Proteolysis Flashcards
What is proteolysis?
The breakdown of proteins into polypeptides or amino acids, through enzymic cleavage of peptide bonds.
What is cellular proteolysis for?
Required for quality control and removal of misfolded or mutant proteins.
Full proteolytic cleaveage is the biological end point for protein activity.
What are examples of needing cellular proteolysis?
Cellular receptors
Transcription Factors
Cell cycle proteins.
What is the major site of proteolysis?
Lysosomes - contain hydrolytic enzymes involved in protein degradation into small polypeptides and proteins.
Degrade lipids, DNA, cells, organelles and pathogens.
Involved in nutrient sensing and recycling of biomolecules.
What is the 26S proteasome?
A macromolecular protein complex involved in the degradation of proteins into small polypeptides, through ubiquitin-mediated proteolysis.
What is the function of the 26S proteasome?
Quality control of protein synthesis - targets misfolded and mutant protein.
Regulates protein activities.
Recycling of peptides and amino acids.
Generation of peptides for antigen presentation.
What is ubiquitin?
A polypeptide which is covalently conjugated via its C-terminal Glycine to a lysine in the target protein or another ubiquitin to form polyubiquitin chains - Ubiquitylation.
Which poly-ubiquitylated chains are important?
Polyubiquitylated proteins with Lysine 11 or 48 ubiquitin linkages are important in being recognised by the 26S proteasome and degraded, and targeting substrates for degradation.
What are the steps of the ubiquitin proteasome system?
Activation
Conjugation
Ligation
Elongation
What is the activation and conjugation of Ubiquitin?
Ubiquitin is activated through ATP.
It is then conjugated by high energy thioester linkages and activated by the E1 enzyme.
The ubiquitin is then transferred to the conjugated enzyme by the thioester linkages.
How is ubiquitin transferred?
Ubiquitin is transferred from E2 to the substrate in the presence of E3.
Or for different types of E3, ubiquitin forms an intermediate with E3, and is then transferred to the substrate.
Allows for ubiquitilation on a lysine residue.
What is elongation of ubiquitin ligase?
The ubiquitin ligase undergoes repeated poly-ubiquitylation to form a poly-ubiquitin chain.
This is then recognised by the proteasome and degraded in an ATP dependent matter into polypeptides and amino acids.
What is the reversal of ubiquitylation?
Enzymes - deubiquitylases (DUBs) and USPs can cleave off the ubiquitin from the substrate.
Rpn 11 cleaves the chain, removes ubiquitin, and released the chain from the substrate.
What are the sub-types of ubiquitin chains?
Mono chains - for protein interactions and localisation
K48 and 11 - for proteasomal degradation.
K63 - activation, DNA repair, Lysosomal targeting
Linear chain - NF-kB activation.
How is the specificity of protein ubiquitylation controlled?
Through the massive diversity of E3 ubiquitin ligases.
There is only 1 or 2 E1.
40 E2, and these can couple with multiple E3 ligases.
There is 800 E3 ligases, which can have many susbtrates.
What is APC/C?
The Anaphase-Promoting Complex/Cyclosome, a multimeric E3 ubiquitin ligase.
APC11 possesses the ligase activity.
Regulates progression through mitosis and G1.
Ubiquitylates cell cycle proteins - cyclins A and B, and securin.
What are the activators of APC?
Cdc20 and Cdh1
Used in recruitment of substrates to APC.
Cdc20 is a proto-oncogene.
What are the E2 ubiquitin conjugating enzymes in APC?
UbcH10, also a proto-oncogene.
Ube2S
Promotes ubiquitylation of the substrate in complex with APC11
How does the APC control sister chromatin segregation in mitosis?
Once the sister chromatins are aligned on the metaphase plate, the APC is activated by Cdc20, which promotes ubiquitylation of Securin.
This allows activation of Separase, which cleaves the cohesing ring which keeps sister chromatin together, so they are pulled into separate daughter cells.
The Cdc20 is degraded by the APC, and CbH1 allows for mitotic exit by promoting degradation of Cyclin B1.
How does ubiquitin-mediated proteolysis drive the cell cycle?
Waves of cyclin synthesis and degradation drives the temporal progression of the cell cycle.
Increases in cyclin levels are controlled transcriptionally.
Reduction in cyclin leves are controlled by ubiquitin-mediated proteolysis.
How is dysregulating of E3 ubiquitin ligases linked to cancer?
Mdm2 proto-oncogenes product is overexpressed in many cancers.
BRCA1 tumour suppressor is inactivated in breast and ovarian cancer.
How is dysregulation of E3 ubiquitin ligases linked to neurodegeneration?
Parkin loss of function by mutations is linked to Parkinson’s disease.
Parkin is also a disease modifier and promotes motor neuron loss in amyotrophic lateral sclerosis (ALS).
How is dysregulation of E3 ubiquitin ligases linked to cardiovascular disease?
MuRF1 and Atrogin 1 expression is misregulated in atrophy and hypertrophy.
Parkin mediates mitrophagy in cardiac injury.
What is the structure of the 26S proteasome?
20S proteolytic core, capped by alpha chains with Beta chains in the centre.
1 or both sides of the proteasome is a 19S regulatory particle, composed of a base and a lid.
What is the function of the 26S protealytic core?
There is a mechanism for recognising the ubiquitylated substrate by the 19S recognising particle.
The substrate is then unfolded and translocated into the 20S protealytic core, where peptidases chop the the substrate into peptides, and release it from the proteasome.
What is the structure of the 20S proteasome core?
Barrel-shaped symmetrical structure
4 heteromeric rings
2 copies of each of 14 subunits - 7 a-subunits on each end, 7 B-subunits in the centre.
Proteolysis occurs in the central channel.
What are the proteolytic enzyme activities associated with the 20S proteasome?
B1 - caspase like - cleaves after acidic residues e.g. aspartic acid and glutamic acid.
B2 - Trypsin like - cleaves after basic residues e.g. lysine and arginine.
B5 - chymotrypsin - cleaves hydrophobic residues - valine and isoleucine.
What are proteasome inhibitors?
Useful in cancer therapeutics:
Induce cell growth and cell death.
Classes - peptide aldehydes, boronates, epoxyketone, B-lactone.
What is the role of the 19S lid?
The lid recognises the poly-ubiquitin chain.
3 receptors on the 26S proteasome - Rpn13, Rpn1 and Rpn10 recognise the chain.
What are chaperone proteins?
Sometimes substrates are recruited through Chaperone proteins.
This has ubiquitylated associated domains, and ubiquitin like domains, to associate with the receptors.
What is the role of the 19S base in substrate unfolding?
Ubiquitin is released and the chain is unfolded in an ATP dependent manner.
The chain is then fed thorugh the proteolytic core of the 20S proteasome.
The ATP ring forms a closed gate, and on binding of the substrate in the presence of ATP, the gate opens.
This allows feeding in of the subtrate and entry into the proteolytic core.
