Protein phosphorylation and other post translational modifications Flashcards
How is the dynamic-rapid response achieved?
Post translational modifications and not gene transcription regulation (takes too long)
What are post translational modifications?
Covalent modifications after translation
What is the eraser of ubiquitination?
Deubiquitinases (DUBs)
What are the 3 ubiquitin enzymes?
E1 - ubiquitin activating enzyme
E2 - ubiquitin conjugating enzyme
E3 - ubiquitin ligase
What are the 3 types of ubiquitination?
- monoubiquitylation
- multi-monoubiquitylation
- polyubiquitylation
What are the four types of conformational change?
Local disruption
Local ordering
Long-range disruption
Long-range ordering
What are the effects of PTMs?
Conformational changes
Protein:protein interactions
Examples of protein:protein interactions
14-3-3 protein
bromodomains
SH2
PHD domains
What is the writer for acetylation?
Acetyltransferase
What is the eraser for acetylation?
deacetylase
What is the writer for methylation?
methyl-transferase
What is the eraser for methylation?
amine oxidase
demethylase
deiminase
What is the writer for proline hydoxylation?
prolyl hydroxylase
What are the roles of PTMs?
Activity Localisation Stability DNA/ RNA binding Complex formation Selectivity
Example of PTM selectivity
PTM control selective activation of particular p53 target genes
Types of PTM crosswalk
Positive crosswalk
Negative crosswalk
What is positive crosswalk?
one PTM is a signal for the addition or removal of a second PTM
What is negative crosswalk?
Mutually exclusive - Direct competition for modifying a single residue
Antagonistic - indirectly by changing the recognition site for another PTM
How are PTMs measured?
Mass spec
Antibodies
What is mass spec?
An analytical tool which ionises chemical species and sorts the ions based on their mass-to-charge ratio.
Used to elucidate the chemical structures of molecules.
How does mass spec work globally and on individual proteins?
Sample undergoes gas/ liquid chromatography
Undergo ionisation interface
Undergo mass spec
How does mass spec work on only individual proteins?
Sample undergoes enzimatic digestion to form peptides
Undergoes gas/ liquid chromatography
Undergo ionisation interface
Undergo mass spec
Advantages of mass spec
- unbiased/ untargetted information
- can differentiate very similar proteins/ isoforms
- can generate a huge amount of information
- quantitative
Disadvantages of mass spec
- expensive and time consuming
- hard to pinpoint the position of the phosphorylation site
- very abundant proteins will mask low abundance proteins
- need expertise e.g. equipment
What do antibodies recognise?
the modified group and part of the peptide surrounding it
What modifications can antibodies recognise?
Methylation
Acetylation
Phosphorylation
What modifications can antibodies not be used for?
Ubiquitylation
SUMOylation
Advantages of using antibodies
- cheap if they are available
- specific
- very sensitive
- can identify subtle differences
- give quantitative information
Disadvantages of using antibodies
- need to already know where it is modified and what type of modification
- not antibodies available for each modified protein
- producing new antibodies is expensive, time consuming and not always successful
What is the basic structure of a protein kinase?
Conserved core
2 lobes
The active site (where the ATP binds) in the cleft between the two lobes
What happens in the inactive form of a kinase?
C-helix out
R-spine disassembled
Activation loop disordered
What happens in the active from of a kinase?
C-helix in
R-spine assembled
Activation loop ordered
What determines kinase specificity?
The depth of the catalytic cleft
Peptide specificity
Distal docking sites
Targetting subunits
How is peptide specificity determined?
depends on the kinase P+1 loop sequence
What is distal docking sites mediated through?
binding domains outside the kinase active site and the substrate phosphorylation site
explain targetting subunits
binding partners that contain docking domains that help to target the kinase to specific substrates
What happens to tyrosine kinases in tumour cells?
Deregulated:
- hyperactivating mutations
- amplifications/ overexpression
- loss of negative regulation
what are the two types of protein kinase inhibitors?
- Non-covalent inhibitors
- Covalent inhibitors
What are the types of non-covalent inhibitors?
Type I - ATP-competitive = bind to the active conformation
Type II - non-ATP-competitive - bind to inactive conformation
Type III and IV - non-ATP- competitive - bind outside the ATP binding site (allosteric inhibition)
Describe covalent inhibitors
Bind to the ATP-binding site
Not ATP competitive
Low selectivity
Describe the use of chemical probes
- ask a specific biological question
- need biological validation
- need specificity
- need to have a define mechanism of action
- bioavailability not needed
Describe the use of drugs
- need to be clinically safe and effective
- need clinical validation
- does not need to be specific
- do not need a define mechanism of action
- need human bioavailability and good pharmacokinetics
What are the two types of resistance?
Primary resistance
Acquired resistance
Explain primary resistance
de novo lack of treatment response
What are the types of acquired resistance?
- Target gene amplification
- Target secondary mutations
- Chromosomal alterations
- Bypass of drug alteration
Explain target gene amplification
Where the target is upregulated e.g. more kinase = drug less effective
Explain target secondary mutations
mutations which result in reduced effectiveness to the drug
Explain chromosomal alterations
Alternative splicing to hyperactive chimeric or truncated kinase
Explain bypass of drug inhibition
Amplification of downstream signalling mediator
