TAR is mediated by replication impairment and that it can
be further enhanced by dysfunction of a number of RNA pro-
cessing enzymes providing a connection between genome dynam-
ics and RNA metabolism. Different studies on the role of
particular proteins involved in double-strand break repair, RNA
biogenesis and replication will be reported in the context of repli-
cation-mediated genome instability events in yeast, Caenorhabd-
itis elegans and human cell lines. In addition, new data will
be discussed about the factors required to assure that replication-
born double-strand breaks are preferentially repaired by sister-
chromatid exchange to prevent instability. The diversity of
mechanisms and factor dysfunction causing recombination-
mediated genome instability and chromosomal rearrangements
will be discussed.
S02.1–2Control over DNA replication in time andspaceZ. Lygerou
University of Patras, Patras, Greece
For genome integrity to be maintained, cells must pass down an
accurate copy of their genome to daughter cells at every cell divi-
sion. DNA replication initiates from hundreds of origins scattered
along eukaryotic chromosomes. Multi-subunit protein complexes
bind onto origins, license DNA for replication and dictate when
and where replication should initiate. Aberrations in these com-
plexes lead to over- or under-replication. Both conditions have
been linked with DNA replication stress and DNA damage and
implicated in tumorigenesis. DNA damage is similarly sensed by
multi-subunit protein complexes which bind onto damaged DNA
and send signals locally for repair and globally for cell cycle arrest
or apoptosis. We study dynamic complexes maintaining genome
integrity by functional imaging in live human cells combined with
modeling and in silico analysis, and investigate the short-term and
long-term consequences of aberrations in the formation and regu-
lation of these complexes. Protein-protein and protein-DNA inter-
actions of origin bound complex subunits were assessed in living
cells by FRAP, FLIP and FRET measurements. Our analysis
reveals multiple steps in the formation of origin-bound complexes,
with dynamic interactions in late mitosis, reiterative loading onto
chromatin during the G1 phase, maximal loading at the G1/S
transition and gradual dissociation from specific subnuclear
regions during S-phase. Following localized DNA damage in live
cells by microsurgery, we observe robust recruitment of the licens-
ing factor Cdt1 to sites of damage mediated by dynamic interac-
tions with PCNA and followed by interactions with the E3
ubiquitin ligase Cul4/DDB1 and subsequent proteolysis. Our data
highlight a dynamic network of interactions maintaining genome
integrity in human cells.
S02.1–3Genomic studies in African populations:approaches and applicationsM. Ramsay
Division of Human Genetics, National Health Laboratory Service
and School of Pathology, Faculty of Health Sciences, University of
the Witwatersrand, Johannesburg, South Africa
The genomic architecture of African populations is poorly under-
stood and the affinities and diversity between ethno-linguistic
groups is beginning to be explored using genomic technologies
including high density SNP and CNV genotyping and whole
exome or genome sequencing. Genome wide association studies
(GWASs) have been extensively applied to search for genetic
associations to complex multifactorial traits in Europeans, but
similar studies in Africans remain scarce. This is largely attrib-
uted to lower levels of funding and thinly distributed resources,
but also to the small pool of trained scientists across the conti-
nent. The high levels of genetic variation and the underlying
structure of African populations present significant challenges,
but the lower levels of linkage disequilibrium and shorter haplo-
type blocks in African populations provide an opportunity for
more effective localisation of causal variants. High throughput
technologies, dense genotyping arrays and plummeting costs are
making GWAS approaches more accessible for African popula-
tions, but ultimately an understanding of the underlying genomic
architecture and environmental influences of each population will
be essential to interpret their contributions to the increase in
non-communicable diseases exacerbated by adverse lifestyle
choices. Two studies will be presented. The first is the genomic
structure of a black South African population in Soweto, just
outside Johannesburg, and comparisons to other African popula-
tions. The second is a fine mapping candidate gene approach in a
case control study searching for genetic associations with rheu-
matoid arthritis in black South Africans. This study has revealed
both common and novel associations. The unique genome
dynamics of African populations have an important role to play
in understanding human health and disease.
S02.1–4DNA topology and SMC complexes inchromosome structure and stabilityC. Sjogren1, J. Kristian1, A. Kegel1, K. Shirahige2 and
T. Kanno1
1Department of Cell and Molecular Biology, Karolinska Institutet,
Stockholm, Sweden, 2Institute of Molecular and Cellular
Biosciences, University of Tokyo, Center for Epigenetic Disease,
Tokyo, Japan
During chromosome replication, the strands of the parental
DNA molecule are pried apart by helicases, leading to DNA
over-winding, or positive supercoiling, ahead of the replication
fork. To allow full and error-free replication, this superhelical
tension has to be removed by topoisomerases which work by
introducing transient breaks in the DNA molecule. Accumulation
of positive supercoils ahead of the fork can also be prevented if
the replication machinery follows the turn of the DNA helix.
This rotation leads to intertwining of sister chromatids, and has
been suggested to occur mainly at sites of fork convergence. We
have shown that in budding yeast, mutants of type I toposiome-
rases trigger a replication delay on longer chromosomes only.
Abstracts S02 Trends in Biochemistry
12 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
This phenotype was also detected in cells lacking a functional
Smc5/6 complex (Smc5/6), member of the family of SMC (Struc-
tural Maintenance of Chromosomes) protein complexes. Further
investigations allowed us to propose that Smc5/6 prevents accu-
mulation of positive supercoils ahead of the replication machin-
ery by facilitating fork rotation through sequestration of nascent
sister chromatid intertwinings. Present studies aim to elucidate
the background and effects of the length-dependent replication
delay, and to challenge the idea that Smc5/6 facilitates fork rota-
tion. Preliminary data indicates that the complex can alter DNA
topology in vitro, and that its chromosomal localization is deter-
mined by transcription and cohesin, another SMC complex.
Results from these and other ongoing studies will be presented.
S02.2 Transcription and Chromatin
S02.2–1Novel cellular players involved in transcriptionelongationS. Chavez
Departamento de Genetica, Universidad de Sevilla, Seville, Spain
Gene regulation research has traditionally focussed on preinitia-
tion complex formation and initiation. More recently, transcrip-
tion elongation has caught the attention of the scientific
community as another important element required to understand
gene control. Our laboratory is interested in understanding the
molecular mechanisms supporting eukaryotic transcription elon-
gation and to clarify the network of functional interactions
between RNA polymerase II and other cell elements. RNA poly-
merase II requires the assistance of numerous general transcrip-
tion factors during the elongation phase. Some of these factors
favour transcription elongation by influencing chromatin dynam-
ics. Some others affect directly RNA polymerase II, modifying
its catalytic properties or its capacity to interact with the RNA
processing machinery. Utilizing yeast genetics, we are identifying
novel cellular players functionally involved in transcription elon-
gation. One example is Sfp1, a regulator of ribosomal protein
genes that we found to increase the tendency of RNA polymerase
II to arrest by backtracking. A second example is the Prefoldin
complex, a co-chaperone involved in the cotranslational assembly
of multimeric complexes. We have found that the Prefoldin com-
plex localize to transcribed genes and facilitates transcription
elongation in a chromatin-related manner. The new perspective
of transcription elongation, integrating these novel players, will
be discussed.
S02.2–2An unexpected link between nucleartopography and chromatin structure regulatesHIV-1 integration and latencyM. Lusic, B. Marini, H. Ali and M. Giacca
ICGEB, Trieste, Italy
Efficiency of HIV-1 integration and establishment of transcrip-
tional latency are the ultimate results of a complex network of
molecular and cellular events, which are still very poorly under-
stood. We have recently explored the relationship between chro-
matin structure, nuclear topography and integration site selection
by HIV-1 in primary CD4+ T cells. 3D-immuno-DNA-FISH has
indicated that the HIV-1 provirus almost exclusively resides at
the periphery of the nucleus in both productive and latent infec-
tion. In particular, specific interactions are formed between the
integrated HIV-1 DNA and the nuclear pore compartment. Of
note, these interactions are also involved in the transcriptional
regulation of the latent provirus. In latently infected, primary
CD4+ T cells the provirus is in close contact with PML nuclear
bodies, which negatively regulate transcription by anchoring the
histone methyltransferase G9a to the proviral DNA, followed by
suppressive H3K9 bimethylation. Transcriptional activation is
concomitant with repositioning from these repressive compart-
ments, which is achieved by active actin polymerization. Taken
together, these results unveil a previously undisclosed link
between the nuclear pore compartment and transcriptional regu-
lation of HIV-1. These findings have important implications con-
cerning the possibility of eradicating HIV-1 disease.
S02.2–3Non-coding RNA as an epigenetic regulator inyeastS. Murray, T. Nguyen, A. S. Barros, D. Brown, J. Ayling and
J. Mellor
Department of Biochemistry, South Parks Road, Oxford, UK
Antisense transcripts in Saccharomyces cerevisiae are initiated at
a promoter chromatin architecture at the 3¢ region of genes,
including a pre-initiation complex (PIC), which mirrors that at
the 5¢ region. Remarkably, for genes with an antisense transcript,
average levels of PIC components at the 3¢ region are ~60% of
those at the 5¢ region. Moreover, at these genes, average levels of
nascent antisense transcription are approximately 45% of sense
transcription. This 3¢ promoter architecture persists for highly
transcribed antisense transcripts where there are only low levels of
transcription in the divergent sense direction, suggesting that the
3¢ regions of genes can drive antisense transcription independent
of divergent sense transcription. Hybrid transcription units, in
which short 3¢ regions are inserted into the middle of other genes,
are capable of both initiating antisense transcripts and terminat-
ing sense transcripts. Antisense transcription can be regulated
independently of divergent sense transcription in a PIC-dependent
manner. In the example shown here, regulated production of an
antisense transcript controls the production of four neighbouring
genes switching the genes on or off reciprocally. Antisense tran-
scription represents a fundamental and widespread component of
gene regulation and the mechanisms by which antisense transcrip-
tion influences chromatin and transcription will be discussed.
S02.2–4Towards the understanding of histone acetyltransferase complexes in transcriptionregulationL. Tora
Institut de Genetique et de Biologie Moleculaire et Cellulaire,
IGBMC, Illkirch, France
Gene expression is a tightly regulated process. Initiation of tran-
scription by RNA polymerase II (Pol II) is believed to be the
outcome of a number of sequential events beginning with the
binding of specific activators to their cognate binding sites. This
initial step will trigger the recruitment of coactivator complexes
and general transcription factors at promoters to allow the load-
ing of Pol II into the preinitiation complex (PIC) to achieve tran-
scription initiation. In this process, coactivators play multiple
crucial roles through enzymatic as well as non-enzymatic func-
tions. GCN5 and PCAF are mutually exclusive histone acet-
lyl transferase (HAT) subunits of two functionally distinct,
but related, multi-subunit coactivator complexes, the SAGA
(Spt-Ada-Gcn5-Acetyltransferase) and the ATAC (Ada-Two-
S02 Trends in Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 13
A-Containing) complexes. These complexes have been shown to
differentially regulate both locus specific gene expression and glo-
bal chromatin structure through their enzymatic activities (HAT
and histone deubiquitinase). I will describe how these human
HAT complexes are targeted to different genomic loci represent-
ing functionally distinct regulatory elements both at broadly
expressed and tissue specific genes. While SAGA can principally
be found at promoters, ATAC is recruited to promoters and enh-
ancers, yet only its enhancer binding is cell-type specific. Further-
more, I will show that ATAC functions at a set of enhancers
that are not bound by p300, revealing a class of enhancers not
yet identified. These findings demonstrate important functional
differences between SAGA and ATAC coactivator complexes at
the level of the genome and define a role for the ATAC HAT
complex in the regulation of a set of enhancers.
S02.3 RNA Biogenesis and Processing
S02.3–1Control of glucose homeostasis byRNA-binding protein HuDE. K. Lee, W. Kim and M. Gorospe
NIA-NIH, Baltimore, MD, USA
Although expression of the mammalian RNA-binding protein
HuD was considered to be restricted to neurons, we recently dis-
covered that HuD was expressed in pancreatic beta cells, where
its levels were controlled by the insulin receptor pathway. We
found that HuD associated with a 22-nucleotide segment of the
5¢-untranslated region (UTR) of preproinsulin (Ins2) mRNA.
Modulating HuD abundance did not alter Ins2 mRNA levels,
but HuD overexpression decreased Ins2 mRNA translation and
insulin production; conversely, HuD silencing enhanced Ins2
mRNA translation and insulin production. Following treatment
with glucose, HuD rapidly dissociated from Ins2 mRNA and
enabled insulin biosynthesis. Importantly, HuD-knockout mice
displayed higher insulin levels in pancreatic islets, while HuD-
overexpressing mice exhibited lower insulin levels in islets and in
plasma. In sum, our results identify HuD as a pivotal regulator
of insulin translation in pancreatic beta cells.
S02.3–2Impact of RNA nuclear experience on proteinsynthesis: the case of the HIV-1 mRNAM. Vallejos1, A. Monette2, J. Deforges3, T.-D. M. Plannk4,
J. S. Kieft5, B. Sargueil3, A. J. Mouland2 and M. Lopez-Lastra1
1Laboratorio de Virologıa Molecular, Instituto Milenio de
Inmunologıa e Inmunoterapia, Escuela de Medicina, Pontificia
Universidad Catolica de Chile, Santiago, Chile, 2HIV-1 Trafficking
Laboratory, Lady Davis Institute for Medical Research-Sir
Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec,
Canada, 3CNRS UMR 8015, Laboratoire de cristallographie et
RMN Biologique, Universite Paris Descartes, Paris, France,4Department of Biochemistry and Molecular Genetics, University
of Colorado Denver School of Medicine, Denver, CO, USA,5Howard Hughes Medical Institute and Department of
Biochemistry and Molecular Genetics, University of Colorado
Denver School of Medicine, Denver, CO, USA
Translation of mRNAs via initiation mediated by Internal Ribo-
some Entry Sites (IRESs) has received increased attention. Genes
bearing IRES elements in their mRNAs are translated in a regu-
lated manner mostly when cap-dependent translation is compro-
mised. In this study we will discuss recent advances in the
understanding of how nuclear events guide IRES-mediated
expression using the capped and polyadenylated unspliced
mRNA of the human immunodeficiency virus type 1 (HIV-1) as
a model system. We present evidence showing how the action of
TRES-transacting factors (ITAFs) plays a pivotal role in IRES-
mediated translation and thereby controls mRNA usage by the
translational machinery. We will discuss how members of a
group of proteins that are involved in gene silencing, transport,
and stabilization, bind to the viral RNA in the nucleus and mod-
ulate IRES-function. The relevance of ribonucleoprotein com-
plexes, assembled in the cell nucleus, on the cytoplasm function
of the RNA will be highlighted.
S02.3–3Assembly, structural dynamics and function ofthe spliceosomeR. Luhrmann
Max Planck Institute for Biophysical Chemistry, Gottingen,
Germany
The spliceosome is a multi-MDa RNP machine that consists of
the small nuclear (sn)RNPs U1, U2, U4/U6 and U5, and numer-
ous non-snRNP proteins. The stepwise interaction of the snRNPs
with the pre-mRNA during spliceosome assembly culminates
with the formation of the so-called B complex which still lacks
an active site. During the subsequent catalytic activation step
major RNA-RNA and RNP remodelling events occur, generat-
ing the activated B complex, which then catalyses the first step
of splicing to yield the C complex. We recently established an
in vitro splicing complementation system that allows us to recon-
stitute both steps of yeast splicing with purified components and
have now extended it to the disassembly stage of the spliceosome.
