Proteomics

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Proteomics
Presenting Yaniv Loewenstein
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Say What?

• Proteome - the entire complement of proteins
  produced in a cell or organism.
• Proteomics an emerging scientific discipline
  encompassing any method for the large scale
  analysis of proteins.

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Why proteomics?

• Proteome analysis is conceptually attractive
  because of its potential to determine properties
  of biological systems that are not apparent by
  DNA or mRNA sequence analysis alone.

• quantity of protein expression.
• the subcellular location.
• the state of modification.
• the association with ligands.
• the rate of change with time of such properties.

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Genome vs. Proteome Complexity
The proteome is much more complex than the genome.

• Alternative Splicing.
• RNA editing.
• Alternative use of different promoters and
  terminators.
• Post Translational Modifications.

1 Gene ? various proteins
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Genome vs. Proteome Complexity (continued)

• Several genomes were solved but no eukaryotic
  proteomes yet.
• Current efforts
• PPAP - the plant proteome annotation project.
• (Arabidopsis thaliana)
• D.melanogaster , M. musculus, S.cerevisiae
  and more.
• Deinococcus radiodurans - proteome was complete
  recently.

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Major Tools

• 2D Gels.
• Pulse-Chase experiments.
• Chromatography.
• Mass Spectrometry.
• Immunoprecipitation.
• Bioinformatics and databases
• YPD, CATH, SCOPE, SWISSPROT, BLAST

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2d gel
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Pulse Chase

• QWhat is meant by a pulse-chase experiment?
• A
• Pulse A brief exposure to some labeling
  condition (i.e. radioactive labeling).
• Chase Removal of the label and observation of
  the labeled material.
• gt follow the fate of the labeled material. 

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Todays Topics

• Identification of in vivo substrates of the
  chaperonin GroEL
• Walid A. Houry , Dmitirij Frishman, Christoph
  Eckerskorn, Friedrich Lottspeich F. Ulrich
  Hartl.
• A sampling of the yeast proteome. Futcher B,
  Latter GI, Monardo P, McLaughlin CS, Garrels JI.
• Correlation between protein and mRNA abundance in
  yeast. Gygi SP, Rochon Y, Franza BR, Aebersold R

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Protein Folding

• 3D fold of a protein is determined by the
  amino-acid sequence.
• Some proteins can reach their folded states
  spontaneously.
• The efficiency of folding is often limited by the
  side reaction of aggregation.
• Misfolding and aggregation of proteins is
  prevented by molecular chaperones.

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Molecular chaperones - what?

• Definition Proteins that interact with partially
  folded or improperly folded polypepetides,
  facilitating correct folding pathways or
  providing microenvironments in which folding can
  occur.

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GroEL - I

• Essential. In E. Coli.

• Structure
• homo-oligomer.
• 2 heptamer rings.

• cofactor GroES ATP.

• some proteins may require several rounds.

• Hydrophobic interactions.

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GroEl cycle Mechanism.
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GroEl cycle Mechanism. II
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Research Goals

• Define a Group of GroEL Subtrates.
• Search for common motifs in the GroEL substrate
  proteins.

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Experiment synopsis

• Pulse-chase labeling.
• At different times of chase, cells were lysed in
  the presence of EDTA.
• GroELsubstrate complexes were isolated by
  immunoprecipitation with anti-GroEL antibodies.
• ATP GroES added in vitro.
• Total soluble cytoplasmic proteins and
  GroEL-bound proteins were separated on 2D gels.
• Control experiments.

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Substrates

• 250300 proteins out of a total of 2,500 were
  observed in complex with GroEL immediately upon
  labeling.
• About half of these proteins were still
  detectable on GroEL after 10 min of chase ,
  though in much lower amounts relative to GroEL.
• Proteins that interact only very transiently
  with GroEL may have escaped detection in this
  analysis.

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A defined set of GroEL substrates Gel
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GroEL substrates - pI and Mr
The pI distributions of total soluble cytoplasmic
proteins and of GroEL substrates were found to be
very similar.
E(total Mr) 37kD E(GroEL sub Mr) 45kD BUT
20kDltMrlt60kD
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GroEL substrates - Kinetics

• a. 160 proteins (Mr lt 60k) released completely
  with time constants between 20s and 2 min.
• b. 100 proteins (Mr lt 60k), a fraction of the
  substrates remained associated.
• c. Several larger proteins (Mr gt 60k) were
  inefficiently released from the chaperonin.

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A defined set of GroEL substrates Gel
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Additional experiments

• Proteins return to GroEL for additional cycles.
• Heat stress conditions.

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GroEL substrates Identification.

• Analyze spots - MS.
• 52 of the most abundant, were unequivocally IDed.
  
• RNA poly subunits
• E.F Tu
• tRNA synthetases
• in accord with previous studies.

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GroEl Substrates Common Motif.

• Run PSI-BLAST for consensus sequence or clusters
  of thereof
• gt 0, null, gurnicht mit gurnicht! ?
• domains were classified independently through
  sequence homology to domains in SCOP CATH.
• gt 18-24 proteins. ?
• and the plot thickens .

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A preferred structural Motif

• GroEL substrates preferentially contain several
  aß domains compared with E. coli proteins.
• Of the multidomain GroEL substrates, 13 of 17
  have at least two aß domains.
• No significant preference for other 3D
  structures was found.
• Several model substrates of GroEL, typically used
  for in vitro studies, belong to this category of
  aß proteins.

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Graph
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Substrate interaction with GroEL

• Preferred domain topology, provides insight into
  why and how these proteins interact with the
  chaperonin.
• Proteins with two or more aß domains, may be
  particularly prone to aggregation.
• Hydrophobic interactions propose possible
  mechanism.

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Possible Motivation

• Biotechnolgy
• Improve the folding efficiency of foreign
  proteins expressed in E. coli.
• Better Understanding of protein Folding
  Mechanisms in eukaryotes.

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Coffee