autophagy Flashcards
what are the three kinds of autophagy?
- Macroautophagy:
Lysosomal, used for bulk digestion - can remove whole organelles, releases molecules to support metabolism - Chaperone mediated autophagy:
also lysosomal, but only degrades individual proteins
‘Turns over’ specific, long-lived proteins
Low capacity - Proteasomes:
Machines in cell where cargo proteins get ubiquitinated and degraded
Non lysosomal, use to degrade individual proteins, major turnover route for short-lived proteins
outline how macroautophagy works
a kind of vesicle known as a phagophore begins to form from scratch (it’s a double membrane) around organelles/some contents of the cytoplasm. membrane expands, membrane expands and closes
fuses with a lysosome (this requires SNAREs)
this forms an autolysosome and the contents are degraded
what proteins are involved and what does this tell us about when autophagy is used?
many proteins are working at each individual step, such as:
HG1 - a kinase that is regulated by AMPK, AMPK responds to the amount of ATP in the cell. HG1 increases production of phagosomes
mTORC1 - a metabolism regulator, which responds to the levels of amino acids
responding to low levels of ATP and amino acids strongly suggests autophagy is activated by starvation
distinguish between phagophore, autophagosome (or just phagosome) and autolysosome
phagophore = the initial part of the vesicle as it is forming/membrane is expanding
autophagosome = the vesicle when the membrane (the phagophore) has elongated and closed
autolysosome = once the lysosome has fused with the phagosome
how is autophagy important in removing damaged/misformed products? what issues are associated with this process?
give 3 brief things
Exercise - causes damage that the body fixes
Mitochondria - most obvious example, often needs replacing?
reduced autophagy is the main cause of age related degeneration (older = reduced lysosomal capacity = less autophagy) and as we age cells accumulate more damage, but this isn’t being removed as quickly
which cells are most likely to be effected by reduced autophagy?
muscle cells and neurons - this is due to these cells being long-lived and highly metabolic so they naturally have more capacity/chance for damage
what experiment showed autophagy is triggered by starvation/fasting can lead to longer lives?
in C~elegans, there were two groups, one group with impaired eating function, this group lived longer (but did not live longer when autophagy genes were knocked out in both groups)
how is autophagy important in recycling nutrients?
how is this relevant in disease?
Upregulated in starvation, results in non-selective bulk degradation of the cytosol to keep the cell alive.
Without autophagy cells that are starving die very quickly - Mice die when they are autophagy-deficient genes via neonatal starvation
cancer cells need autophagy to survive
how is autophagy used in differentiating or remodelling cells? give two examples
Different cells require different structures, so going from one to the other requires getting rid of some things
Erythropoiesis is a key example - red blood cells being made requires removal of organelles like mitochondria
Removal of sperm-derived mitochondria - removed as only females pass on mitochondria
how is autophagy important in killing intracellular pathogens?
Some pathogens escape phagocytes and get inside the cytosol
(macro)Autophagy can destroy pathogens that get inside the host cytosol
Seen with tuberculosis, MRSA and certain viruses
what could we do with autophagy to treat/decrease chances of cancer?
to treat - inhibit autophagy in cancer cells using it to survive
to prevent - upregulate autophagy in healthy cells to increase removal of damaged components
give two other ways in which autophagy is used?
in homeostasis - maintaining a stable state
in signalling - used to remove a signal
explain how autophagy was discovered - Christian de Duve and Oshumi
Christian de Duve - electron microscopy - saw mitochondria surrounded by bigger vesicle. Won nobel prize for discovering lysosomes
1992 Oshumi - showed autophagy in yeast which have one big vacuole instead of multiple lysosomes.
Yeast strains with deficient proteases that get starved show an accumulation of material because stuff in the vacuole can’t be degraded in autophagy
1993 he performed a genetic screen to identify 15 autophagy genes
how does autophagy work selectively, rather than just by bulk degradation?
Adaptor proteins act as a bridge to recruit specific proteins you want to degrade into the autophagosome (before lysosome has fused)
(adaptor proteins need to recognise/bind to the target proteins you want to degrade) Ubiquitin is added to the target protein, so it is then recognised by adaptor protein’s ubiquitin binding domains
(adaptor proteins need to get the now bound target proteins into the autophagosome):
Ateg8 interacting motif (AIM) of the adaptor proteins binds to the atg8 protein on the membrane of the autophagosome, bringing the target into the autophagosome
***Some proteins have their own ateg8 interacting motif (AIM) to directly bind to Ateg8 on the autophagosome membrane and get directly sequestered in autophagosomes
what is a hallmark of neurodegenerative disorders?
visible under a microscope
an accumulation of ubiquitinated protein aggregates that should be degraded in autophagy - seen in Huntington’s, Parkinson’s and Alzheimer’s
what are the genetics of Huntington’s disease?
