Protein Chemistry/Proteomics/Mass spectrometry Uni. of ...research.med.helsinki.fi/corefacilities/proteinc...Protein Chemistry/Proteomics/Mass spectrometry Uni. of Helsinki zThe family

Protein Chemistry/Proteomics/Mass spectrometry Uni. of Helsinki

Towards dynamic protein networks

Maciej Lalowski

Biomedicum Helsinki,Helsinki University

Trasmed course 02/11/2010

Maciej Lalowski

Final


The functional genomic era

Therapy

Development ofcausal therapies for the society´smost devastating disorders

Draft DNAsequence of thehuman genome

~ 35,000 protein-encoding genes

~2000 knownhuman diseasegenes

The Human Genome Project

Functional genomics

approaches

Transcriptomics

Proteomics

Model organisms

Interactomics

Metabolomics

Bioinformatics

Knowledge

Gene function in healthy individuals

Gene dysfunc-tion in disease states

Molecular mechanisms of diseases


The “interactome”

interactomemaps

structure of the ribosome

ccgcctcgcc gcgacacttc acacacagcttcgcctcacc ccattacagt ctcaccacgccccgtcccct ctccgttgag ccccgcgccttcgcccgggt ggggcgctgc gctgtcagcggccttgctgt gtgaggcaga acctgcgggggcaggggcgg gctggttccc tggccagccattggcagagt ccgcaggcta gggctgtcaatcatgctggc cggcgtggcc ccgcctccgccggcgcggcc ccgcctccgc cggcgcagcgtctgggacgc aaggcgccgt gggggctgccgggacgggtc caagatggac ggccgctcaggttctgcttt tacctgcggc ccagagccccattcattgcc ccggtgctga gcggcgccgcgagtcggccc gaggcctccg gggactgccgtgccgggcgg gagaccgcca tggcgaccctggaaaagctg atgaaggcct tcgagtccctcaagtccttc cagcagcagc agcagcagcagcagcagcag cagcagcagc agcagcagcagcagcagcaa cagccgccac cgccgccgccgccgccgccg cctcctcagc ttcctcagccgccgccgcag gcacagccgc tgctgcctcagccgcagccg cccccgccgc cgcccccgccgccacccggc ccggctgtgg ctgaggagccgctgcaccga ccgtgagttt gggcccgctgcagctccctg tcccggcggg tcccaggctacggcggggat ggcggtaacc ctgcagcctgcgggccggcg acacgaaccc ccggccccgcagagacagag tgacccagca acccagagcccatgagggac acccgccccc tcctggggcgaggccttccc ccacttcagc cccgctccctcacttgggtc ttcccttgtc ctctcgcgaggggaggcaga gccttgttgg ggcctgtcctgaattcaccg aggggagtca cggcctcagc

genomic sequences

=> framework that describes all interactions that can take place in the cell


Networks

Examples:

• Internet

• Social Networks

• Neuronal Networks

Properties:

• Scale-free

• Small World


Global view of protein interaction networks:degree distribution / Small world properties

Small world phenomenon: Everyone in the world can be reached through a short chain of social acquaintances.

Six degrees of separation: (Stanley Milgram, 1967): “Two random US citizens are connected on average by a chain of six acquaintances”.

D.J. Watts and S. H. Strogatz, 1998: “Beginning with a regular lattice, the addition of a small number of random links reduces the diameter of the network (the longest direct path between any two vertices, from being very long to being very short)”.


Global view of protein interaction networks:degree distribution / scale free properties

(i) Winner takes all phenomenon: new

vertices attach preferentially to sites that

are already well connected: hubs

(ii) Rich gets richer: generative model

where „page with many in-links attracts

more in-links than a regular page“

(iii) Copy model: new node chooses an

existent node at random and copy a

fraction of the links of the existent node

(iv) However, communities of special

interests resemble normal distribution

Distrubution of links in scale free network of the Web follows a Power law (PL)

Barabasi & Alberts, Science 1999; Barabasi & Oltvai, Nature Rev. Gen. 2004

P(d) d-k, k>1 is a constant

V

E


Using PPI networks to study biological processes

Proteins interact with other proteins in assemblies and pathways to exert their biological function

the majority of cellular proteins form large interaction networks (PPI networks)

PPI networks help to understand protein function and malfunction in health and disease


