Bioinformatic analysis of protein complexes

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Bioinformatic analysis of protein complexes

• Roland Krause
• Cellzome AG, Heidelberg

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Overview

• The Proteome proteins and their interactions
• The yeast proteome project at Cellzome
• Experimental data generation
• Functional analysis
• Obtaining protein complexes
• Comparing protein complexes and interacting
  proteins
• Shared components Building blocks or biochemical
  artifacts?

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Proteomics

• The study of the protein repertoire expressed in
  the cell
• Protein expression levels
• Qualitative
• Quantitative
• Localization
• Protein interactions
• Powerful tool for the elucidation of protein
  function
• Pair-wise interactions
• Protein complexes

• Protein complexes
• Visible structural bodies
• Important players in molecular life

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Acknowledgements

• Cellzome AG
• Dr. Giulio Superti-Furga
• Dr. Gitte Neubauer
• Yeast biology
• Dr. Anne-Claude Gavin
• Dr. Paola Grandi
• Dr. Christina Rau
• Mass spectrometry
• Bernhard Küster, PhD
• Markus Bösche
• Bioinformatics
• Dr. Georg Casari
• Jens Rick
• Julien Gagneur

• EMBL
• Dr. Peer Bork
• Dr. Christian von Mering
• Biozentrum Würzburg
• Prof. Dr. Thomas Dandekar
• Prof. Dr. Jörg Schultz

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The Yeast Proteome Projectat Cellzome

• Tandem Affinity Purification (TAP)
• Mass-Spectrometry (MS)

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Workflow TAP-MS

• Homologous transformation (addition of TAP-tag)
• Test for expression
• Large scale culture

• Purification
• Gel separation of complexes
• Mass spectrometry (MALDI)

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Large scale culture
Transformation
Separation
LIMS system
An integrated workflow
Mass spectrometry
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Handling laboratory informationand annotation

• Selection of protocol according to features of
  gene
• Size, membrane association
• Process information/ user management
• Annotation of new complexes and novel findings
• Collection of information for patent
• Database of known drug targets

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Key figures of the screen

• Purifications 589
• Proteins retrieved 1440, 300 novel
• Complexes discovered 232, 60 appear as novel
• Overlap to the Y2H-data 165 of 1500
  interactions
• 37 percent of proteins in complexes are shared
• Gavin, AC., Bösche, M., Krause, R., et al.
  (2002) Nature

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Functional analysis

• Protein complexes share many components
• The resulting network of complexes builds a
  higher order network
• Highly conserved and essential proteins tend to
  interact with each other
• All localizations are sampled well but for
  membrane proteins
• Small proteins are underrepresented

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Examples of new findings

• New complexes
• 90S Pre-Ribosome
• Gives rise to the primordial, nucleolar ribosome
• Third largest complex in the yeast cell
• Established functionally by Grandi et al, 2002
  (Mol. Cell.), Dragon et al, 2002, (Nature)
• COP9/Signalosome
• Missing complex known in human, fly,
  Arabidopsis
• Known to be related to the 19S regulatory part of
  the proteasome
• Shares components with the proteasome in yeast
• New interactors for known complexes
• Iwr1 with RNA polymerase II
• YFL049w with SWI/SNF complex
• Apparent underestimate of protein complexes in
  the reference literature

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A comprehensive list of protein complexes

• Cluster analysis for protein complexes

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Obtaining complexes


TAP purifications
TAP-tagged protein (entry point)
yTAP-complexes (232)
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A comprehensive list of protein complexes

• Assembly of individual interactions into
  physiological protein complexes
• Allows interpretation and annotation of results
• Manually performed for the publication in Nature
• Used known complexes as a guide
• Contains several inconsistencies
• An automatic procedure would be beneficial
• Cluster analysis
• Should preserve features of real complexes
• Possible clusters
• Of proteins
• Of purifications
• Large number of clusters compared to clustering
  of transcription profiles

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Benchmarking protein complexes

• There is no standard on comparing sets of
  complexes
• How shall we treat the intricate structure of
  protein complexes?