Using this system, we have investigated the factor requirements
and kinetics of the various remodelling steps of the yeast using
fluorescence cross-correlation spectroscopy. We are also employ-
ing electron cryomicroscopy for the investigation of the 3D struc-
ture of yeast spliceosomes at defined stages of assembly. Finally,
I will report on the crystal structure of two proteins involved in
the catalytic activation of the spliceosome.
S02.3–4A new function for CPEB1 coordinatesalternative 3¢ UTR processing withtranslational regulation in cell cycle and cancerR. Mendez
ICREA and Institute for Research in Biomedicine
(IRB Barcelona), Barcelona, Spain
CPEB (for Cytoplasmic Polyadenilation Element Binding pro-
tein) was identified 16 years ago as an RNA-binding protein that
recognizes maternal mRNAs in the cytoplasm of Xenopus Laevis
oocytes and directs their poly(A) tail elongation and translational
activation during meiotic progression. Since then, CPEB has been
shown to regulate the translation of hundreds of mRNAs in both
somatic and germ cells and to drive events as diverse as learning
and memory, cell cycle progression and tumor development.
Now, we have found that the cytoplasm and the translational
regulation is only part of the life of a protein that moonlights as
a nuclear factor responsible for the pre-mRNA processing of the
same mRNAs that, later, is going to regulate at the translational
level. Thus, CPEB is a nucleocytoplasmic shuttling protein that
recognizes the same cis-acting element in the cytoplasmic mature
mRNA as in the nuclear pre-mRNA, recruiting the cleavage and
polyadenylation machinery that mediates both the cytoplasmic
polyadenylation and the nuclear pre-mRNA cleavage and poly-
adenylation at specific polyadenylation sites. In turn, at least in
Abstracts S02 Trends in Biochemistry
14 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
some cases, this affects alternative splicing of the CPEB regulated
transcripts. This is a new function for CPEB, where hundreds of
mRNAs are regulated by alternative processing in the nucleus in
a coordinated manner and associated with cell cycle and tumor
development. A global model for the regulation of gene expres-
sion by the CPEB family of proteins in cell cycle and cell differ-
entiation will be presented.
S02.4 Autophagy and Protein Homeostasis
S02.4–1Protein folding stress in neurodegenerativedisease: an interplay between ER stress andautophagyC. Hetz
Biomedical Neuroscience Institute (BNI), Faculty of Medicine,
University of Chile, and Center for Molecular Studies of the Cell,
ICBM, University of Chile, Santiago, Chile
The most common neurodegenerative diseases, such as Alzhei-
mer’s Disease, Parkinson’s Disease, Amyotrophic lateral sclerosis,
and Huntington’s disease, affect millions of people worldwide,
but there is neither preventive nor curative therapy for them.
These diseases share a common neuropathology, primarily featur-
ing the presence of abnormal protein inclusions containing spe-
cific misfolded proteins. Recent evidence indicates that alteration
in organelle function is a common pathological feature of protein
misfolding disorders. The endoplasmic reticulum (ER) is an
essential compartment for protein folding, maturation, and secre-
tion. Signs of ER stress have been extensively described in most
experimental models of neurological disorders. ER stress is
caused by functional disturbances, which result in the accumula-
tion of unfolded/misfolded proteins at the ER lumen. To cope
with ER stress, cells activate an integrated signaling response
termed the Unfolded Protein Response (UPR), which aims to
reestablish homeostasis through transcriptional upregulation of
genes involved in protein folding, quality control and degrada-
tion pathways. In this talk we overview our efforts to assess the
role of ER stress in protein misfolding disorders, and discuss pos-
sible strategies to target the UPR with therapeutic benefits.
Support from FONDECYT no. 1100176, FONDAP grant no.
15010006, Millennium Institute No. P09–015-F, Muscular Dys-
trophy Association, Alzheimer Association, and the Michael J.
Fox Foundation for Parkinson research (to CH).
S02.4–2Functional characterization of Atg8-interactingproteins in selective autophagyT. M. Roberts1, M. Klijanska1, E. Siergiejuk1,
C. Wilson-Zbinden1, K. Hofmann2, C. Kraft1,* and M. Peter1
1Institute of Biochemistry. ETH-Honggerberg, Zurich,
Switzerland, 2Institute for Genetics, University of Cologne,
Cologne, Germany. *Present address: Max F. Perutz Laboratories,
University of Vienna, Vienna, Austria.
Several lines of evidence suggest the existence of different types
of selective autophagic degradation pathways, which target single
proteins and various cellular structures such as protein aggre-
gates, peroxisomes, ribosomes and mitochondria for degradation
in lysosomes/vacuoles. However, the mechanism of cargo recog-
nition is not well understood. Structural studies have uncovered
a critical role of the ubiquitin-like protein Atg8/LC-3, which spe-
cifically interacts with specific autophagic receptors through con-
served WXXL-like sequences, called the LC3 interacting region
(LIR) (Noda et al., 2007). LIRs function in various autophagic
receptors such as Atg19 in the cytoplasm-to-vacuole targeting
(Cvt) pathway, p62 and neighbour of BRCA1 gene 1 (NBR1) in
autophagic degradation of protein aggregates, and Atg32 and
Nix in mitophagy, and may link the target–receptor complex to
the autophagic machinery.
Here we report on a careful bioinformatic analysis to search the
yeast proteome for potential LIR motif containing proteins.
Promising candidates were then tested biochemically and func-
tionally for their involvement to regulate autophagy or to target
specific cargo for destruction. Interestingly, this approach identi-
fied possible receptors that may specifically be required to target
misassembled pre-60S ribosomes to the vacuole for degradation.
Moreover, we found that yeast Atg1 interacts with Atg8, as has
been found for the mammalian Atg1-homologue Ulk1 (Behrends
et al., 2010). This interaction is abolished by mutations in the LIR
and results in a partial loss of Cvt and autophagy activity. Inter-
estingly, available experiments suggest that Atg8-removes Atg1
from the phagophore assembly sites (PAS), and promotes its asso-
ciation with autophagosomal membranes. Taken together, these
results imply that Atg8 may fulfil multiple roles in autophagy,
including Atg1 regulation and recognition of specific cargo.
S02.4–3Cyclic GMP, colon cell cytostasis andautophagyS. Visweswariah
Indian Institute of Science, Bangalore, India
The intestine is the largest organ in the body, and along with the
microbiome, has a profound influence on the health and well
being of an individual. Cyclic nucleotide signalling mechanisms
are important in regulating fluid and ion secretion in the intes-
tine, but recently, cGMP has emerged as a regulator of cell cycle
progression in intestinal epithelial cells, thereby influencing the
progression of colon cancer. Receptor guanylyl cyclase C (GC-C)
is the target for the gastrointestinal hormones guanylin, urogu-
anylin and the bacterial heat stable enterotoxins (ST) that cause
watery diarrhoea. We now show that activation of this receptor
results in p53-independent regulation of p21, leading to cell cycle
arrest and senescence. GC-C knock-out mice are more susceptible
to carcinogen- induced tumour formation, and the number of
pre-cancerous lesions was not reduced on administration of ST
peptide to these mice, in contrast to that seen in wild type mice.
These results therefore provide a new role for GC-C in maintain-
ing homeostasis in the intestine. Recently, we have characterized
a novel hyperactivating mutation in GC-C that is found in
patients with symptoms of recurrent and frequent diarrhea.
Many patients report bowel obstruction and ileal inflammation,
and some were diagnosed with Crohn’s disease. Given the recent
link between autophagy and Crohn’s disease, we will also present
our recent findings on the regulation of autophagy in intestinal
cells by cGMP, and consequently, GC-C.
S02.4–4Molecular bases for population variation: fromSNP to SAPJ. Wu
Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Shanghai, China
Single-nucleotide polymorphisms (SNPs) are recognized as one
kind of major genetic variants in population scale. However,
polymorphisms at the proteome level in population scale remain
elusive. Recently, we have analyzed amino acid variances derived
from SNPs within coding regions, which is named as single
S02 Trends in Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 15
amino acid polymorphisms (SAPs), and developed a pipeline of
non-targeted and targeted proteomics to identify and quantify
SAP peptides in human plasma at proteomic level. The absolute
concentrations of three selected SAP-peptide pairs among 290
Asian individuals were measured by selected reaction monitoring
(SRM) approach, and their associations with both obesity and
diabetes were further analyzed. Our works revealed that hetero-
zygotes and homozygotes with various SAPs in a population
could have different associations with particular traits. In addi-
tion, the SRM approach allows us for the first time to separately
measure the absolute concentration of each SAP peptide in the
heterozygotes, which also shows different associations with par-
ticular traits. In the light of recent discoveries there are extensive
variations between DNA and mRNA sequences during the tran-
scriptional process, we believe there must be mRNA and protein
sequence differences at proteome-level.
S02.5 Integrated Cell Structure and Function
S02.5–1The wanderings of the proto-oncogeneproduct RasP. Bastiaens
Department of Systemic Cell Biology, Max Planck Institute for
Molecular Physiology, Dortmund, Germany
Oncogenic, gain of function mutations in genes that encode sig-
nal transduction proteins, do not only change the intrinsic activ-
ity of the oncogene product but also change the ‘internal’ state
of the signal transduction network in which the oncogene prod-
uct is embedded. From this point of view, oncogene products
change the collective state of a multi-component network (the
cytoplasmic state) such that the cells adopt a more immature/
embryonic phenotype that responds less or differently to extracel-
lular cues that maintain its original differentiated behavior. Many
oncogene products occur in the early signal transduction machin-
ery at the plasma membrane where they upset the balance of
reactions such that cytoplasmic states are generated that process
the information contained in the extracellular milieu in a differ-
ent way. Because the ability of an oncogene product to couple
into a signaling network is affected by the oncogene product’s
spatial distribution in the cell, and thereby determine the cyto-
plasmic state or activity pattern of growth factor signaling net-
works, it is of great value to investigate how spatially organizing
reaction system affect the oncogene product’s signaling output in
the cytoplasm. Based on our recent finding that the peripheral
membrane proteins of the proto-oncogene Ras family are using
farnesyl-binding solubilizing chaperones that help maintain their
spatial organization on membranes in cells, I will discuss the
underlying principles of the opposed molecular mechanisms of
directional flux and diffusional randomization that pattern Ras
proteins in cells and how the pharmacological modulation of this
spatially organizing system can be exploited to affect the pheno-
type of oncogenic Ras dependent cancer cells.
S02.5–2Regulation of PI3-kinase/Akt signalling byphosphoinositide phosphatasesC. A. Mitchell, E. M. Davies, R. Gurung, J. M. Dyson and
L. M. Ooms
Department of Biochemistry and Molecular Biology, Monash
University, Melbourne, Vic., Australia
Phosphoinositide 3-kinase (PI3-kinase) generates PtdIns(3,4,5)P3
and PtdIns(3,4)P2 signals that recruit to the plasma membrane
and activate many effectors, including the serine threonine kinase
Akt, which regulates cell proliferation and apoptosis. PI3-kinase
is activated by oncogenic mutation and/or gene amplification
leading to increased cancer cell proliferation, survival and inva-
sion. PI3-kinase/Akt signalling is also implicated in angiogenesis
both during embryonic development and in cancer cell growth
and metastasis. PtdIns(3,4,5)P3 is dephosphorylated and its sig-
nalling function terminated or modified via two possible degrada-
tive pathways. PTEN is a tumour suppressor that degrades
PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PTEN inhibits tumour
growth, metastasis and tumour angiogenesis. PtdIns(3,4,5)P3 may
also be hydrolysed by another phosphatase family called the ino-
sitol polyphosphate 5-phosphatases to form PtdIns(3,4)P2.
PtdIns(3,4)P2 acts with PtdIns(3,4,5)P3 to maximally activate Akt
signalling. Ten mammalian 5-phosphatases have been character-
ised which regulate haematopoietic cell proliferation, synaptic
vesicle recycling, and insulin signalling. Specific 5-phosphatases
regulate embryonic development and cancer cell proliferation via
inhibition of PI3-kinase/Akt signalling via distinct molecular
mechanisms. PtdIns(3,4)P2 formed by 5-phosphatase action is
degraded by inositol polyphosphate 4-phosphatases, INPP4A
and INPP4B, in a significant signal terminating reaction.
INPP4A regulates glutamate hypertoxicity in the brain, and
INPP4B is a recently identified tumour suppressor gene in breast
and prostate cancer.
S02.5–3Systematic lipidomic analysis of yeastmutants reveals novel regulation of lipidhomeostasis and plasma membrane/ERcommunicationA. Santos1, I. Riezman1, F. David2, A. Aguilera-Romero1 and
H. Riezman1
1University of Geneva, Geneva, Switzerland, 2EFPL, Lausanne,
Switzerland
We have performed an unbiased, systematic, lipidomics analysis
of two collections of yeast mutantsin the early secretory pathway
previously used for an epistatic miniarray profiling (Schuldiner
et al, 2005) and a collection of yeast kinase and phosphatase
mutants that were previously analysed for their phosphopeptide
profiles (Bodenmiller et al, 2010). Together these two collections
cover approximately 10% of the yeast genome, but probably over
represent those genes involved in lipid homeostasis control. Each
mutant was extracted in duplicate and using different extraction
protocols to optimize for the detection of lipid classes. The lipids
were quantified using mass spectrometry methods using nano-
spray technology and multiple reaction monitoring. Over 200
lipid species were quantified. Bioinformatic handling of the data
was then performed to cluster genes and reveal patterns. Our
results show that systematic lipidomics analysis is a new, rich
source of biological information that can be used to reveal novel
associations between genes, insights into the complex regulatory
networks controlling lipid homeostasis and serve as a basis to
generate hypotheses about connections between lipid homeostasis
and other factors, such as nutritional status or physical proper-
ties of the membrane. Some examples are connections between
the nutritional status of the cells and glycerophospholipid chain
length, the physical properties of the plasma membrane and con-
trol of serine palmitoyl transferase through TOR kinase (Berch-
told et al, 2012) and potential controls of lipid homeostasis
involving membrane compartmentalization and trafficking.
Abstracts S02 Trends in Biochemistry
16 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S02.5–4The role of spindle assembly checkpointproteins in preventing chromosome instabilityand tumorigenesisC. Sunkel
Universidade do Porto, Porto, Portugal
Preventing chromosome missegregation during cell division is
essential to en sure genomic stability and biological organization.
Eukaryotic cells have a surveillance mechanism that monitors
chromosome interaction with the spindle and prevents mitotic
exit when errors are detected. The Spindle Assembly Checkpoint
(SAC) specifically detects absence or abnormal microtubule-ki-
netochore interaction and produces an inhibitor of the Anaphase
Promoting Complex (APC), which cannot promote the degrada-
tion of cohesins delaying anaphase onset. There are a number of
proteins involved in this signal transduction pathway including
Polo, Aurora B, Mps1, Mad2 and BubR1, and how they work in
concert to produce a coherent output that inhibits the APC is
still poorly understood. Using RNAi, chemical inhibitors and
time-lapse confocal microscopy, we have set out to unravel how
these different components of the SAC are involved in the sens-
ing and production of the APC inhibitory complex. We find that
Polo and Aurora B are involved at the early stage of the pathway
giving rise to two separate branches. One involving the protein
kinase Mps1 and the eventual phosphorylation of BubR1 and
the other the kinetochore localization of Mad2 through the RZZ
complex and its binding to Cdc20. The two branches of this
pathway meet when the Mad2-Cdc20 complex interacts with
Bubr1 producing what appears to be the final and more powerful
inhibitor BubR-Cdc20. Our results allow for the first time to inte-
grate all the different components into a coherent sequence
revealing the role of Polo in this process.
S02 Trends in Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 17
S03 Beyond Biochemistry
S03.1 Ageing
S03.1–1Telomerase gene therapy delays aging andincreases longevity in adult and old miceM. Blasco
Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid,
Spain
A major goal in aging research is to improve health during aging.