Principle of the yeast two-hybrid system

Benefits: • In vivo technique• Transient and unstable interactions can be detected

• Independent of endogenous protein expression

• Fine resolution, enabling interaction mapping within proteins

Drawbacks:• Only two proteins tested at a time • Takes place in the nucleus, so many proteins are not in their native compartment

• Predicts possible interactions, but is unrelated to the physiological setting

Yeast two-hybrid system


A first map of the human interactome

• 5632 AD fusion proteins

• 4456 DNA-binding domain fusion proteins

• 25x106 protein pairs tested

• 2005: 3186 unique PPIs between 1705 proteins

• 2008: 6211 unique PPIs between 2948 proteinsStelzl, Worm, Lalowski et al. Cell, 2005

Parkinson‘s disease:


UniHI: An entry gate to the human interactome

HOMOMINTHOMOMINTORTHOLOGY

ORTHOORTHOLOGY

OPHIDORTHOLOGY

COCITTEXT-MINING

BINDLITERATURE

HPRDLITERATURE

CCSB-H1Y2H

MDCMDC--Y2H Y2H Y2H

REACTOMELITERATURE

DIPLITERATURE

253,980 interactions among 22,307 unique human proteins,http://theoderich.fb3.mdc-berlin.de:8080/unihi/?


Development of a novel conceptual framework for interactomemapping

Venkatesan et al. Nature Methods, 2009

The square of the number of genes used to generate a data set, relative to the square of the estimated number of all protein-encoding genes, represents the fraction of all pairwise combinations that can be tested or “completeness”.

Assay sensitivity, or inherent bias of an assay, is the fraction of all biophysical interactions that can possibly be identified by that assay under optimal experimental conditions.

Sampling sensitivity of a data set represents the fraction of all theoretically detectable interactions for a particular assay found under a given sampling condition.

Precision refers to the fraction of observed pairs that are true positives. False positive pairs reflect technical artifacts that are erroneously scored positive in a given assay.


Verification of PPIs by membrane Co-immunoprecipitation

Interactions between huntingtin and known interaction partners

The principle


• Adaptation of Lumier

(Barrios-Rodiles et al., Science

2005)

• Reporter assay format: readouts

for IP and Co-IP

• Determination of binding affinities

• Medium HTP (ELISA IgG-coated

plates)

=> Validation and quantification of binary interactions

Tools: PPIs validation: Quantification of PPIsHEK293 cells


A: The Y3H system can be used

to assay the competitive

binding of three proteins

B: The Y3H detects also the

protein complexes consisting

of three proteins

=> Assaying the competitive binding of three proteins or triple protein complex formation

PPIs validation: Protein complex assembly


Disease networks

• Identification of new disease genes and their prospective networks

• Identification of disease-related subnetworks

• Network-based disease classification


Properties of disease genes: differentially expressed cancer genes tend toward higher network connectivity

Both up- and down-regulated genes show significant positive correlation to protein degree, in contrast to the set of all genes on the microarray

Wachi, S., Yoneda, K., and Wu, R. 2005. Bioinformatics 21: 4205–4208.


Prediction of disease causing genes: a gene-phenotype network

Network-neighbor of a disease-causing gene is likely to cause either the same or a similar diseaseOti, M. and Brunner, H.G. 2007. Clin. Genet. 71: 1–11.


Discriminative subnetworks enriched with hallmarks of cancer

Network-based classifier to the prognosis of breast cancer metastasisChuang, H.Y., Lee, E., Liu, Y.T., Lee, D., and Ideker, T. 2007. Mol. Syst.Biol. 3: 140.

Metastatic (red) Non-metastatic (green) cancer genes.

Diamond- gene is significantly differentially expressed Circle- no significant difference in expression

Susceptibility genes-blue asterisk


PolyQ disorders

Diseases of unstable repeat expansion are a diverse group of disorders that are caused by the expansion of tri-, tetra-, or pentanucleotide repeat sequences.

Repeat expansion disorders include several spinocerebellar ataxias, Huntington disease, spinal and bulbar muscular atrophy, and dentatorubral-pallidoluysian atrophy.

These disorders share many points of pathogenic convergence, such as protein misfolding and accumulation, but also unique aspects that are determined by the normal function of the disease protein.