Caused by polyglutamine (polyQ) expansion in Huntington protein (CAG repeats = prone to expansion and expansion increases when a parent passes it on to a child)
The longer the expansion the higher the risk of disease (explains hereditary aspect)
Long PolyQ = misfolding of protein and aggregation
why is Huntington’s more common with age?
capacity for autophagy decreases, fewer lysosomes etc…
what SHOULD happen to the misfolded proteins?
SHOULD be ubiquitinated and if small enough degraded by proteasome, or large aggregations degraded via macro-autophagy
why does the polyQ expansion - misfolding - protein aggregates etc… actually cause the disease that it does?
poorly understood so what are some theories?
Not clear why neurons die, the function of the protein is poorly understood…
Is the issue loss of protein function?
Toxic oligomer (small aggregates that are sent to the proteasome) damaging the proteasome?
The large aggregates?
Other proteins bound to the Huntington’s protein being lost (it interacts with like 70, which could get incorporated into the aggregates)?
what is Parkinson’s? what about it’s genetics?
a neurodegenerative disorder characterised by the presence of Lewy bodies - aggregates of a-synuclein
mutations involved are often not actually in the a-synuclein gene. the genetics are quite complex and only 5-10% of cases are familial
what is an example of a mutation in a-synuclein causing Parkinson’s?
A-synuclein is normally degraded by chaperone-mediated autophagy
the ‘A53T’ mutation can stop this, causing a-synuclein to block the transporter LAMP2 in the lysosome, preventing a-synuclein from entering to be degraded, as well as other proteins too
aside from looking at a-synuclein, what is thought to potentially be a cause of Parkinson’s?
mitochondrial-derived oxidative damage
Mitochondria produce most of the reactive oxygen species and these damage the cell
If the mitochondria is not functioning correctly, these ROS can damage more cell components
what genetics have been linked to mitochondrial derived oxidative damage causing Parkinson’s?
PINK1 - a mitochondrial kinase, more common loss of function mutation (5-10%) in sporadic early onset parkinson’s
PARKIN - cytosolic E3 ubiquitin ligase in 50% autosomal recessive and 10-15% sporadic early onset parkinson’s
Should - PINK1 and PARKIN should be resulting in ubiquitination and therefore degradation of damaged mitochondria (called mitophagy)
But - loss of function/issues with either gene prevent the damaged mitochondria from being destroyed = more ROS = more damage
how can autophagy be important for cancer cell’s survival?
Tumours - hypoxic due to excessive proliferation but few blood vessels
Inner cells of solid tumours are therefore in hypoxic conditions/starving and so they need autophagy to survive (provide nutrients?(until angiogenesis occurs via HIF pathway…
inhibiting autophagy would target inner cells of tumours but other cells not in hypoxic/nutrients deprived conditions would still survive
what are beclin 1 and BCL2, and what is happening with them in autophagy?
why is this not good in cancer cells?
Beclin 1 forms part of the beclin complex (beclin 1 + lipid kinase) which produces a lipid that stimulates production of autophagosomes (autophagy)/all about cell trying to survive
Bcl2 on mitochondria should prevent activation of caspases in mitochondria, inhibiting apoptosis. So when the two are doing their thing, the cell is committed to surviving / apoptosis is being inhibited - not what you want in cancer cells
what are beclin 1 and BCL 2 doing in apoptosis?
beclin 1 and BCL 2 can directly interact, binding to one another so that neither can do their jobs for stimulating autophagosome production and inhibiting apoptosis, respectively
this means caspases are released and apoptosis occurs
what is the issue with losing beclin 1?
when apoptosis is needed, Beclin 1 and BCL 2 bind, so that caspases can be released
without beclin 1 this interaction cannot occur, so BCL 2 is always inhibiting apoptosis, meanwhile autophagosomes are not produced via Beclin 1 complex to handle damage
when is autophagy good/wanted vs harmful?
Autophagy is anti-oncogenic in healthy cells
But in tumours autophagy allows cells to survive in low oxygen and nutrients, and suppresses apoptosis = pro-oncogenic
what are some potentials and limitations of autophagy-based therapies?
Block autophagy so inner tumour cells cannot survive metabolic stress
Inhibit autophagy to increase apoptosis in chemotherapy
Elevate autophagy to remove damage to PREVENT cancer