In the case of mice, genetic manipulations that shorten or
lengthen telomeres result, respectively, in decreased or increased
longevity. Based on this, we have tested the effects of a telomer-
ase gene therapy in adult (1 year of age) and old (2 years of age)
mice. Treatment of 1- and 2-year old mice with an adeno-associ-
ated virus (AAV) of wide tropism expressing mouse TERT had
remarkable beneficial effects on health and fitness, including insu-
lin sensitivity, osteoporosis, neuromuscular coordination and
several molecular biomarkers of aging. Importantly, telomerase-
treated mice did not develop more cancer than their control lit-
termates, suggesting that the known tumorigenic activity of telo-
merase is severely decreased when expressed in adult or old
organisms using AAV vectors. Finally, telomerase-treated mice,
both at 1-year and at 2-year of age, had an increase in median
lifespan of 24% and 13%, respectively. These beneficial effects
were not observed with a catalytically inactive TERT, demon-
strating that they require telomerase activity. Together, these
results constitute a proof-of-principle of a role of TERT in delay-
ing physiological aging and extending longevity in normal mice
through a telomerase-based treatment, and demonstrate the feasi-
bility of anti-aging gene therapy.
S03.1–2Proteasome activation as a novel anti-agingstrategyS. Gonos
National Hellenic Research Foundation Institute of Biology,
Medicinal Chemistry and Biotechnology, Athens, Greece
Aging and longevity are two multifactorial biological phenomena
whose knowledge at molecular level is still limited. We have stud-
ied proteasome function in replicative senescence and cell sur-
vival. We have observed reduced levels of proteasome content
and activities in senescent cells due to the down-regulation of the
catalytic subunits of the 20S complex (J Biol Chem 278, 28026–
28037, 2003). In support, partial inhibition of proteasomes in
young cells by specific inhibitors induces premature senescence
which is p53 dependent (Aging Cell 7, 717–732, 2008). Stable
over-expression of catalytic subunits or POMP resulted in
enhanced proteasome assembly and activities and increased cell
survival following treatments with various oxidants. Importantly,
the developed ‘proteasome activated’ human fibroblasts cell lines
exhibit a delay of senescence by ~15% (J Biol Chem 280, 11840–
11850, 2005; J Biol Chem 284, 30076–30086, 2009). Our current
work proposes that proteasome activation is an evolutionary con-
served mechanism, as it can delay aging in various in vivo sys-
tems. Moreover, additional findings indicate that the recorded
proteasome activation by many inducers is Nrf2-dependent
(J Biol Chem 285, 8171–8184, 2010). Finally, we have studied the
proteolysis processes of various age-related proteins and we have
identified that CHIP is a major p53 E3 ligase in senescent fibro-
blasts (Free Rad Biol Med 50, 157–165, 2011).
S03.1–3Molecular mechanisms underlying genotype-dependent responses to dietary restrictionM. Kaeberlein
Department of Pathology, University of Washington, Seattle, WA,
USA
Aging is influenced by a complex interaction between environ-
mental and genetic factors. The best-characterized environmental
modulator of longevity is dietary restriction, defined as a reduc-
tion in nutrient availability in the absence of malnutrition. Die-
tary restriction has been shown to increase life span in a wide
variety of species, including the budding yeast Saccharomyces
cerevisiae, the nematode Caenorhabditis elegans, and mice.
Recently, there has been much interest in developing dietary
restriction mimetics, drugs that may provide the health and lon-
gevity benefits of dietary restriction without requiring reduced
food consumption. A major limitation to this appraoch, however,
is the lack of knowledge regarding how different individuals will
respond to dietary restriction. Studies in mice have demonstrated
that a fixed level of dietary restriction can have dramatically dif-
ferent effects on life span in different genetic backgrounds, even
shortening life span in some cases. Thus, it seems clear that geno-
type has a profound effect on individual response to dietary
restriction. Here I will describe our studies aimed at defining the
molecular mechanisms that underlie genotype-specific responses
to dietary restriction in yeast. These studies have allowed us to
identify specific processes that influence this response, including
pH homeostasis of the vacuole/lysozome, mitochondrial superox-
ide dismutase activity, and the mitochondrial proteotoxic stress
response. I will also describe how these findings have led us to
examine the effects of mTOR inhibition in a mouse model
of mitochondrial disease with promising preliminary effects on
survival and health.
S03.1–4Activating conserved longevity pathways:from yeast to humansD. Sinclair
Department of Genetics, Harvard Medical School, Boston, MA,
USA
Over the past 20 years, a number of longevity pathways have
been identified in model organisms. Whether they will lead to
new classes of medicine remains to be seen. Sirtuins are a class of
NAD+-dependent deacetylases that are implicated in the benefi-
cial effects of calorie restriction and possibly exercise. Our lab
studies the role of sirtuins in a variety of age-related diseases,
from Alzheimer’s to type II diabetes. We have recently screened
for molecules that activate Sirtuins by raising NAD+ levels and
will present data on their efficacy in mouse models of age-related
diseases and infertility. We have also found evidence for an
underlying cause of metabolic decline that stems from a decline
in the ability of the mitochondrial and nuclear genomes to
express their genes in synchrony. The latest progress in under-
standing how SIRT1 activators work at the molecular and physi-
ological levels will also be presented.
Abstracts S03 Beyond Biochemistry
18 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S03.2 Global Regulation and CellReprogramming
S03.2–1Landscape of somatic mutations in normaland tumoral genomesA. Camargo
Centro de Oncologia Molecular, – Hospital Sırio Libanes, Sao
Paulo, Brazil
Although patterns of somatic alterations have been reported for
tumor genomes, little is known on how they compare with altera-
tions present in non-tumor genomes. A comparison of the two
would be crucial to better characterize the genetic alterations
driving tumorigenesis. We sequenced the genomes of a lympho-
blastoid (HCC1954BL) and a breast tumor (HCC1954) cell line
derived from the same patient and compared the somatic altera-
tions present in both. The lymphoblastoid genome presents a
comparable number and similar spectrum of nucleotide substitu-
tions to that found in the tumor genome. However, a significant
difference in the ratio of non-synonymous to synonymous substi-
tutions was observed between both genomes. Protein-protein
interaction analysis revealed that mutations in the tumor genome
preferentially affect hub-genes and are co-selected to present syn-
ergistic functions. KEGG analysis showed that in the tumor gen-
ome most mutated genes were organized into signaling pathways
related to tumorigenesis. No such organization or synergy was
observed in the lymphoblastoid genome. Our results indicate that
endogenous mutagens and replication errors can generate the
overall number of mutations required to drive tumorigenesis and
that it is the combination rather than the frequency of mutations
that is crucial to complete tumorigenic transformation.
S03.2–2Gene expression is a circular systemM. Choder1, G. Haimovich1, D. Medina2, G. Millan-Zambrano3,
A. Bregman1, L. Halel-Sharvit1, N. Eldad1, S. Causse4,
O. Barkai1, X. Darzacq4, S. Chavez3 and J. E. Perez-Ortın2
1Department of Molecular Microbiology, Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa, Israel,2Departamento de Bioquımica y Biologıa Molecular, Facultad de
Biologicas, Universitat de Valencia, Valencia, Spain,3Departamento de Genetica, Facultad de Biologıa, Universidad de
Sevilla, Sevilla, Spain, 4Functional Imaging of Transcription, Ecole
Normale Superieure, CNRS, Paris, France
Gene expression is traditionally viewed as a linear process
divided into distinct stages (e.g. transcription, translation). We
have shown that this view is oversimplified. First, RNA polymer-
ase II controls mRNA translation and decay, via a mediator
– Rpb4/7. Second, many transcripts are ‘tagged’ with factors
co-transcriptionally, which later regulate the mRNA localization,
translatability and decay. We name this tagging ‘mRNA imprint-
ing’. Remarkably, promoters, DNA elements known to control
only transcription, regulate also ‘mRNA imprinting’, thus affect-
ing the mRNA fate in the cytoplasm. Third, we found that the
cytoplasmic mRNA ‘decaysome’, known to degrade mRNAs,
also functions as a transcription activator by physically associat-
ing with chromatin. Significantly, the capacity of the decaysome
to function in the synthesis of a certain mRNA in the nucleus
dependends on its ability to complete degrading this mRNA in
the cytoplasm. Our findings demonstrate that gene expression is
a circular process in which the hitherto first and last stages are
interconnected. Finally, we propose that Rpb4/7 represents a
novel class of factors, ‘mRNA coordinators’, which integrate all
stages of the gene expression process into a system. Hence, the
many decisions made during the expression of a given gene seem
to be the result of coordination between all ‘distinct’ stages.
S03.2–3Cloning and engineering bacterial genomes asextra chromosomes in yeastC. Hutchison
The Synthetic Biology Group, J. Craig Venter Institute, La Jolla,
CA, USA
Our Synthetic Biology Group at the J. Craig Venter Institute has
worked on the complete chemical synthesis and installation of
bacterial genomes for a number of years. In the course of this
work we developed several methods for cloning and manipulating
bacterial genomes that can be applied to natural genomes as well
as synthetic ones. First we insert a yeast vector composed of a
yeast centromere, a yeast origin of DNA replication, and a yeast
selectable marker, into the bacterial genome. Then the genome
can be released from the bacterial cell, and introduced into sphe-
roplasts of yeast cells, by PEG mediated transformation. In yeast
the bacterial genome is propagated stably as a Yeast Centromeric
plasmid (YCp), which is essentially a circular Yeast Artificial
Chromosome. This method was developed using mycoplasmas,
which are bacteria with very small genomes and lacking a cell
wall, and we have recently extended it to several other bacterial
species. We also developed a method that we call ‘genome trans-
plantation’, which can be used to reinstall the genome of the
mycoplasma M. mycoides, isolated as free DNA, to produce a
viable bacterial cell. By combining these methods we are able to
bring the power of yeast genetic tools to bear on engineering the
M. mycoides genome. We are using this approach to produce a
minimized M. mycoides genome, and to explore the possibility of
reorganizing bacterial genomes into functional modules.
S03.2–4Tissue-specific epigenetic memory in variouspluripotent stem cellsK. Kim
Sloan-Kettering Institute, Weill Medical College of Cornell
University, New York, NY, USA
Reprogramming somatic cells to generate pluripotent cells not
only offers a means to study mechanisms of cellular epigenetic
reprogramming, but also opens up the possibility of restoring
terminally differentiated cells from adult donors to an embry-
onic-like pluripotent state that can be used clinically for tissue
replacement therapy. Various methods have been used to gener-
ate pluripotent cells: embryonic stem cells (ESC) fused with
somatic cells generates fESC, somatic cell nuclear transfer (nt) to
oocytes generates ntESC, and ectopic expression of four ESC
reprogramming factors in somatic cells generates induced plurip-
otent stem cells (iPSC). However, fESC are tetraploid and ntESC
require the use of oocytes, issues that have limited their clinical
use. In contrast, iPSC are diploid and of somatic origin, and
their use presents few ethical issues.
One challenge in reprogramming somatic cells into pluripotent
stem cells is global epigenetic modification, including DNA deme-
thylation. Through different mechanisms and kinetics, the vari-
ous reprogramming methods ‘reset’ genomic DNA methylation,
an epigenetic modification that influences gene expression. This
observation led us to hypothesize that the pluripotent stem cells
generated using the various reprogramming methods might have
different properties related to differential DNA methylation.
S03 Beyond Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 19
Here, we observed that low-passage iPSC derived from murine
and human tissues harbor residual DNA methylation signatures
characteristic of their somatic tissue of origin, which favors their
differentiation along lineages related to the donor cell, but
restricts alternative cell fates. In contrast, methylation and differ-
entiation patterns of murine ntESC were more similar to those of
classical ESC than were iPSC. Our data indicate that nuclear
transfer more readily establishes the ground state of pluripotency
than factor-based reprogramming, which leaves an epigenetic
memory of the tissue of origin. These properties may influence
efforts to achieve directed differentiation with iPSC for use in
disease modeling or treatment.
S03.3 Artificial Cells and Genomes
S03.3–1Functional analysis for synthetic biology:where is the flywheel?A. Danchin
AMAbiotics SAS, Evry, France
The current avatar of synthetic biology (SB) assumes that we
know enough of what life is to be able to build up synthetic liv-
ing organisms from scratch, or at least program cells and organ-
isms to become cell factories. With this perspective BS combines
two separate entities, a program and a chassis. Most of the work
is performed as if the host cell would accommodate entirely arti-
ficial programs and behave as expected, making the right prod-
ucts, with the right yield, at the right time. There are yet many
obstacles to this dream. Functional analysis reveals unexpected
functions required to make a chassis. Furthermore we must high-
light an essential character of the cell factory: the program repli-
cates (makes copies identical to itself), while the cell reproduces
(makes copies similar to itself). We shall review here some some
of the specific characteristics of the cell frame and some of the
hidden constraints that could jeopardize the success of scaling up
synthetic cells at an industrial scale.
S03.3–2Self-assembled membranes from novelsurfactants for sensing and responseD. Hammer
Departments of Bioengineering and Chemical and Biomolecular
Engineering, University of Pennsylvania, Philadelphia, PA, USA
The construction of bilayer membranes is not limited to lipids, as
many additional surfactants have been shown to self-assemble
into vesicles. We have systematically broadened the pallette of
surfactant molecules that can self-assemble into bilayers, and
have thus created a suite of materials that can impart designer
functionality on cell-like capsules.
In collaboration with M. J. Therien (Duke), we have created a
suite of near IR-emissive polymersomes (NIRPs) that may be
used for imaging and drug delivery. These materials are made by
embedding porphyrin molecules within the polymersome mem-
brane. A notable recent achievement is the incorporation of Dex-
trans in the lumen of polyethylene oxide-based NIRPs. These
capsules release their contents in response to light, with obvious
uses for optical delivery in tumors and other localized regions.
Before failure, the porphyrin configuration and wavelength of
light emission changes. Correspondingly, NIRPs can be used as
stress sensors, since changes in the strain of the membrane
change its internal volume and porphyrin configuration, provid-
ing a link between stress and porphyrin optical emission. In
essence, we have made a color changing stress-sensor that can be
used to monitor stress in soft materials or fluids.
Finally, we have demonstrated the construction of membranes
entirely from surfactant proteins. We recombinantly produced
mutants of the naturally occurring sunflower protein, oleosin,
and studied the phase behavior of self-assembly as a function of
ionic strength and protein structure, observing nanometric fibers,
sheets, and vesicles. We envision the use of recombinant tech-
niques to introduce a host of novel peptidic functionality directly
into these materials.
S03.3–3Expanding the central dogmaV. Pinheiro and P. Holliger
Laboratory of Molecular Biology, Medical Research Council,
Cambridge, UK
All life on Earth relies on DNA and RNA for the storage and
propagation of its genetic information. These polymers are also
uniquely suited to their function, raising the question of whether
there is a fundamental functional constraint or whether their
monopoly reflects evolutionary history.
Because natural nucleic acids rely on polymerases for replica-
tion, we have focused on expanding the substrate spectrum of
these enzymes to enable synthesis and replication of synthetic
nucleic acid polymers (XNAs).
Through a combination of nucleic acid chemistry and polymer-
ase directed evolution, we show that genetic information can be
stored and recovered from eight alternative genetic polymers
based on nucleic acid architectures not found in nature. In doing
so, we have identified a region in the polymerase that regulates
substrate specificity but is distal (>25 A) from its active site.
Such systems expand the central dogma and will conclusively
address questions such as the capacity of different genetic poly-
mers for information storage and propagation. In addition, we
show that for at least one of the XNAs developed, HNA (hexitol
nucleic acids), the replication system is sufficiently robust for apt-
amer development.
