TtgV a key regulator in solvent tolerance

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


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



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


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.



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


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


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



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



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.



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.



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


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


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.



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


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.



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


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


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.



S04.3–4Hedgehog 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-



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-



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.



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


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


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



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



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

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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



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.



TtgV a key regulator in solvent tolerance

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