1
Q
Where is autophagy tightly regulated?
A
- Cell growth
- Development
- Disease
- Homeostasis
2
Q
Homeostasis
A
maintenance of equilibrium or stability within the cell in response to external pressures
3
Q
Basic autophagy cycle
A
- phagophore membrane expands around cargo
- vesicle completed forming an autophagosome
- autophagosome fuses with lysosome
- cargo is degraded
4
Q
What is a lysosome
A
- membranous vesicle in animal cells that digests cellular material
- terminal degradative compartment in endocytosis, phagocytosis and autophagocytosis
- contains hydrolytic enzymes
5
Q
What delivers contents to lysosome
A
endosome
6
Q
Comparison of endosomes and lysosomes
A
- lysosome come from golgi
- endosome come from cell membrane
- endosomes contain M6P receptor
- both travel around cell via microtubular network
7
Q
2 plausible theories of delivery to lysosomes from endosomes
A
- kiss and run
- hybrid model
8
Q
What can lysosomes fuse with
A
- endosomes
- autophagosomes
- phagosomes
- plasma membrane
9
Q
Components of a lysosome
A
- LAMPs
- Proton pump
- ion channel
- cholesterol transporter
- sugar transporter
- nucleoside transporter
- amino acid transporter
- SNAREs
- tethering factors
- GTPases
- motor adaptors
- signalling and transcription factors
10
Q
Role of proton pump
A
maintains acidic pH inside lysosome for enzymes
11
Q
Role of LAMPs
A
makes membrane robust and stable
12
Q
Role of SNAREs and tethering factors
A
allows for membrane attachment
13
Q
Role of motor adaptors
A
allows for movement
14
Q
3 secretion and sorting pathways of proteins
A
- signal mediated diversion to lysosomes
- regulated secretion
- constitutive secretion
15
Q
Signal mediated diversion to lysosomes
A
proteins with M6P receptor are diverted to lysosomes via late endosomes
16
Q
How the cell is protected throughout the lysosomal loading process
A
- cytosol is not the pH for the enzymes to act
- enzymes always contained within a vesicle
- vesicles themselves are resistant to enzymes due to LAMPs
17
Q
Macroautophagy
A
autophagosomes delivers contents to endosomes or lysosomes via fusion
18
Q
Microautophagy
A
contents are directly engulfed by lysosomes via invagination or protrusions
19
Q
Chaperone mediated autophagy
A
- uses chaperones to identify cargo proteins which have a specific motif, these are then translocated into the lysosome
- alternative to UPS
20
Q
UPS process
A
- ubiquitin activated via an E1 activating enzymes
- ubiquitin transferred to target protein via E2 and E3
- protein becomes poly-ubiquitinated and is degraded by the 26s proteasome
21
Q
when does UPS fail
A
when proteins aggregate they then go to CMA
22
Q
Baseline autophagy
A
randomly engulfs the contents within the area its in - IMPORTANT FOR HOUSEKEEPING
23
Q
Induced autophagy
A
selective and usually induced via starvation etc
24
Q
Membrane source from mitochondria
A
- PS translocated from ER to mitochondria
- PS to PE
- PE attached to growing autophagosome membrane from mitochondria
- LC3 attaches to PE = LC3-II
- autophagosome expands and dissociates
25
Membrane source from RER
- cradle on RER buds off and forms autophagosomes
- cradle forms from protein complexes at the surface inducing membrane curvature
26
Membrane source from golgi
ATG 9 vesicles bud off from golgi providing lipids for autophagosome
27
Possible membrane sources
- ER
- mitochondria
- ER-mitochondria contact site
- plasma membrane
- golgi
- ATG 9 vesicle
- recycling endosomes
28
Why is a double membrane important
- proteins on inner membrane can recognise specific targets for selective autophagy
- prevents leakage
29
What is P62
- an adaptor protein found on the inside of phagosomes
- contain LIR and UBA domains allowing for LC3-II and cargo attachment at either end
30
LC3 characteristics
- has a lysine which allows for PE attachment through a ubiquitin like process
- is ubiquitin like
31
Why is LC3-II sometimes found on the outside of vesicles?
