Post-translational-modifications Flashcards
- The relevance of PTMs as a way to increase protein complexity - The use of PTMs as a quick response to stimuli - Different types of PTMs and their regulation. Writers, erasers, readers - Mechanisms of protein regulation via PTMs - PTMs crosstalk
Basic principles of PTMs
- 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
What are PTMs
- 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
Examples of enzyme writers
- kinase (phosphorylation)
- ubiquitin E3 ligase (ubiquitination)
- SUMO E3 ligase (SUMOylation)
- acetyltransferase (acetylation)
- methyltransferases (methylation)
Examples of enzyme erasers
- phosphatase
- deubiquitinase
- deSUMOylase
- deacetylase
- demethylases
Protein phosphorylation
- adding phosphate group alters shape and charge of protein
Effects of PTMs
- conformational changes (long-range disruption/ordering)
- promote interaction with proteins that have affinity for modified residues
PTMs can modulate
- 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
Describe positive crosstalk
one PTM serves as a signal for the addition or removal of a second PTM
Describe negative crosstalk
- direct competition for modification of single residue in protein
- indirectly by masking recognition site for second PTM
Traditional methods of measuring PTMs in cells
- antibodies that recognise specific PTMs
- mass spectrometry
What are modification-specific antibodies
- 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
Advantages of modification-specific antibodies
- cheap
- can provide quantitative information
- can identify subtle differences
- very sensitive (if the antibody is good, enrichment is not needed)
- highly specific
Disadvantages of modification specific antibodies
- 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
Describe mass spectrometry
- 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
Advantages of mass spectrometry
- 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)
Disadvantages of mass spectrometry
- 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)
What are protein kinases
- 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
Structure of protein kinases
- kinase domains are similar
- all kinases have two lobes with the active site in the cleft between them
- active site is where ATP binds
Protein kinases and cancer
- 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
Types of protein kinase inhibitors
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
Activity of protein kinase inhibitors
- 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
Chemical probe
- 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
Drugs
- 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
