Post-translational-modifications

Question 1

Basic principles of PTMs

Answer 1

• maintain homeostasis
• rapid responses
• dynamic-rapid response cannot be achieved using gene transcriptional regulation
• PTMs creates diversity in signalling and is particularly suitable for relaying rapid messages in the cell

Question 2

What are PTMs

Answer 2

• covalent additions introduced to amino acids
• modifications can be a small group or large polypeptide that change the physiochemical property of the modified residue
• highly dynamic and largely reversible

Question 3

Examples of enzyme writers

Answer 3

• kinase (phosphorylation)
• ubiquitin E3 ligase (ubiquitination)
• SUMO E3 ligase (SUMOylation)
• acetyltransferase (acetylation)
• methyltransferases (methylation)

Question 4

Examples of enzyme erasers

Answer 4

• phosphatase
• deubiquitinase
• deSUMOylase
• deacetylase
• demethylases

Question 5

Protein phosphorylation

Answer 5

• adding phosphate group alters shape and charge of protein

Question 6

Effects of PTMs

Answer 6

• conformational changes (long-range disruption/ordering)
• promote interaction with proteins that have affinity for modified residues

Question 7

PTMs can modulate

Answer 7

• activity: e.g. kinase activity by phosphorylation of activation loop
• localisation: e.g. by modifying/masking nuclear localisation signals
• stability: e.g. ubiquitin-mediated proteasomal degradation
• complex formation
• selectivity: e.g. promoter specificity mediated by phosphorylation

Question 8

Describe positive crosstalk

Answer 8

one PTM serves as a signal for the addition or removal of a second PTM

Question 9

Describe negative crosstalk

Answer 9

• direct competition for modification of single residue in protein
• indirectly by masking recognition site for second PTM

Question 10

Traditional methods of measuring PTMs in cells

Answer 10

• antibodies that recognise specific PTMs
• mass spectrometry

Question 11

What are modification-specific antibodies

Answer 11

• generated using modified peptide as antigen
• antibody recongises both the modified group and parts of the peptide surrounding the modified site
• widely used for phosphorylation, acetylation, and methylation analysis

Question 12

Advantages of modification-specific antibodies

Answer 12

• cheap
• can provide quantitative information
• can identify subtle differences
• very sensitive (if the antibody is good, enrichment is not needed)
• highly specific

Question 13

Disadvantages of modification specific antibodies

Answer 13

• only works if you already have information about where your protein is modified and the type of modification
• there are not antibodies available for each modified protein
• producing new antibodies is time consuming, expensive, and not always successful

Question 14

Describe mass spectrometry

Answer 14

• analytical technique that ionises chemical species and sorts the ions based on their mass-to-charge ratio
• spectra are used to elucidate the chemical structures of molecules
• can measure an individual protein or globally

Question 15

Advantages of mass spectrometry

Answer 15

• provides unbiased/untargeted information
• can differentiate very similar proteins/isoforms (i.e. substitution in a single amino acid)
• can generate a huge amount of information - e.g. it can identify different modifications in hundreds of proteins in the same sample
• can be quantitative (e.g. SILAC)

Question 16

Disadvantages of mass spectrometry

Answer 16

• can be expensive and time consuming
• is often difficult to pinpoint the position of the phosphorylation site with single-AA resolution
• very abundant proteins will mask low abundance proteins
• need the expertise (e.g. facility, collab, or company)

Question 17

What are protein kinases

Answer 17

• enzymes that catalyse the transfer of phosphate from ATP to their substrate
• 538 genes coding for kinases
• mainly Ser/Thr or Tyr
• can be clustered into groups, families, and sub-families of increasing sequence similarity and biochemical function
• kinase dendrograms show sequence similarity between kinase domains

Question 18

Structure of protein kinases

Answer 18

• kinase domains are similar
• all kinases have two lobes with the active site in the cleft between them
• active site is where ATP binds

Question 19

Protein kinases and cancer

Answer 19

• tyrosine kinase signalling pathways control the most fundamental processes of cells
• in tumour cells, tyrosine kinase activity is often deregulated due to hyper-activating mutations, amplications, or loss of negative regulation
• half of the known oncogenes are protein kinases
• good drug targets
• ~40 oncology drugs that target kinases have been approved

Question 20

Types of protein kinase inhibitors

Answer 20

Non-covalent inhibitors
- type I: ATP-competitive, bind to active formation
- type II: non-ATP competitive, bind to inactive conformation (maximise benefits, reduce drawbacks)
- type III and IV: non-ATP competitive, bind outside the ATP-binding site (allosteric, more specific)

Covalent inhibitors
- ATP-binding pocket is highly conserved among members kinases -> difficult to find selective agents
- ATP-competitive inhibitors must compete with high intracellular ATP levels

Question 21

Activity of protein kinase inhibitors

Answer 21

• primary source of information from in vitro assays
• need to be careful using this data as the specificity and potency of inhibitors in vitro do not reflect their activity and potency in cells
• cannot assume that inhibitors that work great in cells will work in vivo -> must reach target in the body in sufficient concentration and remain there long enough to see expected biologic events

Question 22

Chemical probe

Answer 22

• ask a specific biological question
• need biological validation (works in cells)
• need specificity (one target)
• need to have a define mechanism of action
• bioavailability not needed

Question 23

Drugs

Answer 23

• need to be clinically safe and effective
• need clinical validation
• do not need to be specific
• do not need to have a define mechanism of action
• needs human bioavailability and good pharmacokinetics