Everett and Wood, Brain 2004


Huntington‘s Disease

Progressive neurodegenerativedisorder with autosomal dominantinheritance

Affects one in 10,000 individuals

Characterized by personality changes,motor impairment and subcortical dementia

Generally midlife age of onset

Duration of 15-20 years

Selective neuropathology in theputamen and caudate nucleus, including intranulear inclusions

Several functions of htt can be inferred from PPI networks

HD


The first disease protein interaction network

• 35 baits and 51 preys with186 interactions

• 167 potential interaction are novel

• 16 of the novel interactions were independently verified

• 16 uncharacterized proteins were functionally annotated

•15 novel Htt-IP identified

Systematic interactionmapping is a valuabletool for the assessmentof protein function providing a basis for the understanding of human diseases

Goehler, Lalowski et al. Mol Cell, 2004


Identification of GIT1 - a novel stimulator of huntingtinaggregation

3 273 374 432 483 652 692124 231137

ARF Ank SHD1 CC PBS


ARF GTPase activating protein GIT1


GIT1 and huntingtin form complex in mammalian cells and colocalize in neuronal inclusions


SH-SY5Y cells

COS1

Human brain

R6/1 transgenic mice

HD patient

PC12 cells


Function of GIT1

• Multidomain protein

• Localizes to PSD

• Directly associates with FAK

• Regulates Arf GTPases

• Binds to paxillin, PIX and PAK

• Role in vesicle trafficking, adhesion and cytoskeletal organization

3 273 374 432 483 652 692124 231137

ARF Ank SHD1 CC PBS


GIT1 forms a complex with PIX and regulates actin dynamics

Ser/ Thr p21-activating kinase; Rac1 effector

PAK3 PAK

Oligophrenin 3

PAK3

Rho family GAPOligophrenin 1OPHN1

GEF for Rac/ Cdc42;activator of PAK

PIXARHGEF6

FunctionProteinGene

X-linked mental retardation is associated with abnormalities in dendritic spines

GIT1PBSSHD1AnkGAP

SH3 DH PH

PIX

PAKKinasecribPP PP

Rac Rac*

GDP GTP

Actin dynamics

Spine morphogenesis Synapse formation


Loss of GIT1 function may contribute to the development of HD

Exocytosis

Endocytosis

AMPARNMDR AMPAR

RecyclingRecycling

ExocytosisEndocytosis



Network-based characterization of brain-specific Huntington's disease modifiers

Interactome Transcriptome Phenome Foldome

Functional maps of disease processesIdentification of disease modulators


Hodges A et al.Hum Mol Genet.2006, 15(6):965-77

Network-based strategy for selection HD brain-specific modifiersGene Atlas: Su et. al. PNAS, 2006: ~15,000 genes

Stroedicke et al. submitted


Huntington‘s disease relevant candidate genes

Up-regulated, p<0.01 Down-regulated, p<0.01Stroedicke et al. submitted


The family is composed of five cytoplasmic phosphoproteins of high sequence homology, named CRMP1-5CRMPs are highly expressed in brain, where they form tetramersCRMPs are important for axon formation and neurite differentiationCRMPs are mediators of semaphorin induced growth cone collapse, and part of Akt/Rho/Rac and GSK3signalling

Wang & Strittmatter, 1996

D-box

1 149 157 572317 321 380 413 503 528

CRMP-binding site

T-box Proline rich extension

NLS

D-box

1 149 157 572317 321 380 413 503 528

CRMP-binding site


D-box

1 149 157 572317 321 380 413 503 528

CRMP-binding site


NLSD-box: Signal for proteolysis

T-box: Putative DNA binding domain

HA-CRMP1HA-Vector

TAP-VectorTAP-HD510Q17 kDa

64

82

64

64

- -++- - ++

-- ++

IB: Anti-HA

IB: Anti-Htt

IB: Anti-CRMP1

IB:Anti-HA

Out

put

Out

put

Inpu

tIn

put

-+- +

++++++

Anti-H

tt (Pa

b)

Anti-H

tt (NIS)

No abInp

ut(10

%)

64

220

Mouse cortex

IB: Anti-CRMP1

IB: Anti-Htt (Mab)

++

++

Anti-s

ynaptob

revin

CRMPs are multidomain proteins

A B

D-box: Signal for proteolysis

T-box: Putative DNA binding domain

HA-CRMP1HA-Vector

TAP-VectorTAP-HD510Q17 kDa

64

82

64

64

- -++- - ++

-- ++

IB: Anti-HA

IB: Anti-Htt

IB: Anti-CRMP1

IB:Anti-HA

Out

put

Out

put

Inpu

tIn

put

-+- +

++++++

Anti-H

tt (Pa

b)

