Protein Intrinsic Disorder, Cell Signaling and Alternative Splicing

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Protein Intrinsic Disorder, Cell Signaling and
Alternative Splicing
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Outline of Talk

• Examples of intrinsically disordered proteins
• Prediction of natural disordered regions
• Disorder and cell signaling
• Disorder and molecular recognition
• Disorder and alternative splicing
• Protein isoforms and functional diversity via the
  linkage of alternative splicing and intrinsic
  disorder

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Molecular Recognition Element (MoRE)
CDK
Cyclin A
p27kip1
3D structure from Russo A et al., Nature
382325-331 (1996)
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Disorder and Function
Category Change Examples Descriptions
Molecular Recognition D ? O 113 Inter- and Intra-protein, ssDNA, dsDNA, tRNA, rRNA, mRNA, nRNA, bilayers, ligands, co-factors, metals
Protein Modification Variable 36 Acetylation, fatty acylation, glycosylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, proteolytic digestion
Entropic Chains Variable 17 Linkers, spacers, bristles, clocks, springs, detergents, self-transport
Dunker AK et al., Adv Protein Chem 62 25-49
(2002)
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Prediction of Disorder
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www.disprot.org
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p53 MoREs
PONDR VL-XT Score
Oldfield et al., Biochemistry 44 12454-12470
(2005)
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Protein Interaction Domains
http//www.mshri.on.ca/pawson/domains.html
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GYF Domain and CD2 Chain B
Freund et al., (2002) Embo J. 215985-5995
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GYF Domain of CD2 Binding Protein
Freund et al., (1999) Nat. Struct. Biol. 6656-660
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CD2 Binding Partner of GYF Domain
Consensus sequence (GYF binding sites) has the
sequence ppppghr. The peptide in the crystal
structure has the aa sequence shrppppghrv.
Freund et al., (1999) Nat. Struct. Biol. 6656-660
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Analysis of Signaling Interactions

• Examined each interaction on Pawsons website.
• Almost all of the interactions involved ordered
  regions binding to disordered partners.
• Conclusion if Pawsons examples are typical,
  then a very significant proportion of
  protein-protein signaling interactions use
  disordered regions.

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Parallel Paradigms

• Catalysis
• AA seq ? 3-D Structure ? Function
• Signaling
• AA seq ? Disordered ? Function
• Ensemble

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Alternative Splicing and Intrinsic Disorder

• Find proteins with both ordered and disordered
  regions.
• Find mRNA alternative splicing information for
  these proteins and map to the ordered and
  disordered regions.
• For alternatively spliced regions of mRNA, do
  they code for ordered protein more often or do
  they code for disordered protein more often?

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Alternative Splicing
5 UTR
3 UTR
Coding Sequence
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Alternative Splicing
5 UTR
3 UTR
Coding Sequence
mRNA
Transcription
Translation
Protein sequence
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Alternative Splicing
5 UTR
3 UTR
Coding Sequence
mRNA 2
mRNA 1
Transcription
Translation
Isoform 1
Isoform 2
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Alternative Splicing
5 UTR
3 UTR
Coding Sequence
mRNA 2
mRNA 1
Transcription
Translation
Isoform 1
Isoform 2
AS region
Folding
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Structural Studies of AS
Structured AS regions
Pyrophosphorylase
RAC1 Tumor necrosis factor
Sulphotransferase
Glutathione S-transferase
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Studying the Relationship ID?AS
ASG (AS Gallery)
DisProt
?
SwissProt (VarSplic)
Database of proteins with experimentally
determinedstructure and disorder www.disprot.org
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Results on ASED
Distribution of structurally characterized AS
regions
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Enlarging the Dataset
ASED dataset
PONDR VSL1 ID predictor (gt 80 accuracy)
Validation
ASSP dataset 558 AS human proteins
fromSwissProt 1,266 AS regions
Analysis
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Global Results
AS regions disorder distributions in ASED and ASSP
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Alternative Splicing and Disorder

• Ordered Proteins active site residues non-local
  in sequence, become associated by protein folding
  
• Disordered Proteins and regions functional
  residues localized in squence
• Functional regions for signaling and regulation
  are located one after another
• Alternative splicing edits functional sets and
  thereby leads to regulatory and signaling
  diversity

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Breast Cancer Protein 1 (BRCA1)
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Summary

• Protein signaling interactions involve intrinsic
  disorder (ID) a high percentage of the time.
• Alternative splicing (AS) often occurs in regions
  of pre-mRNA that code for intrinsic disorder.
• AS ID facilitate regulatory and signaling
  diversity.
• Is AS ID the critical combination for the
  evolution of multi-cellular organisms?

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Acknowledgements

Temple University Zoran Obradovic Slobodan
Vucetic Vladimir Vacic Kang Peng Rockefeller
University Lilia Iakoucheva Sebat University of
Wisconsin John Markley Chris Oldfield UCSF Ethan
Garner PNNL Richard Smith Eric Ackerman
Indiana University Predrag Radivojac Pedro
Romero Marc Cortese Gerard Go Amrita Mohan Jie
Sun Siama Zaida Jack Yang University of
Idaho Celeste J. Brown Chris Williams Molecular
Kinetics Vladimir Uversky Yugong Cheng
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Support

• NSF CSE II 9711532
• NIH R01 LM007688
• USDA 2000 1740
• INGEN, Lilly Endowment
• Molecular Kinetics