References
1. Pinheiro et al. (2012) Science, 336 (6079), 341–4.
2. Cozens et al. (2012) PNAS, 109 (21), 8067–72.
S03.3–4Chemical synthesis and installation of abacterial genomeH. Smith
1,2
1The Synthetic Biology Group, J. Craig Venter Institute, San
Diego, CA, USA, 2Rockville, MD, USA
Every living cell runs off of a set of genetic instructions written
as a four letter code in its DNA. By analogy to computers, a
cell’s genome (its DNA) is the software and the rest of the cell
(the cytoplasm) is the hardware. The genome contains the operat-
ing system for the cell and the cytoplasm is needed to express the
DNA operating system code. At the Venter Institute, we have
chemically synthesized an entire bacterial genome and then
brought that genome to life by ‘transplanting’ it into the cyto-
plasm of a closely related bacterial species, thus making a ‘syn-
thetic cell’. We believe that the future lies in learning how to
rewrite the genetic instructions of synthetic cells so that they can
make useful products such as biofuels and pharmaceuticals.
Abstracts S03 Beyond Biochemistry
20 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S03.4 Computing with Molecules and Cells
S03.4–1Population-based bacterial computingM. Amos
Manchester Metropolitan University, Manchester, UK
Interest in synthetic biology has recently expanded from single-
cell solutions to encompass population-based approaches. The
benefits of this include the potential for distributed computation,
improved error-resistance and noise tolerance, and the availabil-
ity of a richer ‘instruction set’ through compartmentalisation. In
this talk we review recent work in this area, and describe several
of our own contributions.
S03.4–2Mechanisms of antigen degradationA. Gabibov
The Institute of Bioorganic Chemistry, Moscow, Russia
Degradation machinery of biopolymers and physiologically active
low molecular weight compounds may serve a key target for
development of new therapeutics in a verity of severe pathologies.
We studied how several autoantigens are degraded by antibodies,
enzymes and by proteasome.One of the key quality control sys-
tems in the cell is closely related to the protein degradation ma-
chineryby proteasome pathway. Its dramatic failure may occur
either on the level of regulation of ubiquitination and/or catalytic
specificity of proteasome due to the shifting of proteasome-im-
munoproteasome balance. The peculiarities of cleavage of myelin
basic protein (MBP) by proteasome/immunoproteasome com-
plexes are described. Antibody catalysis was proved to have inti-
mate links with development of immune pathologies and
neurodegeneration. The mechanisms of degradation of DNA,
MBP and HIV viral surface antigen, gp120 are disclosed. Anti-
bodies as potential ‘catalytic vaccines’ toward pathological pro-
teins and low molecular weight intoxicants are described.
Combinatorial approaches with elements of rational design based
on X-ray analysis followed by QM/MM and MD approaches
demonstrated good prediction capacities to improve catalytic effi-
ciency.
S03.4–3Uncovering the Human cell lineage tree: thenext grand scientific challengeE. Shapiro
Weizmann Institute of Science, Rehovot, Israel
The cell lineage tree of a person captures the history of the per-
son’s cells since conception. In computer science terms it is a
rooted, labeled binary tree, where the root represents the primary
fertilized egg, leaves represent extant cells, internal nodes repre-
sent past cell divisions, and vertex labels record cell types. It has
approximately 100 trillion leaves and 100 trillion branches
(�100,000 bigger than the Human genome); it is unknown.
We should strive to know it, as many central questions in biol-
ogy and medicine are actually specific questions about the
Human cell lineage tree, in health and disease: Which cancer cells
initiate relapse after chemotherapy? Which cancer cells can
metastasize? Do insulin-producing beta cells renew in healthy
adults? Do eggs renew in adult females? Which cells renew in
healthy and in unhealthy adult brain? Knowing the Human cell
lineage tree would answer all these questions and more.
Fortunately, our cell lineage tree is implicitly encoded in our
cells’ genomes via mutations that accumulate when body cells
divide. Theoretically, it could be reconstructed with high preci-
sion by sequencing every cell in our body, at a prohibitive cost.
Practically, analyzing only highly-mutable fragments of the gen-
ome is sufficient for cell lineage reconstruction. Our lab has
developed a proof-of-concept method and system for cell lineage
analysis from somatic mutations. The talk will describe the sys-
tem and results obtained with it so far, and future plans for this
project.
S03.4–4Synthetic cellular devices: expanding thespace of biological computationR. Sole
Biology Department - Universitat Pompeu Fabra, Barcelona, Spain
Computation is an intrinsic attribute of biological entities. All of
them gather and process information and respond in predictable
ways to an uncertain external environment. Are these computa-
tions similar to those performed by artificial systems? Despite the
similarities between molecular networks associated to information
processing and the wiring diagrams used to represent electronic
circuits, major differences arise. Such differences are specially rel-
evant while engineering molecular circuits in order to build novel
functionalities. Among others, wiring molecular components
within a cell becomes a great challenge as soon as the complexity
of the circuit becomes larger than simple gates. An alternative
approach has been recently introduced based on a non-standard
approach to cellular computation. It allows the synthesis of mul-
ticellular engineered circuits able to perform complex functions
and open a novel form of computation.
S03.5 Dealing with Errors and Evolution
S03.5–1The fitness landscape of Escherichia coli in itsnatural ecosystemI. Gordo
Instituto Gulbenkian de Ciencia, Oeiras, Portugal
Adaptation is one of the main processes responsible for diversity
in the natural word. Yet the rules and the genetic basis underly-
ing it, remain poorly understood. Here we show that, contrary to
observation of in vitro studies, adaptation in vivo does not pres-
ent a signature of diminishing returns. We determine for the first
time, the rate and distribution of effects of mutations responsible
for adaptation of Escherichia coli to the mammalian gut. The
adaptive process in this ecosystem shows a remarkable signature
of clonal interference, the simultaneous competition of clones
carrying different adaptive mutations. Consistent with this adap-
tation in vivo is characterized by the accumulation of large effect
mutations. Importantly, adaptive mutations are not depleted
along the adaptive walk and their dynamics are supportive of
theoretical fitness landscape models beyond those classically con-
sidered. These results have major consequences for our under-
standing of strain diversity in the human microbiome.
S03 Beyond Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 21
S03.5–2The importance of Sonic hedgehog, the5¢ Hoxd genes and Meis2 in the evolutionof the bat wingM. Mason1, D. Hockman1, L. M. Curry1, D. Jacobs2,
M. P. O. Logan3 and N. Illing1
1Department of Molecular and Cell Biology, University of Cape
Town, Rondebosch, South Africa, 2Department of Zoology,
University of Cape Town, Rondebosch, South Africa, 3MRC
National Institute for Medical Research, Mill Hill, London, UK
A change in the regulation of genes known to be important for
patterning the vertebrate limb underpins the evolution of the bat
wing. Bat wings are characterized by the retention of interdigital
webbing and the extended embryonic growth of the metacarpals
and phalanges for digits II to V. In contrast, bat hindlimbs have
relatively short, free, symmetrical digits. Sonic hedgehog (Shh) is
famous as the signaling molecule expressed in the zone of polar-
izing activity in the developing limb, which is important for pat-
terning along the posterior-anterior axis. We have shown that the
activation of a second wave of Shh expression in the interdigital
membrane is a unique feature of bat limb development. A micro-
array analysis of gene expression in developing autopods from
the bat wing and hindlimb relative to the mouse forelimb identi-
fied Meis2 and Hoxd11 transcriptsas being differentially
expressed across all comparisons. Our analysis was extended to
include the 5¢Hoxd genes revealing that Hoxd10–12 are differen-
tially expressed between the bat fore and hindlimb. In situ experi-
ments show that Hoxd10–12 are strongly expressed in the
elongating digit rays of the bat forelimb, but have low expression
in the hindlimb. Meis2, a gene known to be important for pat-
terning the proximal domain of the developing limb bud, is also
expressed in the interdigital region of bat forelimbs and mouse
autopods. The novel expression domain of Shh and Meis2 in the
expanding interdigital region of the bat forelimb and the reduced
expression of Hoxd12-Hoxd10 in the bat hindlimb gives fresh
insight into how tetrapod limbs are patterned, and how different
limb morphologies may evolve.
S03.5–3Inferring the history of species using manygenesD. Posada
Universidad de Vigo, Vigo, Spain
The unprecedented amount of data resulting from next-genera-
tion sequencing techniques has opened a new era in phylogenom-
ics, or phylogenetics at large. However, although large datasets
should in theory increase resolution, multilocus data has also
uncovered a great deal of phylogenetic incongruence among dif-
ferent genomic regions, revamping the interest in the (old) ‘spe-
cies trees/gene trees’ debate. In this talk I will offer an overview
of some the most important challenges evolutionary biologists
will have to face to reconstruct the history of species in the XXI
century, and of some of the tools we have developed in this
regard. This will include dealing with unprecedented amounts of
information, that will often force us to trade-off between model
complexity and analytical feasibility, and to somehow deal with
evolutionary process of gene tree incongruence like incomplete
lineage sorting, gene duplication and loss and horizontal gene
transfer.
S03.5–4Mutations, error thresholds and recombinationE. Szathmary
Dept of Plant Systematics, Ecology and Theoretical Biology,
Biological Institute, Eotvos University, Budapest, Hungary
Although mutations are inevitable, their rates have been subject
to evolution. Beyond a certain mutation rate most organizations
experience an error threshold above which functional information
cannot be selectively maintained. Some mechanisms, thought to
rival nucleic acid replication in early evolution, can be shown to
be insufficient because of excessive mutation rates rendering
directional selection virtually impossible. For nucleic acid tem-
plates the error threshold now seems less severe than before,
because the phenotypic error threshold is less stringent than the
genotypic one, and internal recombination also alleviates the
problem somewhat. Early replicators presumably had survived as
a result of selection on mineral surfaces and group selection of
protocells, despite the fact that the component processes were
highly stochastic. The genetic code has an organization that sub-
stantially minimizes the effects of replication and translation
errors. In contemporary biology sometimes mutation rates are
selected to increase for good reasons. Whereas mutations are
ubiquitous, recombination is not. Yet, recombination is fairly
widespread. The reasons for this are likely to include: (i) the
jumping far in sequence space, (ii) faster response to directional
selection and (iii) solving search problems on rugged fitness land-
scapes much faster than clonal reproduction.
Abstracts S03 Beyond Biochemistry
22 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S04 Molecular Bases of Diseases
S04.1 Neurodegenerative and OrganDegenerative Diseases
S04.1–1Disruptions on the highways of cellularsignallingD. Alessi
MRC Protein Phosphorylation Unit, College of Life Sciences,
University of Dundee, Dundee, UK
My laboratory focuses on unravelling the roles of components
that regulate protein phosphorylation or ubiquitylation pathways
emerging from the genetic analysis of human disease. I will talk
about how we have studied poorly characterised components of
signal transduction pathways that are mutated in human disease
such as LKB1 (Cancer), WNK1 (hypertension disorder) and Par-
kinson’s disease (PINK1 and LRRK2). I will present data that
demonstrates how these pathways are organised, how they recog-
nise signals, how the signal moves down the pathway to elicit
physiological responses and to comprehend what goes wrong in
human disease. I will also show examples of how our research
findings enable us to play the engineer and devise new strategies
to treat human disease.
S04.1–2From aminoacyl-tRNA synthetase mutations toperipheral neuropathies – a flying perspectiveA. Jordanova
Laboratory of Neurogenetics, Institute Born-Bunge, Antwerp,
Belgium
Aminoacyl-tRNA synthetases (ARS) are ubiquitously expressed,
essential enzymes involved in the initial step of protein synthesis.
They catalyze the charging of tRNA with its cognate amino acid
and in this way are the central players in ensuring the fidelity of
translation from genetic code to amino acid sequence. Reflecting
their fundamental importance for cellular life, ARS are present
in all known species ranging from bacteria to humans. Recent
data from human and mouse disease models unexpectedly
revealed their important role in human pathology. Mutations in
tyrosyl-, glyclyl-, alanyl- and lysyl-tRNA synthetases cause differ-
ent subtypes of Charcot-Marie-Tooth disease (CMT), the most
common form of inherited peripheral neuropathy. It is challeng-
ing to understand how mutations in these primordial enzymes
lead to neurodegeneration restricted to the peripheral nerves
only. We and others established that the CMT phenotype caused
by mutant tyrosyl- and glycyl-tRNA synthetases is not due to
impaired aminoacylation activity, but to a gain-of-function alter-
ation of the mutant enzymes or interference with an unknown
function of the wild type proteins. We are applying genetic and
functional genomics approached in CMT patients and Drosophila
models in order to unravel the non-canonical role of these pri-
mordial enzymes in neurodegeneration and to translate this
knowledge into treatment opportunities for CMT.
S04.1–3The reactivation of the epithelial-mesenchymaltransition in organ degenerationM. A. Nieto
Instituto de Neurociencias, CSIC-UMH, San Juan de Alicante,
Spain
The epithelial-mesenchymal transition (EMT) occurs during
embryonic development for the formation of many tissues and
organs, but also occurs in the adult as a physiological response
to injury and during the progression of cancer and other patholo-
gies (Nieto, Ann Rev Cell Dev Biol, 2011). The EMT was
recruited during evolution to define embryonic territories and to
control epithelial plasticity. Therefore, the embryo holds the clues
to the molecular and cellular mechanisms operating after its reac-
tivation in the adult, despite the peculiarities associated with dif-
ferent pathological EMTs. Although the relevance of the EMT in
human disease has been debated until recently, it is now estab-
lished as an important step in the metastatic cascade of epithelial
tumors and it is emerging as fundamental in organ fibrosis. The
development of renal fibrosis is an excellent model to study the
contribution of EMT to organ degenerative diseases and very
importantly, it is the link between progressive loss of renal func-
tion and primary diseases such as glomerulonephritis, diabetes,
toxic injury, congenital abnormalities, urinary tract obstruction
and chronic rejection of transplanted kidneys. Renal fibrosis has
been associated with the activation of interstitial fibroblasts to
give rise to collagen secreting myofibroblasts. In addition, data
from mouse models have shown that myofibroblasts can also
originate from renal tubular epithelial and endothelial cells that
undergo EMT. Among the transcription factors that trigger
EMTs, Snail genes are a good example to understand both physi-
ological and pathological EMTs. I will discuss the pleiotropy of
this gene family and its involvement in the reactivation of the
EMT during the development of organ fibrosis.
S04.1–4Clearance of misfolded proteins in systemicamyloidosis: experience with transthyretinM. J. Saraiva
Institute for Molecular and Cell Biology, Porto, Portugal
Increasing evidence indicates that accumulation of misfolded pro-
teins in the form of oligomers, protofibrils or amyloid fibrils, and
their consequences in intracellular signaling cascades represent uni-
fying events in many of slowly progressive neurodegenerative dis-
orders. Extracellular protein misfolding and aggregation occurring
in systemic amyloidosis triggers inflammation, oxidative stress,
matrix remodeling, the unfolded-protein-response and ER path-
ways that resemble in many aspects, including common molecular
players and scenarios, to those described in local amyloidoses,
affecting for example the central nervous system (CNS), such as
Alzheimer Disease. Thus, similarities and dissimilarities in toxicity
found between the CNS and the periphery are very useful to pin-
point and guide us to the treatment of aging-associated neurode-
generative disorders. Studies with small compounds or molecules,
known to recognize and disrupt amyloidogenic structures, have
proven efficient in removing and promoting clearance of protein
aggregates in studies with experimental models of misfolding disor-
ders. However, the mechanisms and key players in these processes
are largely unknown. Different types of molecules efficient in
removing aggregates in pre-clinical studies in a transgenic model
S04 Molecular Bases of Diseases Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 23
for transthyretin amyloidosis affecting the peripheral nervous sys-
tem and possible mechanisms behind their effects will be discussed.
