Module 1 Flashcards
Where is autophagy tightly regulated?
- Cell growth
- Development
- Disease
- Homeostasis
Homeostasis
maintenance of equilibrium or stability within the cell in response to external pressures
Basic autophagy cycle
- phagophore membrane expands around cargo
- vesicle completed forming an autophagosome
- autophagosome fuses with lysosome
- cargo is degraded
What is a lysosome
- membranous vesicle in animal cells that digests cellular material
- terminal degradative compartment in endocytosis, phagocytosis and autophagocytosis
- contains hydrolytic enzymes
What delivers contents to lysosome
endosome
Comparison of endosomes and lysosomes
- lysosome come from golgi
- endosome come from cell membrane
- endosomes contain M6P receptor
- both travel around cell via microtubular network
2 plausible theories of delivery to lysosomes from endosomes
- kiss and run
- hybrid model
What can lysosomes fuse with
- endosomes
- autophagosomes
- phagosomes
- plasma membrane
Components of a lysosome
- LAMPs
- Proton pump
- ion channel
- cholesterol transporter
- sugar transporter
- nucleoside transporter
- amino acid transporter
- SNAREs
- tethering factors
- GTPases
- motor adaptors
- signalling and transcription factors
Role of proton pump
maintains acidic pH inside lysosome for enzymes
Role of LAMPs
makes membrane robust and stable
Role of SNAREs and tethering factors
allows for membrane attachment
Role of motor adaptors
allows for movement
3 secretion and sorting pathways of proteins
- signal mediated diversion to lysosomes
- regulated secretion
- constitutive secretion
Signal mediated diversion to lysosomes
proteins with M6P receptor are diverted to lysosomes via late endosomes
How the cell is protected throughout the lysosomal loading process
- 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
Macroautophagy
autophagosomes delivers contents to endosomes or lysosomes via fusion
Microautophagy
contents are directly engulfed by lysosomes via invagination or protrusions
Chaperone mediated autophagy
- uses chaperones to identify cargo proteins which have a specific motif, these are then translocated into the lysosome
- alternative to UPS
UPS process
- 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
when does UPS fail
when proteins aggregate they then go to CMA
Baseline autophagy
randomly engulfs the contents within the area its in - IMPORTANT FOR HOUSEKEEPING
Induced autophagy
selective and usually induced via starvation etc
Membrane source from mitochondria
- 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
Membrane source from RER
- cradle on RER buds off and forms autophagosomes
- cradle forms from protein complexes at the surface inducing membrane curvature
Membrane source from golgi
ATG 9 vesicles bud off from golgi providing lipids for autophagosome
Possible membrane sources
- ER
- mitochondria
- ER-mitochondria contact site
- plasma membrane
- golgi
- ATG 9 vesicle
- recycling endosomes
Why is a double membrane important
- proteins on inner membrane can recognise specific targets for selective autophagy
- prevents leakage
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
LC3 characteristics
- has a lysine which allows for PE attachment through a ubiquitin like process
- is ubiquitin like
Why is LC3-II sometimes found on the outside of vesicles?
allows for attachment to kinesin so movement can occur
UPR response
- in response to cellular stress
- halts protein translation
- degrades misfolded proteins
- activating signalling pathways for protein folding
- restores normal function of ER
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
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
What affects transformation
environment - hypoxia, carcinogen etc
Hypoxia produces cancer survival strategies via
- increasing autophagy
- survival in low nutrient microenvironments by adaptation
- adapted cells are often more aggressive and resistant
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
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
What are PROTACs
- proteolysis targeting chimera
- utilises UPS
Components of PROTAC
- POI ligand binding domain
- E3 ligase binding domain
- linker which has to be a specific length
PROTAC E3 ligase considerations
- shape complementarity
- binding strength and affinity
- subcellular localisation for region of degradation
- cell type specific expression
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
Disadvantages of PROTAC
- possible toxicity
- linker has to be correct = difficult
- can only degrade intracellular, soluble and short lived proteins
What are LYTACs
- lysosome targeting chimera
- utilises endosome-lysosome pathway
Components of LYTACs
- POI ligand binding domain
- lysosome shuttling receptor binding domain (M6P)
- flexible linker
Possible problems with LYTAC
- possible toxicity
- immune responses
What does LYTAC target
extracellular and membrane-bound proteins
What can POI binding domains be made of
- small molecule
- antibody
- peptide
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
What are the domains of autophagy adapter proteins?
- LIR = LC3-II interaction region
- UBA = ubiquitin recognition protein - recognises polyubiquitinated cargo
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
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
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
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