• Variant complexes
• RSC complex
• Lsm1-Lsm7 vs Lsm2-Lsm8
• Cyclin dependent kinases
• Megacomplexes
• Assemblies of complexes
• Transient interactions
• Definitions vary
• Kinetics
• Cell cycle
• Functional associations

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Ribosomal biogenesis
From Schafer et al, EMBO Journal (2003)
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Clustering of proteins

• Clustering of proteins
• Shared components are not preserved
• Each protein is assigned to a single complex
• Megacomplexes did not allow for a good separation
• Very few data points 80 of the proteins have
  less than 3 identifications
• Simpler approach Cluster of purifications
• A purification should contain complexes already

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Clustering of purifications
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Similarity indices for comparing complexes or
purifications

• Dice-Index
• Jaccard-Index
• na, nb Number of components in group a or b

• Geometric index
• Simpson-Index
• ni Number of components in the intersection

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Experimental complications

• Sensitive identification of background proteins
• Ribosomal proteins
• Heat shock proteins
• Abundant enzymes
• Filtering by class and detection frequency
• Missing identifications
• Differences in expression levels
• Small proteins
• Membrane associated proteins

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Refinements of similarity indices

• Normalized Dice-like index (by column)
• Normalized Simpson-like-index

f Frequency of detection
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Comparing clustering results

• Manually refined the MIPS and YPD complex sets
  for benchmark
• Parameter exploration, comparing the results to
  the benchmark set
• 80 complexes are contained in the curated
  complex set
• No increase when expanding beyond 250 complexes
• 252 complexes from the TAP set using means
  clustering and a threshold of 0.3

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Results and conclusions

• Combined HMS-PCI and the TAP set
• 494 clusters (a reasonable total number of
  complexes)
• 46 of 94 identical entry points occur in the same
  cluster
• Refining the distance index is crucial to the
  clustering
• Future work
• Clustering of proteins (bi-clustering) and
  classification of proteins
• Different clustering algorithms
• Including more information into distance measure
• Bait protein
• Refine benchmarking
• Krause R., et al. (2003) Bioinformatics.

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Comparison of protein-protein interaction screens

• Differences between individual methods and
  reference sets

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Comparison of different data sets

• Biochemical purifications
• Gavin et al. (2002) (TAP)
• Ho et al. (2002) (HMS-PCI)
• Yeast-two hybrid
• Ito et al. (2000, 2001), Uetz et al. (2000)
• mRNA-co-expression
• Eisen et al., (1998) Marcotte (2000)
• In silico predictions
• STRING (von Mering et al., (2003)
• Synthetic lethals

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Interaction density
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Functional biases
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Comparison
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Conclusions

• The overlap between the individual methods is
  surprisingly small
• Different methods complement each other
• Individual methods are not exhausted
• Single experimental methods can be as reliable as
  combined sets
• Integration
• Bader, G. and Hogue, C. (2002) Nat. Biot.
• Kemmeren H., et al. (2002) Mol. Cell
• Von Mering C., Krause, R., et al. (2002) Nature

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Shared components of protein complexes

• Biochemical artifacts or versatile building
  blocks?

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Shared components in the Cellzome screen
Co-activator of Pol II transcription
SAGA complex
Cytoskeleton
NuA4 histone acetylase
Chromatin remodelling
Histone deacetylase complex
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Motivation and approach

• Artifacts or structural principle?
• Relevance to medical target discovery
• Target to several processes
• Understanding side effects
• Evolutionary insights
• Study of known shared components

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Functional arrangements

• Dihydrolipoamide dehydrogenase (Lpd1)
• 2-Oxoglutarate dehydrogenase
• Glycine decarboxylase
• Pyruvate dehydrogenase
• Common enzymatic function

• RNA polymerases
• Shared proteins are not the business end
• Regulatory structural roles

Cramer, P., et al. (2000) Science
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Structural arrangements for shared components
Examples Spt6 Tethers exosome to the RNA
polymerase for surveillance
Examples Lsm1-7 complex Lsm2-8 complex
Examples Signaling networks
Manuscript in preparation
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Research interests
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Research interests

• Improve clustering approaches
• Find a sensible structure for protein complexes
  and their interactions
• Benchmark set of protein complexes in yeast
• Functional properties of protein complexes
• Conquering the human proteome and experimental
  planning
• Hypothesis-free research

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Thank you!
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Thank you!