S04.2 Inflammation and Diseases
S04.2–1Macrophage polarization and metabolic fateM. Cascante, P. Martın-Sanz, P. Traves, M. Pimentel-Santillana,
P. Prieto, M. Fernandez, P. Martın-Sanz, M. Cascante and
L. Bosca
Instituto de investigaciones Biomedicas Alberto Sols (CSIC-
UAM), Madrid, Spain
Macrophages have a wide variety of locations and functions that
are determined by its origin and the type of activation imposed by
the environment. Under a more academic than functional point
of view macrophage activation can be classified as pro-inflamma-
tory (M1 polarization), anti-inflammatory (M2) or pro-resolution/
deactivation (M0), these profiles coexisting in the course of the
immune response. A line of interest has been the characterization
of signaling pathways that determine the polarization and its
effect on the release of mediators of inflammation. In addition to
this, these mediators affect the function and gene expression in
differentiated cells, such as hepatocytes, cardiomyocytes and myo-
fibroblasts. We recently analyzed the metabolic aspects associated
with macrophage activation trying to answer the question about
what changes in the regulation of energy metabolism and precur-
sors (NADPH, riboses, etc.) accompany the different types of
polarization and to what extent these changes are necessary for
the activation phenotype. To get an idea of the magnitude of
changes involved, for example after M1 activation through TL4
challenge, there is an alteration in the expression of over a thou-
sand of genes. The interest of these studies is to envisage the pos-
sibility to regulate macrophage function by altering their
metabolic activity as a complementary strategy to regulate their
participation in the inflammatory response. We could show that
regardless of the stimulus used and the availability of energy sub-
strates, the macrophage is in more than 90% glycolytic, with lim-
ited use of other fuels for energy purposes; however, the pathways
to generate metabolites from the TCA and glutaminolysis are
fully functional and these molecules are used for other purposes.
Supported by BFU2011-024760, RECAVA and CIBERehd.
S04.2–2Transfer of cargo from exosomes to bonemarrow progenitors promotes melanomametastasisH. Peinado1, D. Lyden1 and J. Bromberg
2
1Children‘s Cancer and Blood Foundation Laboratories,
Departments of Pediatrics, Cell and Developmental Biology, Weill
Cornell Medical College and Memorial Sloan-Kettering Cancer
Center, New York, NY, USA, 2Department of Medicine,
Memorial Sloan-Kettering Cancer Center and Weill Cornell
Medical College, New York, NY, USA
Exosomes are membrane-derived microvesicles constitutively
released by cells that play important roles in cell communication,
signaling, immunomodulation and more recently tumorigenesis
through the horizontal transfer of mRNAs, microRNAs, and
proteins. We explored the function of melanoma-derived exo-
somes in the formation of primary tumors and metastases in mice
and human subjects. We determined that treatment of mice with
tumor-derived exosomes could increase both the metastatic bur-
den and the number of sites of metastatic disease; which was
associated with increased endothelial permeability and expression
of inflammation and extracellular matrix remodeling genes. Exo-
some administration reprogrammed bone marrow progenitors
toward a pro-vasculogenic phenotype that was positive for c-Kit,
the receptor tyrosine kinase Tie2 and Met. Reducing Met expres-
sion in exosomes diminished the pro-metastatic behavior of bone
marrow cells. Consistent with a role for exosome-packaged MET
in metastatic disease, higher amounts of MET were found in cir-
culating exosomes and hematopoietic progenitor cells (CD45-C-
KITlow/+TIE2+) isolated from patients with late stage mela-
noma compared with healthy controls. Furthermore, reduction
RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane
trafficking and exosome formation, were highly expressed in mel-
anoma cells. Rab27A RNA interference decreased exosome pro-
duction, preventing bone marrow education and reducing, tumor
growth and metastasis. Furthermore, both protein content (e.g.
expression of the melanoma specific tyrosinase-related protein 2)
and protein concentration was predictive of progression of dis-
ease and poorer outcome. Our data show that exosome produc-
tion, transfer and education of bone marrow cells supports
tumor growth and metastasis, has prognostic value and offers
promise for new therapeutic directions in the metastatic process.
S04.2-3A complex secretory program orchestrated bythe inflammasome controls paracrinesenescenceJ. C. Acosta1, A. Banito1, T.-W. Kang2, P. Janich3,
A. P. Snijders4, T. Longerich5, O. J. Sansom6, S. A. Benitah3,
L. Zender2 and Jesus Gil1
1Cell Proliferation Group, MRC Clinical Sciences Centre, London,
UK, 2Helmholtz Centre for Infection Research, Braunschweig,
Germany, 3Center for Genomic Regulation and UPF, Barcelona,
Spain, 4Proteomics Facility, MRC Clinical Sciences Centre,
London, UK, 5Institute of Pathology, University of Heidelberg,
Heidelberg, Germany, 6The Beatson Institute for Cancer Research,
Garscube Estate, Glasgow, UK
Oncogene-induced senescence (OIS) is crucial to tumor suppres-
sion. Profound changes occur in senescent cells such as the imple-
mentation of a complex pro-inflammatory response termed the
senescence-associated secretory phenotype (SASP). The SASP
reinforces senescence, activates immune surveillance and para-
doxically has pro-tumorigenic properties. Here, we present evi-
dence that the SASP can also induce a ‘‘paracrine senescence’’
response in normal cells. This paracrine senescence response is
also observedin mouse and human models of OIS in vivo. Quanti-
tative proteomic analysis using stable isotope labeling with amino
acid in cell culture (SILAC) identified 103 components of the
secretome significantly upregulated in senescent cells. A screen
using chemical inhibitors to target these factors identified multi-
ple SASP components mediating paracrine senescence. Amongst
them we can mention several TGFb family ligands, VEGF,
CCL2 and CCL20. The mechanisms by which these factors con-
tribute to senescence seem to be diverse, with for example TGFbligands playing a major role by inducing p15INK4b. Expression of
paracrine SASP components is controlled by inflammasome-
mediated IL-1 signaling, which behaves as a master regulator of
the SASP. The inflammasome and IL-1 signaling are activated in
senescent cells and IL-1a expression can reproduce SASP activa-
tion, resulting in senescence. Importantly, using a mouse model
of senescence induction in liver hepatocytes, we showed that inhi-
bition of IL1R or a combination of the identified factors blunted
hepatocyte senescence and the elimination of the premalignant
hepatocytes by the immune system. Our results demonstrate that
SASP members cause paracrine senescence and its manipulation
can have significant effects in vivo.
Abstracts S04 Molecular Bases of Diseases
24 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S04.2–4Intravascular immunosurveillance andperipheral tolerance by resident perivascularcellsF. Sanchez Madrid
Servicio de Inmunologıa, Hospital Universitario de la Princesa,
Universidad Autonoma de Madrid and Centro Nacional de
Investigaciones Cardiovasculares, Madrid, Spain
Immune regulation in peripheral tissues is essential to maintain
tissue homeostasis. Macrophages are immune cells highly special-
ized in antigen capture and clearance of pathogens in different
tissues where they are part from the first immune barrier against
exogenous injuries. To investigate the potential pro-inflammatory
or suppressive role of tissue resident myeloid subsets in the skin,
we first aimed to develop a non-invasive imaging model to visual-
ize the spatio-temporal organization of immune interactions lead-
ing to a tolerogenic response. Direct visualization of mouse ear
epidermis and upper dermis with minimal invasiveness was per-
formed using a standard confocal microscope. We identified a
subset of Perivascular Macrophages (PVM), associated to vessels,
that are thought to exert immune tolerance functions. These cells
are able to extend projections into vessel lumen, crossing the
pericyte sheath, basement membrane, and endothelial layer. This
phenomenon was observed in structured small vessels in the skin,
where paracellular permeability was preserved. By this mean,
PVM sample and capture particulate antigens directly from the
bloodstream. Furthermore, mainly under inflammatory condi-
tions, PVM are able to establish contacts with intravascular leu-
kocytes that vary from transient second-range interactions to
extravasation events. The nature of these contacts, the pheno-
typic and functional characterization as well as the ontogeny of
PVM subpopulations from skin will be also addressed.
S04.3 Stem Cells and Their Niches
S04.3–1From colon stem cells to colorectal cancerE. Batlle
ICREA & Oncology Program, Institute for Research in
Biomedicine (IRB), Barcelona, Spain
The inner layer of the intestinal tube, the intestinal epithelium,
is in a constant process of renewal. Hundreds of millions of
terminally differentiated intestinal cells are replaced by new cells
every day during the life of an adult organism. This tremendous
regenerative power is ultimately sustained by a small population
of intestinal stem cells. It is believed that alterations in the biol-
ogy of human colon stem cells (CoSCs) account for the patho-
physiology of various large-bowel disorders, including colorectal
cancer (CRC). Yet, the identification of human CoSCs remains
elusive. We have recently achieved for the first time the isolation
of stem cells of the human colonic epithelium. Differential cell
surface abundance of the receptor EPHB2 allows the purification
of different cell types from human normal colon mucosa biopsies.
Colon epithelial cells with highest EPHB2 levels exhibit the lon-
gest telomeres and express markers characteristic of intestinal
stem cells. Using culturing conditions that recreate the intestinal
stem cell niche, a substantial proportion of EPHB2-high cells can
be expanded in vitro as an undifferentiated and multipotent pop-
ulation. Furthermore, we have also discovered that most human
CRCs are constituted by cell populations with phenotypes similar
to either CoSCs or intestinal differentiated cells organized into
well-defined compartments. CoSC-like cells purified from primary
CRCs generate tumors in immunodeficient mice with high effi-
ciency and display both self-renewal and differentiation capacity.
These results imply that CRC shares a common hierarchy with
the intestinal mucosa and that the acquisition of an intestinal
stem cell gene program is a central process in the development of
metastatic and recurrent CRC.
S04.3–2Niche cell-stem cell conversion regulated bythe Snail class transcriptional repressor,EscargotG. Hime1, J. Voog2, S. Sandall3, G. Hime1, M. Loza-Coll3,
M. Fuller4 and L. Jones3
1University of Melbourne, Vic., Australia, 2University of
California- San Diego, La Jolla, CA, USA, 3The Salk Institute for
Biological Studies, La Jolla, CA, USA, 4Stanford University
School of Medicine, Stanford, CA, USA
Stem cells reside within specialized microenvironments, or niches,
that control many aspects of stem cell behaviour, including the
decision between self-renewal and initiation of differentiation.
Somatic hub cells at the apical tip of the Drosophila testis regulate
the behaviour of both the cyst stem cells (CySCs) and germline
stem cells (GSCs) and, as such, are a primary component of the
stem cell niche in the testis. Here we demonstrate that hub cells
depleted of the transcription factor Escargot (Esg) acquire CySC
characteristics and undergo differentiation as cyst cells, resulting
in complete loss of all hub cells and eventually, CySCs and GSCs.
We identified Esg-interacting proteins and confirmed an interac-
tion between Esg and the co-repressor C-terminal binding protein
(CtBP), which is also required for maintenance of hub cell fate.
Our results indicate that differentiated niche cells can acquire stem
cell properties upon removal of a single transcription factor in
vivo, revealing the importance of defining networks that maintain
the balance of cell fates within the stem cell niche.
S04.3–3Stem cells from the mammalian blastocyst –How similar are mouse and human?J. Rossant
Departments of Molecular Genetics, and Obstetrics and
Gynecology University of Toronto, Toronto, Canada
The mammalian blastocyst contains about 100 cells and only
three distinct cell types. One cell type, the epiblast progenitor,
gives rise to all cell types of the body and to pluripotent embry-
onic stem (ES) cells, while the other two cell types give rise to
placental and other support tissues. By studying both the embryo
and its derived stem cells in the mouse, we have been able to
identify signaling pathways and transcription factors specifying
cell fate in the mouse blastocyst. FGF signals from the epiblast
progenitors act to maintain the proliferation of trophoblast stem
cells and to promote the differentiation of primitive endoderm.
Given that FGF is required to maintain human ES cells in the
undifferentiated state, this suggests that the blastocyst niche may
differ between mouse and human. Understanding these differ-
ences is key to successful generation, maintenance and differenti-
ation of human ES and induced pluripotent stem cells.
S04 Molecular Bases of Diseases Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 25
S04.3–4Hedgehog signalling and cancer initiationR. Toftgard, C. Finta, A. Cherry, A. Fullgrabe, M. Kasper and
L. Jovine
Center for Biosciences and Department of Biosciences and
Nutrition, Karolinska Institutet, Stockholm, Sweden
The skin hair follicle is a mini-organ cycling between phases of
active growth, regression and quiescence and is home to a num-
ber of diverse epithelial stem- and progenitor cell populations
localized to different niches. Genetic lineage tracing combined
with transplantation assays in the mouse reveals striking differ-
ences between the normal function during tissue maintenance
and an ability to serve as multipotent stem cells during regenera-
tion induced by stress or physical injury. Stem cells marked by
expression of Lgr5 normally maintain the hair follicle itself
whereas Lgr6 expressing stem cells contributes to the sebaceous
gland and the interfollicular epidermis.
Basal cell carcinoma of the skin (BCC) is the most common
malignancy with aberrant activation of Hedgehog (Hh) signalling
as a pathognomonic feature. However, the cell of origin has been
a long-standing issue of debate with such cells suggested to reside
either in the hair follicle germinative compartment or in the inter-
follicular epidermis. We have addressed this question in mouse
models with conditional activation of the Hh-pathway induced
by inactivation of the Ptch1 gene and find that stem cell popula-
tions marked by Lgr5 and Lgr6 expression are cells of origin for
BCC and that wounding and tissue regeneration has a major
influence on cancer development.
Suppressor of fused (SUFU) is an essential repressor of Hh
signalling in mammals having a tumour suppressor function and
with ability to inhibit GLI-mediated transcription of target genes
independent of intact primary cilia. Deciphering the mechanisms
underlying the key role of SUFU in intracellular Hh-signal trans-
duction at the molecular level is an important research aim. To
this end we have determined the 3D structure of full length
SUFU revealing the presence of a unique structural properties
and pinpointing key regulatory domains.
S04.4 Cancer Genomics and Biomarkers
S04.4–1Human cancer epigenomicsM. Esteller
Bellvitge Biomedical Research Institute, – IDIBELL, Barcelona,
Spain
An altered pattern of epigenetic modifications is central to many
common human diseases, including cancer. Many studies have
explored the mosaic patterns of DNA methylation and histone
modifications in cancer cells on a gene-by-gene basis, among
them the seminal finding of transcriptional silencing of tumor
suppressor genes by CpG island promoter hypermethylation. Epi-
genetic gene inactivation in transformed cells involves many ‘belts
of silencing’. We are in the process of completing the molecular
dissection of the entire epigenetic machinery involved in methyla-
tion-associated silencing, such as DNA methyltransferases,
methyl-CpG binding domain proteins, histone deacetylases, his-
tone methyltransferases, histone demethylases and Polycomb pro-
teins. The first indications are also starting to emerge about how
the combination of cellular selection and targeted pathways leads
to abnormal DNA methylation. In addition to classical tumor-
suppressor and DNA repair genes, epigenetic gene silencing
includes ncRNAs with growth inhibitory functions. Recent tech-
nological advances, such as whole genome bisulfite sequencing,
are now enabling cancer epigenetics to be studied genome-wide.
It is time to ‘upgrade’ cancer epigenetics research and put
together an ambitious plan to tackle the many unanswered ques-
tions in this field using genomics approaches to unravel the epige-
nome.
