Proteolysis - Controlling activity in cells Flashcards
What is classical proteolysis?
Way of degrading a protein
From protein - peptide - amino acid
What is used for cleaving proteins in classical proteolysis?
Digestive enzymes
Pepsin, trypsin, chymotrypsin
What is cellular proteolysis used for?
Down regulation/desensitisation of pathways
Activation
cell death
What are the major sites of proteolysis?
Proteome - exclusively involved in degradation of proteins and polypeptides
Lysosome - Involved in protein degradation and also degradation of cells, organelles and pathogens
What is ubiquitin?
Short 76 amino acid regulatory protein found in most eukaryotic tissues
What is the function of ubiquitin?
targeting proteins for degradation by the 26S proteasome by polyubiquitinating them
How does ubiquitylation occur?
Conjugated via Gly-76 to a lysine in either the target protein or another ubiquitin molecule
What are the main steps in the ubiquitin-Proteasome pathway?
- E1 enzyme is activated and ubiquitin added to E1
- Ubiquitin is transferred to E2 (conjugating enzyme)
- E3 ubiquitin ligase presence causes transfer of ubiquitin to substrate via ubiquitylation
- This happens a number of times causing elongation of ubiquitin chain
- Proteasomal degradation of polyubiquitylated substrate in ATP to ADP manner to inactivate the proteins function
What are the function of K11 and K48 ubiquitin linkages?
Target proteins for proteasome mediated degradation
What is important about the E3 ubiquitin ligase?
Many different E3 types allow for large diversity
What does the diversity of E3 ubiquitin ligase control?
Controls the specificity of protein ubiquitylation
What are the 3 types of E3 ubiquitin ligases?
RING E3 - allows for direct ubiquitin transfer from E2 to substrate
HECT/RBR E3 - Ubiquitin transferred to E3 before substrate
Give an example of an E3 ligase and how it is activated
Anaphase promoting complex (APC):
Multi-subunit E3 Ubiquitin ligase
Ubiquitylates key cell cycle proteins - Cyclins A & B, Securin and SKP2
What does the APC do?
Regulates progression through mitosis and G1 phase
What activates the APC?
Cdc20 and Cdh1
How does the APC control regulation of mitotic progression through ubiquitin-mediated proteolysis?
- Sister chromatics attach to mitotic spindle where the checkpoints become satisfied and recruit Cdc20 to activate APC
- APC ubiquitylates securin which inhibits separase
- Degradation of Securin allows separase to activate
- Separase can now cleave Cohesin
- The sister chromatids are separated to daughter cells
- APC converts to Cdh1 form allowing for Cyclin B1 degradation and therefore the mitotic exit
What are the E2 conjugating enzymes?
UbE2C/UbcH10
UbE2S
What do UbE2C and UbcH10 do?
Initiates ubiquitin chain formation
What does UbE2S do?
Elongates Ubiquitin chains
How does the 26S proteasome degrade a protein?
- The lid recognises ubiquitylated species
- The protein is then unfolded and translocated into the 20S proteasome core
- Here the peptidases cleave and produce small peptide products which then exit the proteome
What is the structure of the 26S proteasome?
Lid for Ubiquitnated protein recognition
20S proteasome Core
19S regulatory particle
What is the structure of the 20S proteasome Core?
Barrel shaped symmetrical structure
2 copies of each 14 distinct subunits:
7 alpha subunits on each end
7 beta subunits in the centre
Proteolysis occurs in the central channel
What does the 19S regulatory particle of the 26S proteasome do?
Recognises ubiquitylated proteins
Targets proteins to proteasome and acts as chaperones by substrate unfolding
What are the proteolytic activites associated with the 20S proteasome?
Beta1 - Caspase: cleaves after acidic residues
Beta2 - Trypsin: Cleaves after basic residues
Beta3 - Chymotrypsin: Cleaves after hydrophobic residues
How can the proteome be inhibited?
Inhibitors bind to peptidase to inhibit activity and promote apoptosis
How does the 19S base unfold the substrate before it enters to the 20S proteasome?
- After recognition ubiquitin is bound
- Ubiquitin chain is removed and protein unfolded in ATP manner
- The protein is then fed into the 20S core
What are the Deubiquitinating enzymes of the 26S proteasome?
Ubiquitin is recycled
RPN11, UCH37, USP14 cleave ubiquitin
What is the role of the immunoproteasome?
Recognises proteins made by pathogens and generates antigenic peptides to be presented on surface of an APC to CD8 T-cells
What is the role of the 11S regulatory particle of the immunoproteasome?
Forms a heptameric ring
May activate 20S proteasome activity
May act as a molecular sieve
What is the role of the Ubiquitin-Proteasome pathway?
Targeted protein degradation in a range of biological processes
What is the effect of inactivation or overexpression of E2 and E3 enzymes?
Has deleterious effects on the cells
Uncontrolled cell growth - cancer
Cell death - Neurodegeneration
What is the role of lysosomes?
Involved in protein degradation
Organelle degradation
Lipid recylcing
What is the structure of lysosomes and where are they found?
Membrane Hydrolytic enzymes (40 types)
Where are primary lysosomes formed?
From the Golgi apparatus
Where are Secondary lysosomes formed?
Formed by fusion
Which cellular pathways are coupled with lysosomes?
Phagocytosis
Autophagy
Endocytosis
What are the functions of lysosomes in endocytosis?
Lysosomes degrade ligands and receptors or aid in recycling or proteins to plasma membrane
What are the main roles of lysosome in cellular pathways?
Cellular clearance
Lysosomal exocytosis
Transcriptional Regulation
What is the role of the K63 link?
K63 polyubiquitin linked chains help in targeting proteins to lysosomes
What is the role of parkin in ubiquitylation and autophagy pathway?
PINK1 phosphorylates ubiquitin at S56 to recruit and activate parkin to damaged mitochondria
PINK1 and parkin are mutated in Parkinson’s disease
