L2 : Role of Interactions Domains in Signalling

Question 1

What defines a protein domain?

Answer 1

Recurring functional and evolutionary module
Compact, semi-independent unit of a protein that can fold autonomously (smallest unit of function)
40-300aa length

Question 2

What types of motifs do protein interaction domains recognise?

Answer 2

1. PTMs
2. Peptides/short motifs
3. Other domains
4. Phosphplipids (eg. DAG, PI3P)
5. Nucleic acids

Question 3

What domains recognise modified protein sequences (PTMs)?

Answer 3

SH2 (Src homology 2) - binds pTyr
Conserved pY pocket recognises pY
Binding specificity achieved by variable 3-6 residue pocket downstream of pY
PI3K p85 SH2 domain binds pYXXM sequence in PDGFR

PTB (phosphotyrosine binding) - binds pTyr
Often found in signal transduction proteins
Binding specificity determined by upstream (Nt) residues
IRS-1 binds NPXpY sequence

Bromodomain - binds acetylated Lys (remember ABC)
Generally on histones
~110 aa length

Chromodomain - binds methylated proteins
Generally on histones
40-50 aa length

Question 4

What domains mediate protein-protein interactions?

Answer 4

SH3 - recognise protein rich regions
Small (~60 aa)
Positioning of proline defines specificity of interactions
Often found in signalling pathways regulating cytoskeleton

WW domains
PDZ domains - binds S/T-X-Phi motif (phi = hydrophobic residue)

Question 5

What domains are involved in protein-phospholipid binding?

Answer 5

PH (Plekstrin homology)
Binds phosphoinositides with high affinity
Specificty for PI3P, PI4P, PIP2, PIP3
Sequestered to various cell membranes so usually associated with membranes

PX domain
C2 domain

Question 6

What is the domain organisation of signalling proteins?

Answer 6

Generally contain multiple domains (some of same nature)
Enzymatic domains mean presence of regulatory interaction domain also

SH1 = catalytic kinase domain

Question 7

What domains are involved in DNA recognition?

Answer 7

Helix-turn-helix

Zinc fingers
- Short fold stabilised by Zn2+
- Cys4 ZF
- Cys2 His2 ZF

Basic domains
1. Leucine zippers factors (bZIP)
- very 7th AA is Leu
- binding to major groove always sequence specific
2. Basic helix-loop-helix (bHLH)

High mobility group (HMG)
- bind to minor groove in non sequence specific manner
- function as enhancers instead of TFs (not initiation)

Question 8

What is the role of DNA-binding domains in TFs?

Answer 8

TFs generally have 1 or 2 DBD to ensure high sequence specificity for promoter regions

Question 9

Protein interaction domains (writers, erasers, readers)

Answer 9

• Writers:
  Add PTMs (kinases, acetylases, methylases)
• Erasers:
  Remove PTMs when no longer necessary (phosphatases, deacetylases, demethylases)
• Readers:
  Read/recognise PTMs (SH2, SH3, PH, bromo/chromodomains etc.)

Question 10

What are roles of protein domains in cellular functions?

Answer 10

• Signal transduction
• Metabolic pathways
• Cellular biosynthetic complexes
• Proteasomal degradation machinery
• Spliceosome

Question 11

What techniques are used to study PPIs and regulatory networks?

Answer 11

• Computational approaches (6BIND, DIP, MINT)
• In vitro methodologies (pull down assay, 1phage display, biacore studies, x-ray diffraction)
• In vivo methods (Y2H screens, Y3H screens, 2Split ubiquitin system)

Question 12

What are yeast hybrid (YH) screens used for?

Answer 12

Y1H = protein-DNA interactions
Y2H = PPIs
Y3H = protein-RNA interactions

Question 13

What is the principle of co-immunoprecipiation?

Answer 13

Protein complexes are isolated from solution using antibody
Antibody is specific for one protein within complexes
Can analyse by WB/mass spec (what is protein complexed with?)

Question 14

What is the principle of Y2H screens?

Answer 14

Used to detect PPIs
EXPAND

Question 15

What is the principle of pull down assays?

Answer 15

EXPAND

Question 16

What is proteomics?

Answer 16

Study of proteomes

1. Protein separation techniques
   (gel filtration, subcellular fractionation, SDS-PAGE)
2. Protein detection
   (silver staining, fluorescent dyes, immunoblotting, radiolabelling)
3. Protein identification
   (immunoblotting, sequencing by Edman degradation, mass spec)
4. Bioinformatics

Question 17

What are the applications of proteomics?

Answer 17

Understanding:
- Signalling/metabolic pathways
- PPIs in multienzyme complexes
- Protein profiles in cells/tissue under physiological/pathological conditions

Discovering:
- Novel targets for drug discovery
- Novel markers for developing diagnostic markers

Question 18

What is the link between proteomics and heart disease?

Answer 18

Heart disease is leading cause of morbidity
Molecular mechanisms of failure largely unknown
significant changes in myocardial gene and protein expression observed during cardiac dysfunction
- Cytoskeletal and myofibrillar proteins
- Proteins associated with mitochondria, metabolism, stress responses