S04.4–2DNA polymorphisms and the DNA mismatchrepair genes in oesophageal cancerM. I. Parker1,2
1International Centre for Genetic Engineering and Biotechnology
(ICGEB), Cape Town, South Africa, 2Division of Medical
Biochemistry, University of Cape Town, Cape Town, South Africa
Oesophageal squamous cell carcinoma (OSCC) has a high preva-
lence in the Black and Mixed Ancestry populations of South
Africa. Our previous studies have detected association of DNA
variants in several genes with OSCC in the Mixed Ancestry pop-
ulation, but no associations in the Black population. Recently,
three genome-wide association studies in Chinese populations
identified five new OSCC susceptibility loci, including variants at
PLCE1, C20orf54, PDE4D, RUNX1, and near UNC5CL. In this
study, we tested SNPs from these five loci for association with
OSCC in 1256 and 1117 subjects from the South African Black
and Mixed Ancestry populations, respectively.
The DNA mismatch repair (MMR) enzymes repair errors in
DNA that occur during normal DNA metabolism or are induced
by certain cancer-contributing exposures. We assessed the associ-
ation between 10 single-nucleotide polymorphisms (SNPs) in five
MMR genes and oesophageal cancer risk in South Africans.
Prior to genotyping, SNPs were selected from the HapMap data-
base, based on their significantly different genotypic distributions
between European ancestry populations and four HapMap popu-
lations of African origin. In the Mixed Ancestry group, the
MSH3 rs26279 G/G versus A/A or A/G genotype was positively
associated with cancer (OR = 2.71; 95% CI: 1.34–5.50). Similar
associations were observed for PMS1 rs5742938 (GG versus AA
or AG: OR = 1.73; 95% CI: 1.07–2.79) and MLH3 rs28756991
(AA or GA versus GG: OR = 2.07; 95% IC: 1.04–4.12). In
Black individuals, however, no association between MMR poly-
morhisms and cancer risk was observed in individual SNP analy-
sis. The interactions between MMR genes were evaluated using
the model-based multifactor-dimensionality reduction approach,
which showed a significant genetic interaction between SNPs in
MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry
subjects, respectively. The data also implies that pathogenesis of
common polymorphisms in MMR genes is influenced by expo-
sure to tobacco smoke. In conclusion, our findings suggest that
common polymorphisms in MMR genes and/or their combined
effects might be involved in the aetiology of oesophageal cancer.
S04.4–3Autophagy in cell death and cancerK. Ryan
Beatson Institute for Cancer Research, Glasgow, UK
Macroautophagy (autophagy) is a catabolic membrane-trafficking
process that serves to deliver cytoplasmic constituents to lyso-
somes for degradation. The process functions in the majority, if
not all, cells as a means to turnover damaged proteins and
organelles. Autophagy therefore functions as a cellular homeo-
static mechanism which when perturbed leads to accumulation of
misfolded proteins and non-functional organelles and loss of cel-
lular integrity. The levels and cargoes of autophagy can respond
to various extracellular and intracellular cues to bring about
selective and sometimes contrasting effects. For example, in rela-
Abstracts S04 Molecular Bases of Diseases
26 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
tion to cell death, autophagy has, depending on context, been
reported to have both positive and negative effects on cell viabil-
ity. Due the multiple roles of autophagy in regulating cellular
homeostasis, it is not a surprise that changes in autophagy can
have effects on tumour development. It is not yet clear, however,
when autophagy is tumour-promoting or tumour-suppressive in
any given context. We have therefore been addressing this issue
by studying the role of autophagy at different stages of tumour
development in vivo. Data from these studies will be presented
alongside our in vitro studies to understand the role of autophagy
in tumour suppression.
S04.4–4E-cadherin disfunction in gastric cancer.Celullar consequences and clinical applicationsR. Seruca
Institute of Molecular Pathology and Immunology of the
University of Porto, Porto, Portugal
GC has a high mortality rate, since it is mostly detected at
advanced stage. The only chance for cure lies in the complete
resection of the tumour within healthy boundaries. This can only
be achieved if there is limited local disease without any metastatic
spread. In most of the cases a complete gastrectomy is necessary,
albeit depending on the location of the tumour subtotal resection
of the stomach can be performed. The benefit of adjuvant radio-
chemotherapy is still discussed.
It is a major challenge to define markers that allow enrolment
of patients in individualized treatment and follow-up regimes.
There is only evidence for clinico-pathological criteria that can
be applied, whereas up to today, no molecular marker has been
identified.
One of the most basic characteristics of cancer cells is that they
adhere poorly to each other, being this fact usually associated
with their ability to invade the surrounding tissues. E-cadherin
plays a pivotal role in cell–cell adhesion. The critical importance
of E-cadherin to normal development is demonstrated by the
lethality in the very early stage of embryogenesis. In cancer, the
study ofsporadic tumours and early hereditary diffuse gastric
cancer (HDGC) lesions in germline CDH1 mutation carriers sug-
gests that E-cadherin loss can be an early or initiating event in
tumorigenesis but also an important marker of progression.
Besides mediating cell–cell adhesion, E-cadherin also exerts its
tumour suppressor function by acting as a cell membrane recep-
tor. Increasing evidences indicate that the role of E-cadherin in
tumor progression depends on the activation of signaling path-
ways related to migration and cell survival. We have performed
several in vitro studies and in vivo studies to clarify E-cadherin
mediated signaling pathways, associated cellular effects and its
involvement in tumorigenesis and its relevance as a marker of
poor prognosis in gastric cancer.
S04.5 Role of Hypoxia in Pathogenesis ofInflammation in Cancer
S04.5–1Metabolomics approaches for cancer research:from metabolic networks to targetidentificationE. Gottlieb
The Beatson Institute, Glasgow, UK
In order to engage in fast replicative division, a cancer cell must
duplicate its genome, synthesise proteins and lipids, and assemble
these components to form daughter cells. These activities require
increased uptake of nutrients to be used as biosynthetic precur-
sors and an energy source. However, rapid tumour growth sur-
passes the required blood supply and exposes cancer cells to
extreme conditions of metabolic deficit and stress. Therefore, can-
cer cells undergo many metabolic changes (collectively known as
‘metabolic transformation’) that support their growth and sur-
vival. The extent to which metabolism plays a role in tumorigene-
sis cannot be overstated and drugs that selectively target these
processes are likely to at least delay, if not halt tumour progres-
sion. Our work utilizes analytical chemistry and system biology
approaches to study metabolic transformation. We investigated
cells deficient in the mitochondrial tumour suppressor fumarate
hydratase (FH). FH is a tricarboxylic acid (TCA) cycle enzyme
and a tumour suppressor which is lost in some severe cases of
renal cell cancer. Using genetically-modified primary mouse renal
cells we collected metabolomics data and applied a computa-
tional model, generated to study their unique metabolome. We
identified several important metabolic pathways which are spe-
cific and crucial for the survival of cells deficient in FH. These
include the heme biosynthesis and degradation pathway as well
as mechanisms of alleviating TCA cycle carbon stress. These
technologies are not only important for understanding the basic
biochemistry of cancer cells but they can inform us on future
clinical management of cancer and may lead to new therapeutic
approaches to target cancer-specific metabolic pathways.
S04.5–2Contrasting effects of hypoxia on peripheraland central neurogenesisJ. Lopez-Barneo
Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario
Virgen del Rocıo/CSIC/Universidad de Sevilla, Seville, Spain
The carotid body (CB) contains a population of peripheral neural
progenitors, which upon in vivo exposure to sustained low O2
tension (PO2) are activated to proliferate and to differentiate into
neuron-like and O2-sensitive glomus cells. Neurogenesis also per-
sists throughout life in the subventricular zone (SVZ) and the
hippocampus. However, the response of central neural progeni-
tors to hypoxemia is unknown. We have performed a compara-
tive study of the effect of O2 tension on neurogenesis in the CB
and SVZ. In vivo experiments were done on adult rodents
exposed for 3–28 days to low PO2 (~10%). In vitro CB and SVZ
neurosphere assays were also performed. Systemic hypoxia
induced CB growth due to conversion of GFAP+ stem cells to
nestin+ proliferative progenitors, which in turn differentiated
into TH+ glomus cells. In contrast, hypoxia induced arrest of
SVZ neurogenesis, which was characterized by a narrowing of
the germinal layer and increased angiogenesis in the neighboring
brain parenchyma. The inhibitory response of SVZ to hypoxia
was due to a decrease of transient amplifying C cells as well as
immature neuroblasts (A cells). Apparent recovery of CB and
SVZ structures were observed two weeks after animals were
returned to a normoxic environment. In vitro experiments showed
that both CB and SVZ stem cell proliferation are little affected by
low PO2 (up to 1%). Lower PO2 values (0.5%) resulted in
decreased proliferation capacity of SVZ progenitors. Hypoxia
enhanced in vitro differentiation of CB precursors to TH+ glo-
mus cells. In contrast, survival of newly generated central neurons
in vitro was drastically reduced when O2 tension was <1%. These
data suggest that central and peripheral neural stem cells are rela-
tively resistant to hypoxia. The opposite responses to hypoxia of
CB and SVZ in vivo suggest that niche factors, up-regulated by
lowering PO2, may differentially influence peripheral and central
stem cell function. These observations could have major patho-
S04 Molecular Bases of Diseases Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 27
physiological and medical implications, as chronic hypoxemia is a
highly prevalent condition in the human population.
S04.5–3Hypoxia signalling and tumour metabolism.Novel therapeutic approchesI. Marchiq, R. Le Floch, J. Chiche, D. Roux and J. Pouysssegur
University of Nice, IRCAN, Centre A. Lacassagne, Nice, France
In metazoan, sensing the availability of oxygen and key nutrients
is integrated with growth factor signaling. This nutrient check-
point control is essential for cells to receive the order to progress
through the division cycle. Therefore, rapidly growing cells have
developed sophisticated regulatory systems to rapidly respond to
oxygen and nutrient fluctuations in the microenvironment.
Early on in evolution, oxygen sensing emerged, as a central
control mechanism of energy metabolism and vasculogenesis. At
the heart of this regulatory system is the Hypoxia-Inducible Fac-
tor, HIF-1, which controls the expression of, among other gene
products, VEGF-A, Angiopoıetin-2 and Notch-ligand, three key
angiogenic factors in vertebrates. This finding has placed the
hypoxia-signaling pathway at the forefront of nutritional control.
HIF-1 can induce a vast array of gene products controlling gly-
colysis, intracellular pH (pHi), angiogenesis, cell migration and
invasion, and so has become recognized as a strong promoter of
tumor growth. The pro-invasion feature of HIF-1, measured by
stimulation of Epithelial-Mesenchyme-Transition, could be seen
as an integrated program ‘designed’ for migration-induced nutri-
ent-search, as in microorganisms. It is therefore not surprising
that HIF-1 also promotes access to another source of nutrients
by inducing macro-autophagy. In this presentation, we will high-
light some of the HIF1-induced gene products – carbonic anhyd-
rases IX and XII (CAs) and monocarboxylate transporters
(MCTs) – which regulate pHi by controlling export of metaboli-
cally-generated acids (carbonic and lactic acids). We report that
targeting pHi-regulated processes severely restricts tumour
growth, a process that compromises glycolysis-generated ATP
levels. We propose that membrane-bound carbonic anhydrases
(CAIX, CAXII), monocarboxylate transporters (MCT1 and
MCT4) as well as their chaperon Basigin/EMMPRIN/CD147),
which are associated with exacerbated tumor metabolism repre-
sent new potential targets for anticancer therapy.
S04.5–4The cytokine macrophage migration inhibitoryfactor (MIF), an HIF-1a target gene, is aregulator of CLL survivalI. Shachar
Department of Immunology, Weizmann Institute of Sciences,
Rehovot, Israel
Chronic lymphocytic leukemia (CLL), the most common Western
adult leukemia, is characterized by the progressive accumulation
of small mature CD5+
B lymphocytes in the peripheral blood,
lymphoid organs, and bone marrow (BM). The main feature of
the disease is decreased apoptosis, resulting in the pathologic
accumulation of these malignant cells. Appropriate cellular
responses to changes in oxygen tension during normal develop-
ment or pathological processes, such as cardiovascular disease
and cancer, is ultimately regulated by the transcription factor,
hypoxia-inducible factor (HIF). Unlike their normal counter-
parts, CLL cells express HIF-1a even under normoxia. In addi-
tion, overexpression of HIF-1a has been observed in leukemic
cells in BM specimens from CLL patients. HIF transcription fac-
tor has been implicated in controlling the expression of a wide
variety of genes implicated in apoptosis, angiogenesis, invasion,
and metastasis. It was previously shown that HIF-1a is a potent
inducer of the expression and secretion of the proinflammatory
cytokine, macrophage migration inhibitory factor (MIF). Our
studies show an upregulation of MIF expression in CLL cells.
Binding of MIF to its receptor, CD74, induces NF-kB activation,
resulting in Bcl-2 expression and CLL cell survival. In addition,
MIF induces the expression of the immunoreceptor CD84 from
the early stages of the disease. Activation of cell surface CD84
initiates a signaling cascade that enhances CLL cell survival.
Both immune-mediated neutralization or blockade of MIF,
CD74 or CD84 induce cell death in vitro and in vivo. Thus, our
findings characterize MIF and its target genes as regulators of
CLL cell survival.
Abstracts S04 Molecular Bases of Diseases
28 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S05 Enviromental Biochemistry
S05.1 Oxidative Stress: Dealing with Oxygen(Portuguese-Brazilian Conference)
S05.1–1Protection of the photosynthetic apparatusagainst oxygen stressE.-M. Aro
Molecular Plant Biology, Department of Biochemistry and Food
Chemistry, University of Turku, Turku, Finland
Evolution of oxygenic photosynthesis in cyanobacteria required
novel ways to cope with oxygen stress. Cyanobacteria generated
a new group of flavodiiron proteins (FDP) for this purpose. In a
model cyanobacterium, Synechocystis sp. PCC 6803, four FDPs
(Flv1, Flv2, Flv3 and Flv4) function as two distinct heterodimers
is protection of photosystem (PS) II and PSI. Expression of the
flv4-sll0218-flv2 operon is strictly regulated and becomes active at
protein level only in severe stress conditions to provide protection
to PSII. Another heterodimer Flv1-Flv3 functions in accepting
electrons from PSI without formation of reactive oxygen species.
This heterodimer is crucial for protection of PSI against oxidative
damage.
The function of flavodiiron proteins in protection of the photo-
synthetic apparatus against oxidative stress is, however, energeti-
cally costly, even futile, in forwarding electrons released in water
splitting PSII further to molecular oxygen, forming water again.
In the course of evolution, the flv4-sll0218-flv2 operon disap-
peared first whereas the flv1 and flv3 orthologs have remained in
the genomes of green algae, mosses and lycophytes but are absent
from higher plant genomes. Concomitantly with disappearance of
the flavodiiron proteins, other protective mechanisms of the pho-
tosynthesic apparatus have appeared and will be discussed.
S05.1–2 (Portuguese-Brazilian Conference)Dietary interventions, mitochondria, oxidantsand lifespanA. Kowaltowski
Instituto de Quımica – IQ-USP, Sao Paulo, Brazil
Mitochondrial energy metabolism and mitochondrially-derived
oxidants have, for many years, been recognized as central toward
the effects of aging. Calorie restriction (CR) enhances animal life-
span and prevents age-related diseases, including neurological
decline. Recent evidence suggests a mechanism involved in
CR-induced lifespan extension is NO•-stimulated mitochondrial
biogenesis. We examine here the effects of CR on brain mito-
chondrial content. CR increased eNOS and nNOS and the
content of mitochondrial proteins in the brain. We established an
in vitro system to study the neurological effects of CR using
serum extracted from animals on this diet. In cultured neurons,
CR serum enhanced nNOS expression and increased nitrite levels
(a NO• product). CR serum also enhanced the levels of cyto-
chrome c oxidase and increased citrate synthase activity and
respiratory rates. CR serum effects were inhibited by L-NAME
and mimicked by the NO· donor SNAP. Furthermore, both CR
sera and SNAP were capable of improving neuronal survival.