Anti-H

tt (NIS)

No abInp

ut(10

%)

64

220

Mouse cortex

IB: Anti-CRMP1

IB: Anti-Htt (Mab)

++

++

Anti-s

ynaptob

revin

CRMPs are multidomain proteins

A B

The collapsin response mediator protein family


CRMP1 binds to Htt and is reduced in HD brain


Endogenous complex in the brain

Downregulation/cleavage at the protein level

Binary interaction quantified


CRMP1 reduces huntingtin aggregation and toxicity



CRMP1 expression rescues a Htt336Q128 induced eye phenotype


INCLUSIONS

Impair proteasomefunction

OLIGOMERS

Full length HttQn

Qn

Toxic Htt fragment

Protection by sequestering oligomers

Toxic by blocking axonal transport

Increase Htt levels

Metabolic effects

TranscriptionCBPSp1core transcription machinery PGC 1

Mitochondrial function

Excitotoxicity

CalpainsROS

?

?

?

?

? ?

?

?

Systematic prediction of HD modifiers



Combination of different protein interaction techniques to increase the level of confidence

Figeys, Cell Res, 2008


Gateway-Entryvector

pBTM116-D9

pcDNA3.1-PA-D57

pcDNA3.1-YFP

pcDNA3.1-GFP

pGEX-6p-D21

pDESTcopGAD426-D3

pcDNA3.1-Luc

Shuttling of human cDNAs from an entry vector into different destination plasmids using the GATEWAY® cloning technology.

Four libraries currently available:

1) Mammalian Gene Collection (MGC, USA)human ORFeome v5.1 contains app. 18,500 Full ORF Shuttle Clones http://horfdb.dfci.harvard.edu/(2) ImaGenes (Berlin, Germany):Full ORF Shuttle Clones contain the complete coding sequences for genes of interest (Human, mouse, rat).www.imagenes-bio.de/ (purchasable)3) NBRC (Japan):Human Gateway Entry Clones (33,275 clones) www.hgpd.jp/ (purchasable)4) MDC (Prof. Erich E. Wanker):The library contains ~16,000 unique genes and covers approximately 75% of the human genome (available on collaborative basis).

PPIs screening technologies: “GATEWAY recombinational cloning”


Mass spectrometry of purified complexes

FLAG affinity purificationand mass spectrometry

GST (His6)- affinity purificationand mass spectrometry

Tandem affinity purificationand mass spectrometry


Affinity support

Affinity support

Affinity support

Tag Tag

Tag

Protease cleavage

Polyacrylamide gel electrophoresis

Protein identification bymass spectrometry

Mass spectrometry of purified complexes

Affinity chromatography and mass spectrometry

•Tandem affinity purification (TAP)•High-throughput mass-spectrometricprotein complex identification (HMS-PCI)Benefits: •Several members of a complex can be tagged, giving an internal check for consistency•Detects real complexes in physiological settings

Drawbacks:•Might miss some complexes not present under the given conditions•Tagging may disturb complex formation•Loosely associated components may be washed off during purification


PPI/Genetic interactions/pathways databases• UniHi Unified Human Interactome

(http://theoderich.fb3.mdc-berlin.de:8080/unihi/?)• PINA (http://csbi.ltdk.helsinki.fi/pina/interactome.tutorial.do)• IntAct (http://www.ebi.ac.uk/intact/main.xhtml)• HPRD Human Protein Reference Database (www.hprd.org/)• BioGRID (http://thebiogrid.org/)• Reactome (http://www.reactome.org/)• BIND (www.bind.ca)• DIP (http://dip.doe-mbi.ucla.edu/dip/Main.cgi)• Others…..

• Compendium: (http://ppi.fli-leibniz.de/jcb_ppi_databases.html)Databases & Data Collections

1. Experimental2. Predictions3. Related Domain, Pathway and Network Databases4. Webtools5. Books

Protein Chemistry/Proteomics/Mass spectrometry Uni. of ...research.med.helsinki.fi/corefacilities/proteinc...Protein Chemistry/Proteomics/Mass spectrometry Uni. of Helsinki zThe family

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