Lect5: Protein degradation Flashcards
Pulse chase experiments:
- Explain the protocol:
- What did it find?
- Explain the protocol:
See picture
- What did it find?
How long degradation took, averaged accross all proteins (not looking at any one in particular)
Pulse chase experiments and protein degradation curves:
- Explain two lines on image:
- Compound testing:
- Explain two lines on image:
Proteins are either short lived or long lived, implies two pathways, or two different categories of stability.
- Compound testing:
Can test whether a compound slows or quickens the rate of protein degradation.
- Changing pulse length:
Shorter pulses result in a larger portion of the proteins being from quickly degraded category.
Autophagy (delivery of proteins to lysosomes):
- When are all three used more often?
- Microautophagy:
- Macroautophagy:
- Chaperone assisted autophagy:
- When are all three used more often?
Starvation
- Microautophagy:
1: Invagination of the lysosome membrane, consuming non-selectively regions of the cytoplasm
2: Constitutive process - Macroautophagy:
1: Double membrane (from ER or Golgi likely) wraps around an organelle, intracellular parasite or cytoplasm of interest. Form an Autophagosome.
2: Autophagosome merges with lysosome, fusing to make autophagolysosome.
3: Digestion - Chaperone assisted autophagy:
1: highly specific process, with many proteins
2: Can only target 30% of cytosolic proteins
3: A complex (hsc70) binds to specific proteins and translocates them accross the membrane
4: helps: selectively remove proteins during starvation, remove oxidatively damaged proteins, regulate cell metabolism (breaks down fat for example, build up in fat if chaperone autophagy halts)
Macroautophagy:
- Degradation is performed by?
- Control:
- Roles:
- Degradation is performed by?
acid lysosomal hydrolases
- Control:
Ø Initiated by signals leading to inhibition of mTOR signaling (signals cell has enough energy) and/or activation of AMPK signaling (signals cell lacks energy)
Ø Sequence of ubiquitin-like conjugation reactions, mediated by proteins
called ATGs (AuTophaGy-related)
- Roles:
Kill intracellular parasites
Retrieve nutrients
Kill damaged mitochondria
Upregulated after childbirth (recoup energy, remove unnecessary machinery)
Active in apoptosis
Methods to keep proteosomes from killing the cell:
They are only activated in low pH (lysosomal proteases (cathespins)
They are proenzymes, activated on excretion
They are activated by high Ca2+ (which exists extracellularly, and only transigently intracellularly)
Associates with inhibitors (seen in caspases, called proenzyme state, activated during apoptosis)
Lysosomal proteases:
Ø Detected within all vesicles of the endocytic pathway, activated by low pH
Ø Have different pH-optima and stability at various pH
Ø Can be secreted to extracellular milieu
Ø Most are ubiquitously expressed, while some have tissue specific distribution
Functions of lysosomes:
Ø Bulk protein degradation within lysosomes
Ø Antigen processing within early endosomes
Ø Proprotein and Prohormone processing at locations such as secretory vesicles
Ø Degradation of matrix constituents in the extracellular space
Ø Suspected contribution to the initiation of apoptotic processes within the
cytosol
Ubiquitin-Proteosome System (ups):
Ubiquitination:
- Monoubiquitination
- Multiubiquitination
- Polyubiquitination
- What signal marks for proteosome degradation?
- Monoubiquitination
attachment of single ubiquitin at one site
- Multiubiquitination
attachment of single ubiquitins at multiple sites sites
- Polyubiquitination
attachment of multiple ubiquitins forming a chain
- What signal marks for proteosome degradation?
Lys48 polyubiquitination
How is ubiquitination achieved:
- Bond type:
- Process:
- What gives E3 their specificity?
- Bond type:
Isopeptide, Ub must be added to Lys
- Process:
See picture.
Note:
of E1 < E2 < E3.
Adding Ub to E1 requires energy, the rest do not, as it is transfered from one to the next.
- What gives E3 their specificity?
Specific degradation signals are recognized on target proteins (may only be visible if protein is misfolded) by specific E3
Deubiquitination:
- Two times it occurs:
- Two times it occurs:
By deubiquitanating enzymes (DUBs) before they reach the proteosome
By a DUB associated with the proteosome, which allows Ub to be recycled
Ubiquitin-like proteins (UBLs)
- ATG12 conjugation system and ATG8 conjugation system:
- SUMO (small ubiquitin-like modifier):
- ATG12 conjugation system and ATG8 conjugation system:
Passed by E1 and E2 proteins to be placed on lipids, marking them for degradation.
- SUMO (small ubiquitin-like modifier):
SUMO can be added on Lys48, this stops ubiquination from occurring, preventing degradation
Proteosome:
- 20s subunit (Barrel):
- 19s subunit (Base and Cap):
- Immuninity change:
- 20s subunit (Barrel):
Open on both ends
Made of 7 alpha and 7 beta on each side.
Entrance is small (10 angstroms) so only unfolded proteins can enter
3 unique types of cleavage, 6 sites total in enzyme
Cut into little peptide chains, which are degraded or displayed as antigens
- 19s subunit (Base and Cap):
Base: which unfolds protein using ATP
Cap: removes ubiquitin for recycling
- Immuninity change:
Barrel gets some new unique beta subunits which can make different peptides for antigen display
Ways to inhibit the UPS for experimental study:
Ø Small inhibitors of the proteasome (PSI, MG132, lactacystin, epoxomycin, bortezomib etc.)
Ø RNAi of proteasome subunits or specific enzymes of the E1-E2-E3 cascade
Ø Thermosensitive mutants of E1 and E2 enzymes in yeast
Ø Overexpression of lysinless ubiquitin
Ø Small inhibitors of the proteasome (PSI, MG132, lactacystin, epoxomycin, bortezomib etc.)
Self explanatory
Ø RNAi of proteasome subunits or specific enzymes of the E1-E2-E3 cascade
See which enzyme is breaking it down
Ø Thermosensitive mutants of E1 and E2 enzymes in yeast
Allows better control of the same thing
Ø Overexpression of lysinless ubiquitin
Only one ubiquitination, can see which site it is being added to, and if its being added at all (if ubiquitin is labelled or antibody quantified)
Dysregulated activity – the wrong stuff is degraded:
- two general examples:
- How does this work in HPV?
- two general examples:
Ø Autoimmune diseases
Ø Viral infections, when viral proteins hijack the UPS
- How does this work in HPV?
ups is hijacked, and proteosomes break down p53, a tumor suppressor protein!
ERAD (ER-associated degradation):
- Define:
- CFTR:
- Define:
ER proteins which cannot fold are translocated accross membrane to proteosomes for degradation.
- CFTR:
In cystic fibrosis this happens to CFTR. Can be treated by drugs which inhibit the proteosome. This allows the CFTR to survive and make it to the surface.