Since eNOS is the main source of NO· involved in mitochondrial
biogenesis, we investigated the mechanism of NOS activation by
treating vascular cells with serum from CR rats and found
increased Akt and eNOS phosphorylation, in addition
to enhanced nitrite release. Inhibiting Akt phosphorylation or
immunoprecipitating adiponectin (found in high quantities in CR
serum) completely prevented the increment in nitrite release and
eNOS activation. Overall, we demonstrate that adiponectin in the
serum from CR animals increases NO· signaling by activating
the insulin pathway, resulting in enhanced mitochondrial biogen-
esis and neuronal survival.
S05.1–3Oxidative stress, antioxidants and apoptosis:impact on cancer therapyT. Ozben
Dept. of Biochemistry, Medical Faculty, Akdeniz University,
Antalya, Turkey
Numerous in vitro studies have demonstrated that a wide range
of anticancer agents generate Reactive Oxygen Species (ROS) in
malignant cells. Damage to the mitochondrial membrane via
ROS may activate the apoptotic pathway. There are conflicting
views on the concurrent use of antioxidants with conventional
cancer treatments. This argument is based on the fact that some
chemotherapy drugs generate ROS and antioxidants may inhibit
ROS and prevent cancer cells to be killed by ROS induced apop-
tosis. In order to clarify the roles of antioxidants in chemother-
apy, we investigated Quercetin, N-acetylcysteine (NAC), and
Curcumin in different cells treated with different anticancer
drugs. We studied cytotoxic activity of Topotecan and Quercetin
in human breast cancer cell lines, MCF-7 and MDA-MB-231.
We investigated the effect of NAC on doxorubicin and vincristine
cytotoxicity in MRP1 transfected (293MRP) cells. We studied the
effects of Curcumin and NAC on Bleomycin induced apoptosis
in NTera-2 and NCCIT human testicular cancer cells. Our data
indicated increased oxidative status in MCF-7 and MDA-MB-
231 cells exposed to Topotecan. Quercetin didn’t inhibit ROS
generation and enhanced cytotoxicity of Topotecan in both cells.
In contrast, NAC enhanced resistance against doxorubicine and
vincristine in MRP1 overexpressing cells. Our data showed that
Curcumin and NAC inhibit oxidative stress generated by bleomy-
cin and diminish apoptosis in testicular cancer cells. We conclude
that Quercetin, NAC and Curcumin have diverse effects in the
cytotoxicity of chemotherapeutic drugs. These studies provide a
better understanding of the apoptotic pathways which may lead
to the development of new therapies involving induction of apop-
tosis to kill cancer cells selectively.
S05.1–4Control of adrenal steroidogenesis viaH2O2-dependent, reversible inactivation ofperoxiredoxin III in mitochondriaS. G. Rhee
Ewha Womans University, Seoul, Korea
Peroxiredoxins (Prxs) catalyze the reduction of H2O2 to water,
with a conserved cysteine residue serving as the site of oxidation
by H2O2. H2O2 oxidizes the peroxidatic Cys to Cys–SOH, which
then reacts with another cysteine residue to form a disulfide that is
subsequently reduced by an electron donor. The mammalian Prx
family comprises six isoforms (PrxI–PrxVI). Prx I–IV are unique
in that the peroxidatic Cys undergoes hyperoxidation during catal-
ysis to cysteine sulfinic acid (Cys–SO2H), resulting in inactivation
of peroxidase function. Sulfinic 2-Cys Prxs are reduced back to
the active form by sulfiredoxin (Srx) in a process that consumes
S05 Enviromental Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 29
ATP and cellular thiols. The physiological significance of the
reversible formation of sulfinic 2-Cys Prx has not been known.
We now show that PrxIII in mouse adrenal cortex is inacti-
vated by H2O2 produced by cytochrome P450 enzymes during
corticosterone production stimulated by adrenocorticotropic hor-
mone. This inactivation of PrxIII triggers a sequence of events
including the accumulation of H2O2, activation of p38 mitogen-
activated protein kinase, suppression of the synthesis of steroido-
genic acute regulatory protein, and inhibition of steroidogenesis.
The levels of inactivated PrxIII, activated p38, and sulfiredoxin
undergo circadian oscillations. Steroidogenic tissue–specific abla-
tion of sulfiredoxin in mice resulted in the persistent accumula-
tion of inactive PrxIII and suppression of the adrenal circadian
rhythm of corticosterone production. The seeming imperfections
of electron leakage by cytochrome P450 and PrxIII inactivation
by its own substrate thus appear to represent an evolutionary
adaptation for feedback inhibition of steroidogenesis.
S05.2 Dealing with Osmotic Stress
S05.2–1From the biochemical lab to the field andback: development of transgenic plants withaugmented tolerance to environmentalhardshipsN. Carrillo
Faculty of Biochemical and Pharmaceutical Sciences, University of
Rosario, Rosario, Argentina
Part of the damage undergone by plants exposed to adverse
environments is caused by perturbation of electron delivery in
chloroplasts, resulting in adventitious transfer to oxygen and
build-up of reactive species that inactive all types of biomole-
cules. Plants and the microorganisms from which they evolved
(algae and cyanobacteria) display different strategies to cope with
adverse situations. Plants resort to multigenic responses involving
avoidance, scavenging, repair and reprogramming of nutrient and
water uptake. Microorganisms, instead, respond by substituting
sensitive targets by isofunctional, stress-resistant counterparts.
Here we describe a novel strategy involving introduction of a cy-
anobacterial gene encoding for a chloroplast-targeted flavodoxin
(Fld) into model plants, which led to transgenic lines with
increased tolerance to multiple sources of stress. Fld is induced
under stress situations in microorganisms but is absent in plants.
The mechanism of tolerance was studied in vitro and in vitro,
using nuclear and plastid transformation, antisense and interfer-
ence RNA technology. The results indicate that cyanobacterial
Fld is able to productively interact with chloroplast systems and
enzymes, despite eons of evolutionary divergence, restoring
proper electron delivery in plastids. The tolerant phenotype
largely stems from functional replacement of endogenous ferre-
doxin, which is stress-sensitive and declines under hostile environ-
mental situations. Both the dose and the redox state of Fld
were shown to be critical elements of tolerance, and addi-
tional manipulation of the electron transfer machinery in Fld-
expressing plants strengthened the protective effect, leading to
even higher levels of tolerance with strong biotechnological
potential.
S05.2–2How aquaporins help microbes to adapt andsurvive environmental stressesK. Lindkvist
Department of Cell and Molecular Biology, Goteborg University,
Lund, Sweden
Aquaporins are transmembrane proteins which facilitate the flow
of water through cellular membranes. Aquaglyceroporins belong
to the family of aquaporins, but they facilitate flow of uncharged
solutes, such as glycerol. An unusual characteristic of yeast aqu-
aporins and aquaglyceroporins is that they frequently contain an
extended N-terminus.
The yeast aquaglyceroporin Fps1 is particularly important for
osmo-adaptation by controlling intracellular glycerol levels dur-
ing changes in external osmolarity. Upon high osmolarity condi-
tions, yeast accumulates glycerol by increased production and by
restricting glycerol efflux through Fps1. We propose that glycerol
flux through the channel is controlled by interplay between the
trans-membrane helices and the termini. This mechanism enables
yeast cells to fine-tune intracellular glycerol levels. Furthermore,
we have studied the three-dimensional structure of the water
transporting aquaporin, Aqy1. Our structure reveals that the
water channel is closed by the N-terminus. Nevertheless, func-
tional assays show that Aqy1 has appreciable water transport
activity which aids survival during rapid freezing of yeast. These
findings establish that Aqy1 is a gated water channel.
Rapid freezing or thawing and sudden osmotic changes are fre-
quently encountered by micro-organism. For example, actions of
warm blooded animals in cold environments, such as breathing
and coughing can expose micro-organisms to large temperature
shocks. Likewise, dramatic changes in osmolarity arise when
microbes encounter ripe fruit or rainwater. Thus the evolution of
gated aquaporins and aquaglyceroporins would provide an eco-
nomic solution to numerous stresses associated with rapidly
changing environments, aiding the organism’s quest to adapt and
survive.
S05.2–3Control of adaptive responses to stress byHog1/p38 SAPKsC. Sole, M. Nadal, A. Duch, J. Jimenez, A. Gonzalez, N. Conde,
A. Gubern, M. Joaquin, E. Nadal and F. Posas
Cell Signaling Unit, Departament de Ciencies Experimentals i de
la Salut, Universitat Pompeu Fabra, Barcelona, Spain
Exposure of cells to increases in extracellular osmolarity results
in the activation of the Hog1/p38 family of stress-activated pro-
tein kinases. Activation of these MAP kinases is required to gen-
erate a set of osmoadaptive responses essential to survive under
high osmolarity conditions. Adaptation to osmostress requires
the induction of a large number of genes, which indicates the
necessity to regulate several aspects of the cell physiology. Induc-
tion of gene expression is highly dependent on the presence of
the MAP kinase, which suggests a key role for the HOG signal-
ing pathway in the regulation of gene expression in response to
osmostress.
In response to stress, the MAPK controls several mechanisms
related to transcription initiation and elongation as well as chro-
matin organization. The MAPK also controls cell cycle. Here,
the MAPK is able to modulate cell cycle delay in different phases
which highlight the relevance of cell cycle control in response to
stress.
Abstracts S05 Enviromental Biochemistry
30 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
S05.2–4Dynamic regulation of hyperosmotic stresssignaling in the budding yeastH. Saito
Institute of Medical Science, University of Tokyo, Tokyo, Japan
When challenged with high external osmolarity, the budding yeast
Saccharomyces cerevisiae initiates an adaptive program that
includes: (i) synthesis and accumulation of the compatible osmo-
lyte glycerol; (ii) transient cell cycle arrest; (iii) transient inhibition
of protein synthesis; and (iv) a global change in gene expression
pattern. These responses are controlled by the Hog1 MAP kinase
(MAPK), which is activated by high osmolarity stimulus through
the HOG (High Osmolarity Glycerol) signal pathway. The HOG
pathway can be activated by two alternative osmosensing mecha-
nisms, termed the SLN1 branch and the SHO1 branch. A signal
emanating from either branch converges on the Pbs2 MAPK
kinase (MAPKK) that activates Hog1.
The SLN1 branch employs the Histidine kinase-based signaling
mechanism that is homologous to the bacterial two-component
systems. In contrast, the working principle of the SHO1 branch
remains relatively obscure. We have found that the activity of the
SHO1 branch is regulated by dynamic interactions among four
transmembrane proteins, namely Hkr1 and Msb2 (the putative
osmosensors), Sho1 (the membrane anchor for the Pbs2 MAP-
KK), and Opy2 (the membrane anchor for the Ste11 MAP-
KKK), as well as between these membrane proteins and their
cytoplasmic partners. In particular, I will discuss the roles of
transmembrane segments in organizing protein-protein interac-
tions that are important for activating the SHO1 branch.
S05.3 Life in Extreme Environments(in Memoriam of Costas Drainas)
S05.3–1Deciphering the role of large ATP-independentpeptidases complexes in extremophilicArchaeaA. Appolaire1, S. Gribaldo2, M. A. Dura1, E. Rosenbaum1,
V. Marty1, F. Veilleux1, E. Girard1, F. Gabel1, G. Zaccai1 and
B. Franzetti1
1Institut de Biologie Structurale, Grenoble, France, 2Institut
Pasteur, Paris, France
Intracellular proteolysis is a pivotal function in extremophiles. It
controls proteins and polypeptides breakdown for metabolic
adaptation and protein quality control under environmental
stress conditions. Among extremophiles, halophiles, are naturally
adapted to cope with multiples stresses. Neutron spectrometry
studies showed that the proteome of Halobacterium cells exhibits
a high molecular rigidity that can be associated with the peculiar
salt-dependent solubility process that was described in vitro for
several halophilic proteins. Interestingly, a moderate decrease in
external salt concentration was found to be sufficient to provoke
important perturbations in the molecular dynamics properties of
the cellular proteome (in preparation). In these conditions, we
found that the proteasome function is significantly up regulated.
Under such stress conditions, anti-proteasome drugs treatments
induced compensatory peptidase activities in high molecular
weight fractions of total cell extracts. A proteomic analysis of
these fractions led to the discovery of several self-compartmental-
ized peptidases, including a 12-subunits tetrahedral shaped com-
plex called TET. The combined X-ray and cryo-EM structural
studies of PhTET1, a homologous complex from Pyrococcus hori-
koshii, allowed to determine the internal structure of the TET
particle and to propose a novel mode of peptide processing
mechanism. The taxonomic distribution and phylogenetic analy-
sis of TET homologues revealed that the TET system is con-
served in the tree kingdom of life. It also allowed us to specify
the evolutionary history of this ancient class of enzymes and their
relationships with another large ATP-independent peptidase com-
plex call Tricorn (TRI) (In preparation). Interestingly the
genomes of hyperthermophilic euryarchaeota contain up to four
TET-like proteins. Their structural and enzymatic characteriza-
tions showed that they form an integrated peptide destruction
system. A site directed mutagenesis study revealed that the TET
can form multisubunits complexes and display different peptidase
activity depending on their oligomerization state [in preparation].
Preliminary in vivo data suggest that this may represent a way to
regulate proteolysis under limiting growth conditions.
S05.3–2TtgV a key regulator in solvent toleranceJ. L. Ramos, A. Segura, L. Molina, S. Fillet, T. Krell, P. Bernal
and E. Duque
Consejo Superior de Investigaciones Cientıficas, Estacion
Experimental del Zaidın, Department of Environmental Protection,
Granada, Spain
Bacteria have been found in all niches explored on Earth, their
ubiquity derives from their enormous metabolic diversity and
their capacity to adapt to changes in the environment. Some bac-
terial strains are able to thrive in the presence of high concentra-
tions of toxic organic chemicals, such as aromatic compounds,
aliphatic alcohols and solvents. The extrusion of these toxic com-
pounds from the cell to the external medium represents the most
relevant aspect in the solvent tolerance of bacteria, however, sol-
vent tolerance is a multifactorial process that involves a wide
range of genetic and physiological changes to overcome solvent
damage. These additional elements include reduced membrane
permeabilization, implementation of a stress response pro-
gramme, and in some cases degradation of the toxic compound.
We discuss the recent advances in our understanding of the
mechanisms involved in solvent tolerance, in particular the 3D
structure of TtgV, an IclR-family regulator that controls the
main solvent extrusion pump.
S05.3–3Ionic compatible solutes of hyperthermophiles:how do they protect cells against heat stress?H. Santos
New University of Lisbon, Oeiras, Portugal
Hyperthermophiles grow optimally at temperatures above 80�C.Most of the isolates originate from marine geothermal areas and
are slightly halophilic. Like other halophiles, they developed
strategies to balance the external osmotic pressure and the accu-
mulation of organic solutes appears to be the most common one.