allows for attachment to kinesin so movement can occur
32
UPR response
- in response to cellular stress
- halts protein translation
- degrades misfolded proteins
- activating signalling pathways for protein folding
- restores normal function of ER
33
ER autophagy Sec62
- counteracts ER growth from UPR
- conformational change in Sec62 translocation complex exposing its LIR domain
- LIR becomes phosphorylated at the serine or threonine residues increasing it binding affinity for LC3-II
- LC3-II binds and autophagosome forms engulfing ER material
34
ER autophagy FAM134B
- when ubiquitinated within RHD domain then a conformational change occurs causing clustering
- clustering causes membrane curvature
- has LIR domain allowing for LC3-II binding
35
What affects transformation
environment - hypoxia, carcinogen etc
36
Hypoxia produces cancer survival strategies via
- increasing autophagy
- survival in low nutrient microenvironments by adaptation
- adapted cells are often more aggressive and resistant
37
Reasons autophagy may suppress cancer
- stress induces P62 = increased selective autophagy = removal of damaged proteins and organelles
- prevents accumulation of genetic defects
- prevents ROS production via removal of dysfunctional mitochondria
- maintenance of normal stem cells
- preserves genomic stability
- oncogene degradation
- anti inflammatory
38
Reasons autophagy may promote cancer
- delays apoptosis via providing nutrients for metabolism
- promotes adaptation
- allows survival of dormant cells
- resistance to hypoxia and starvation
- maintenance of cancer stem cells
39
What are PROTACs
- proteolysis targeting chimera
- utilises UPS
40
Components of PROTAC
- POI ligand binding domain
- E3 ligase binding domain
- linker which has to be a specific length
41
PROTAC E3 ligase considerations
- shape complementarity
- binding strength and affinity
- subcellular localisation for region of degradation
- cell type specific expression
42
Advantages of PROTAC
- has greater specificity than some kinase inhibitors
- can be recycled so can be administered in low doses
- can target proteins which are not normally targetable by inhibitors due to lack of active sites
43
Disadvantages of PROTAC
- possible toxicity
- linker has to be correct = difficult
- can only degrade intracellular, soluble and short lived proteins
44
What are LYTACs
- lysosome targeting chimera
- utilises endosome-lysosome pathway
45
Components of LYTACs
- POI ligand binding domain
- lysosome shuttling receptor binding domain (M6P)
- flexible linker
46
Possible problems with LYTAC
- possible toxicity
- immune responses
47
What does LYTAC target
extracellular and membrane-bound proteins
48
What can POI binding domains be made of
- small molecule
- antibody
- peptide
49
why does the linker length matter for PROTACs
need to have the correct length to specifically reach the lysine residue on the POI so it can be ubiquitinated
50
What are the domains of autophagy adapter proteins?
- LIR = LC3-II interaction region
- UBA = ubiquitin recognition protein - recognises polyubiquitinated cargo
51
Computational modelling of FAM134B
- measured the time taken for vesicle completion
- compared un-ubiquitinated vs ubiquitinated
- Ubiquitinated had increased speed of vesicle completion - due to increased membrane curvature
52
Liposome experiment FAM134B
- Liposomes carried either GST-Ub, GST-RHD or GST-RHD-Ub
- GST was a proxy protein and a protein purification tag
- analysed and diameters were measured via TEM
- GST-RHD-Ub had smallest diameter due to increased membrane curvature
53
How does autophagy contribute to cellular renewal and survival
- Cellular house keeping = quality control, constant membrane renewal
- Cell survival during starvation, cell can use components released from degradation as nutrients
- Renewal of membrane proteins
54
Importance of ubiquitination process in autophagy
- selective autophagy = polyubiquitin tag is recognised via adapter proteins such as p62
- ER phagy = FAM134B, membrane curvature
- LC3 = becomes lipidated by PE at lysine residue via ub like process
Biosci 353
flashcards
Decks in class (4)
# Cards