In contrast to the solutes found in mesophiles, solutes from hy-
perthermophiles are generally negatively charged, and most fall
into two categories: glycosides and polyolphosphodiesters. The
most representative compound in the first category is mannosyl-
glycerate (MG) while di-myo-inositol phosphate (DIP) is the
most widespread member of the second group. Our team charac-
terised several new compatible solutes and assessed their efficacy
to protect enzymes against heat inactivation and/or aggregation
[1]. Moreover, several analogues were synthesised chemically
using the natural solutes as lead compounds [2]. The superior
protective effect of ionic solutes against thermal denaturation of
model proteins together with the increase in the intracellular level
S05 Enviromental Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 31
of some of these solutes in response to heat stress suggests their
implication in thermoprotection. The genes involved in the syn-
thesis of MG and DIP have been identified and suitable deletion
mutants were used to prove this hypothesis. Moreover, the evolu-
tionary history of the biosynthetic enzymes was investigated [3].
Finally, we used several techniques to probe the mechanisms
responsible for protein stabilization. In particular, NMR was
used to seek correlation between protein stabilization and restric-
tion of backbone and side-chain motions at different time scales
[4].
References
1. Santos et al., (2011) In Extremophiles Handbook. Chapter 4,
pp 497–520.
2. Faria et al., (2008) Carbohydr Res 343:3025–3033.
3. Goncalves et al., (2012) Environ Microbiol 14:691–701.
4. Pais et al., (2012) Prot Sci, in press.
S05.3–4Microbial biodiversity in the hyperaridAtacama DesertR. Vicuna, A. Azua-Bustos and C. Urrejola
Departamento de Genetica Molecular y Microbiologıa, Facultad de
Ciencias Biologicas, Pontificia Universidad Catolica de Chile,
Santiago, Chile
The Atacama Desert, located between 17� and 27� S latitude in
northern Chile, is the driest and probably the oldest desert on
Earth. Climate studies in its hyperarid core have shown that
rains are very scarce, averaging less that 1 mm/year. In turn, its
soils have elevated salt content and are exposed to high solar
radiation. Thus, to thrive in this region, microbial life has had to
adapt to particular niches where environmental conditions are
less severe. Caves in the Coastal Range of the desert represent
one of such niches. In one of them, we have recently described a
member of the eukaryote red algae Cyanidium, whereas in a dif-
ferent cave we found the first Dunaliella algae able to grow in a
subaerial habitat. At present we are characterizing a hypolithic
biofilm obtained from the underside of a quartz translucent
stone, also from the Coastal Range, where fog represents the
only regular source of moisture. The biofilm consists of a com-
plex association of cyanobacteria, algae, archaea and heterotro-
phic bacteria and its development relies on a positive feedback
between fog availability and the higher thermal conductivity of
the quartz rock. From this biofilm we have isolated an unre-
ported Chroococcidiopsis strain that we are using as a model to
study adaptation to low water availability. By setting a novel
controllable experimental assay, we are characterizing the physio-
logical response of this strain to desiccation. Desiccated Chroo-
coccidiopsis cells maintain their internal ultrastructure and the
integrity of their DNA and RNA. They also exhibit higher viabil-
ity than other desert strains from the same genus. Moreover, they
synthesize increasing levels of sucrose and trehalose, suggesting
active metabolism under this severe condition.
S05.4 Responding to EnvironmentalPerception
S05.4–1What’s new in Polerovirus transmission andmovement?B. Bencharki1, Z.-G. Veronique2, B. Sylvaine3 and B. Veronique3
1Faculte des Sciences et Techniques, Settat, MA, Morocco,2IBMP, Strasbourgf, France, 3INRA Colmar, Colmar, France
Polerovirus are icosaedric plant viruses with a positive RNA gen-
ome, localized in phloem cells and obligatory transmitted by
aphids in a circulative and non propagative mode. In order to
look for plant proteins potentially involved in virus transmission,
we developed two different screens (in vitro and in vivo) to iden-
tify partners of virus particles or structural viral proteins.
Using Far-Western blot on protein extracts from non-infected
cucurbit sap, we identified nine proteins able to bind in vitro
purified particles of polerovirus. Most of the proteins were
defence proteins but we also found among the candidates the
major phloem protein 2 of cucurbits, a mobile phloem lectin able
to bind viroids in vitro and in vivo. This protein could potentially
be involved in virus transport in the plant or in virus acquisition
by aphids.
The second method is based on yeast two hybrid system to
screen Arabidopsis thaliana cDNA libraries using structural viral
baits. Several candidates were identified among them cytoskele-
ton related proteins, a kinase, and a protease. The cytoskeleton
proteins could be involved in intracellular virus transport or in
cell to cell movement of the virus.
Work is in progress to confirm some of the interactions
observed between viral proteins and plant proteins. A. thaliana
knock-out mutants of the candidate gene are being tested for
viral accumulation to assess the importance of these genes in the
viral cycle. If infected, these mutants will be used as virus source
in aphid transmission experiments to evaluate the role of the can-
didates in virus acquisition by aphids. The results will be pre-
sented and discussed.
S05.4–2The function of plastid redox homeostasis forcoordinating development of plantphotosynthetic and non-photosynthetic organsF. J. Cejudo
Instituto de Bioquımica Vegetal y Fotosıntesis, Universidad de
Sevilla and CSIC, Seville, Spain
Plastids are organelles present in photosynthetic and non-
photosynthetic plant tissues. Whilst it is well known that thiore-
doxin-dependent redox regulation is essential for leaf chloroplast
function, little is known of the redox regulation in plastids of
non-photosynthetic tissues, which cannot use light as direct
source of reducing power. Thus, the question remains whether
redox regulation operates in non-photosynthetic plastid function
and how it is integrated with chloroplasts for plant growth. In
this talk I will show that NADPH-thioredoxin reductase C,
NTRC, previously reported to be exclusively expressed in green
tissues, is also expressed in non-photosynthetic tissues of Arabid-
opsis thaliana, where it is localized to plastids. Moreover, NTRC
is involved in maintaining the redox homeostasis of plastids also
in non-photosynthetic organs. To test the relationship between
plastids of photosynthetic and non-photosynthetic tissues, trans-
genic plants were obtained with redox homeostasis restituted
exclusively in leaves or in roots, through the expression of NTRC
under the control of organ-specific promoters in the ntrc mutant
Abstracts S05 Enviromental Biochemistry
32 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
background. Our results show that fully functional chloroplasts
are necessary and sufficient to support wild type rate of root
growth and lateral root formation. In contrast, fully functional
root amyloplasts are not sufficient for root, or leaf, growth unless
chloroplasts are functional. The signaling function of the chloro-
plast to coordinate growth of photosynthetic and heterotrophic
tissues during plant development will be discussed.
S05.4–3Can plants ‘think and memorize’?Exponentially integrated quantum-molecularoverall regulation of growth, photosynthesis,defence and acclimatory responses inArabidopsisS. M. Karpinski
Warsaw University of Life Sciences-SGGW, Warsaw, Poland
In a simplified model of photosynthesis, light energy absorbed
by chlorophylls of photosystem II is distributed between
photochemistry, fluorescence, and heat. Spectrally and time-
resolved fluorescence combined with foliar heat dynamics mea-
surements demonstrates that higher plants evolved genetic and
physiological overall regulatory system, which optimizes photo-
system II quantum-molecular functions and the fate of photons
absorbed in excess [1, 2]. This in turn specifically influence overall
electrochemical signalling [3] that regulate growth, acclimatory
and defense responses in Arabidopsis [4–6]. Moreover, changes in
photochemistry, water use efficiency, hormonal and reactive oxy-
gen species cellular homeostasis, and seed yield of Arabidopsis
can be defined by the exponential function and simple equation
with natural logarithm (y = y0*e-Kx), that depends on molecu-
lar regulators: Lesion Simulating Disease 1 (LSD1), Enhanced
Disease Susceptibility 1 (EDS1) and Phytoalexin Deficient 4
(PAD4) (4–6). The LSD1 recessive null mutant (lsd1) regardless
of permissive laboratory or non-permissive laboratory and field
conditions demonstrates constant seed yield, but significant varia-
tion in photochemistry and water use efficiencies, and in foliar
transcriptomes that depend on EDS1 and PAD4. Obtained
results suggest that LSD1/EDS1/PAD4 constitute at least tree
component molecular machinery regulating plant Darwinian fit-
ness. This processing allows to reach the best possible seed yield
and Darwinian fitness in multivariable natural environment.
References
1. M. Kulasek et al., submitted (2012).
2. S. Karpinski, H. et al., Science 284: 654–657 (1999).
3. M. Szechynska-Hebda et al., Plant Cell 22: 2201–2218 (2010).
4. A. Mateo, et al., Plant Physiol. 136: 2818–2830 (2004).
5. P. Muhlenbock, et al., Plant Cell 20: 2339–2356 (2008).
6. W. Wituszynska et al., submitted (2012).
S05.4–4Role of transporters in photosyntheticacclimationC. Spetea
University of Gothenburrg, Gothenburg, Sweden
Plants convert sunlight energy into chemical energy by photosyn-
thesis in chloroplasts. Since they are sessile life forms, fluctua-
tions in their immediate environment may cause inactivation of
the photosynthetic apparatus, affecting their daily productivity.
A multilevel network of acclimation strategies (at organism, cel-
lular and molecular level) helps plants to maintain an efficient
photosynthesis despite environmental changes. Important players
in photosynthetic acclimation are the solute transporters located
in the chloroplast thylakoid (photosynthetic) membrane. They
mediate exchange of solutes between the chloroplast stroma and
the thylakoid lumen. Research on thylakoid transporters is a rel-
atively young field since only a few such proteins have been char-
acterized so far [1]. In this talk, the molecular characterization of
a thylakoid ATP/ADP carrier will be presented, together with
insights into its function and evolution emerging from phyloge-
netic studies [2–4]. An outlook of photosynthetic activities that
occur inside the thylakoid lumen [1,5] and await identification of
responsible thylakoid transporters will also be provided.
References
1. Spetea and Schoefs. Comm Integr Biol 2010; 3: 122–129.
2. Thuswaldner et al. J Biol Chem 2007; 282: 8848–8859.
3. Yin et al. Plant Physiol 2010; 153: 666–677.
4. Spetea et al. Front Plant Sci 2012; 2: e110.
5. Spetea. Progress Bot 2012; 73: 207–230.
S05.5 Molecular Clocks and Cell Cycling
S05.5–1The cell cycle and the circadian clock:dynamics of two coupled cellular rhythmsA. Goldbeter
Universite Libre de Bruxelles, Brussels, Belgium
This presentation will focus on the dynamics of the cell cycle and
its entrainment by the circadian clock. I will first discuss a
detailed computational model for the network of cyclin-depen-
dent kinases (Cdks) that controls the dynamics of the mamma-
lian cell cycle [C. Gerard & A. Goldbeter (2009) PNAS 106:
21643]. The model contains four Cdk modules regulated by phos-
phorylation-dephosphorylation, association with Cdk inhibitors,
and cyclin synthesis or degradation. Growth factors trigger the
transition from a quiescent, stable steady state to self-sustained
oscillations in the Cdk network. These oscillations correspond to
the repetitive, transient activation of cyclin D/Cdk4–6 in G1, cy-
clin E/Cdk2 at the G1/S transition, cyclin A/Cdk2 in S and at
the S/G2 transition, and cyclin B/Cdk1 at the G2/M transition.
The model accounts for major properties of the mammalian cell
cycle such as continuous cell cycling in the presence of supra-
threshold amounts of growth factor, control of cell cycle progres-
sion by the balance between antagonistic effects of the tumor
suppressor pRB and the transcription factor E2F, existence of a
restriction point in G1, and endoreplication. The model for the
mammalian cell cycle shows how the regulatory structure of the
Cdk network results in its temporal self-organization, leading to
the repetitive, sequential activation of the four Cdk modules that
brings about the orderly progression through the cell cycle
phases. I will next show that the coupling of the cell cycle to the
circadian clock can lead to synchronization of these two major
cellular rhythms. Entrainment of the cell cycle by the circadian
clock may occur through several modes of coupling based on the
circadian control of cell cycle proteins such as cyclin E, kinase
Wee1, and the Cdk inhibitor p21 [C. Gerard & A. Goldbeter
(2012) PLoS Comp. Biol. in press].
S05.5–2Regulatory networks at the core of thearabidopsis circadian oscillatorP. Mas
Centre de Recerca en Agrigenomica (CRAG), Barcelona, Spain
The molecular circuitry underlying circadian rhythms relies on
the reciprocal regulation of clock components forming negative
S05 Enviromental Biochemistry Abstracts
FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS 33
feedback loops at the core of the oscillator. However, deciphering
the oscillator transcriptional regulatory code is a major challenge
due to the complex interplay among clock activators and repres-
sors, which are primarily responsible for the generation of the
loops. In our studies, we have used genome-wide binding analysis
to define the Arabidopsis circadian network structure and the
role of TOC1, a key circadian component. Analysis of TOC1
deployment across the entire genome shows binding to nearly all
oscillator genes, indicating that the feedback circuitry occurs
through an unexpectedly complex wiring of direct binding events.
Arrhythmic and constant binding of TOC1 suppresses morning
and evening oscillator expression suggesting that in contrast to
prevailing assumptions, TOC1 functions as a global oscillator
repressor. The use of steroid-inducible transgenic lines, lumines-
cence assays and mathematical modeling mechanistically validate
the direct repressive function of TOC1. Our systems-biological
approach has therefore uncovered the widespread repression
of oscillator expression by TOC1. Together with evidence in
mammals and insects showing that core components also control
a large group of oscillator genes, our results suggest that despite
the evolutionary divergence, the widespread effect of master oscil-
lator components is a conserved paradigm defining the structure
and dynamics of the eukaryotic circadian clock.
S05.5–3Linking metabolism to epigenetic controlP. Sassone-Corsi
Department of Biological Chemistry, University of California,
Irving, CA, USA
S05.5–4Novel factors linking the regulation ofpremrna splicing and circadian rhythms inArabidopsisG. Schlaen, S. P. Santangelo and M. Yanovsky
Fundacion Instituto Leloir, Buenos Aires, Argentina
Circadian clocks allow organisms to time biological processes to
appropriate phases of the day. There is increasing evidence that
proper regulation of clock function involves alterations in alter-
native splicing (AS) of clock genes, but little is known about the
mechanisms linking AS and the clock. We have recently shown
that defects in PRMT5, which transfers methyl groups to argi-
nine residues present in Sm spliceosomal proteins, impair circa-
dian rhythms in Arabidopsis. This phenotype is caused, at least
in part, by an alteration in AS of the clock gene PRR9. In mam-
mals, lack of methylation of Sm proteins leads to severe defects
in spliceosome assembly. To further explore the link between
splicing and the clock in plants we analyzed the effect of muta-
tions in genes whose homologues are known to regulate assembly
of small nuclear ribonucleopreteins (snRNPs) in mammals. In
vertebrates, snRNP assembly is mediated by the SMN complex,
composed of the proteins SMN and Gemins2–8. Mutations in
SMN and GEMIN2 like genes in Arabidopsis caused photoperi-
odic dependent developmental defects. Developmental and physi-
ological alterations included changes in leaf shape, petiole length
and flowering time. In addition, gemin2, but not smn-like mutants
displayed altered circadian rhythms. Genome-wide studies
showed that GEMIN2 contributes to the regulation of a small
subset of AS events, with minor effects on constitutive splicing.
The circadian phenotype of gemin2 mutants is consistent with
increased retention of intron 4 of the clock gene TOC1, an AS
event regulated by temperature changes. These results provide
novel mechanisms linking the circadian network to the regulation
of AS, and suggest that GEMIN2 regulation of AS of TOC1
could contribute to adjustment of the plant circadian clock.
Abstracts S05 Enviromental Biochemistry
34 FEBS Journal 279 (Suppl. 1) (2012) 6–34 ª 2012 The Authors FEBS Journal ª 2012 FEBS
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