Autophagy Flashcards
what is autophagy?
Autophagy is a housekeeping process in cleaning up cells
- A mechanism to digest intracellular material
what is the basic process of autophagy?
- Membrane is formed in cytosol which expands and engulfs proteins/organelles of the cell
- Double-membrane cup chape expands to engulf
- The membrane captures the contents in vesicles which can fuse with lysosomes to be degraded and recycled
why do cells need degradation?
- Homeostasis – equilibrium of anabolism and catabolism
- Signalling – change protein composition of cell for signalling
- Removing damaged components – avoid DNA damage
- Recycling nutrients
- Reprogramming cells – differentiation (change cytosolic components)
what are the 4 main mechanisms of degradation?
- ubiquitin/proteasome system (UPS)
- macroautophagy
- microautophagy
- chaperone-mediated autophagy
what is ubiquitin/proteasome system (UPS)?
- Proteins in the cell are targeted for degradation by the binding of ubiquitin to amino acids on the proteins
- Proteosome recognises the ubiquitin and then degrades the proteins
- non-lysosomal
- degrades individual proteins
- major turnover route for short-lived proteins, as many proteosomes are available
what is macroautophagy?
- vesicles form in cytosol, engulf, fuse with lysosome and degrade contents
- Lysosomal
- Bulk digestion pathway
- Can remove whole organelles
- Molecules released can support metabolism e.g. amino acids, sugars
what is microautophagy?
lysosomes directly invaginate the proteins and degrade material
what is chaperone-mediated autophagy?
receptor LAMP2a on surface of lysosome recognises specific target proteins with specific amino acids, so proteins directly enter lysosome
what are the characteristics of microautophagy and chaperone-mediated autophagy?
- Lysosomal
- Only degrades individual proteins
- Turns over specific, generally long-lived proteins
- Relatively low capacity
what are the functions of macroautophagy?
- nutrient recycling
- cellular remodelling
- removal of damaged components
- killing intracellular pathogens
how is macroautophagy important in nutrient recycling?
- Autophagy is rapidly upregulated under nutrient starvation
- Causes non-selective bulk degradation of the cytosol
- Cells lacking autophagy genes die under starvation - autophagy-deficient mice die during neonatal starvation
- Cancer cells in solid tumours need autophagy to survive
how is autophagy important in cancer?
Cancer cells lack organelles and are in hypoxic conditions, so if autophagy is inhibited, cancers die
- Cancer cells in solid tumours need autophagy to survive
how is macroautophagy important in cellular remodelling?
Autophagy is the only mechanism to degrade organelles, so is essential to form some specific cell types:
- Erythropoiesis – red blood cell differentiation
- Red blood cells have no organelles, and are made up of mostly protein
- They lose their nucleus and other organelles via autophagy so that they can capture oxygen - Removal of sperm-derived mitochondria in the fertilised egg
- Mitochondrial DNA is always maternally inherited (comes from the egg)
- Paternal mito from the sperm has to be removed to prevent double mito
how is macroautophagy important in removal of damaged cellular components?
Cellular components accumulate damage over time, e.g. misfolding of proteins, oxidative damage to proteins, which must be removed
example: after exercise, muscle has suffered mechanical damage, leading to disruption of muscle tissue and swollen mito
- This triggers autophagy to remove damaged mitochondria
- Important to maintain function of cells and tissues
how is autophagy involved in ageing and development of neurodegenerative diseases?
As we age, the rate of accumulation of damage is greater than the ability of the organism to remove it by autophagy
- Lysosomal activity decreases as we age
- Expression of lysosomal genes decreases, so damage accumulates
- Reduced autophagy is the major reason for age-related degeneration
- Long-lived or highly metabolic cells such as neurons and muscle are the most susceptible t reduced autophagy and thus ageing
what is the dietary-restriction hypothesis? can autophagy make humans live longer?
Starvation/exercise -> increased autophagy -> increased damage repair and nutrient recycling
- Wildtype C. elegans live for around 20 days
- Mutant C. elegans eat-2 cannot eat the bacteria that they normally consume, and they live for longer (30 days)
- Life span increase 2-3 fold
this does not work in humans, as an inappropriate diet can lead to age-related pathologies
how is macroautophagy important in killing intracellular pathogens?
How immune cells work – engulf pathogens via phagocytosis
- many pathogens escape into the cytoplasm e.g. TB, salmonella and thus avoid phagocytosis
- They prevent phagosomes fusing with lysosome
- They cannot be engulfed again as they are now in the cell, so can replicate
But, pathogen is now susceptible to autophagy which can engulf them, fuse with lysosome and kill them (plan B)
summary of autophagy in physiology and disease - diverse roles
- Recycling nutrients - Important to survive starvation
- Used by cancer to survive low-nutrient conditions - Damaged protein/organelle removal/degradation
- Ageing, muscular dystrophy, neurodegeneration
- Too much organelle damage leads to cancer - Cellular remodelling
- Erythrocyte differentiation – removal of organelles
- Removing sperm-derived mitochondria - Intracellular pathogen removal: Tuberculosis, MRSA, viruses
why is it difficult to utilise autophagy against cancer?
- To treat cancer, we may want to inhibit autophagy to prevent its ability to survive in starving conditions
- But, to prevent cancer we may want to increase autophagy to prevent DNA damage
how can autophagy be used to prevent ageing and neurodegeneration?
In the context of ageing, muscular dystrophy and neurodegeneration, we want to boost autophagy to remove damaged proteins/organelles
how were autophagosomes identified?
- lysosomes were observed under EM
- Oshumi showed that autophagy occurred in yeast via their vacuole - a mutant yeast which was nitrogen-starved and protease deficient had a vacuole filled with damaged organelles
- 1993 genetic screens identified 15 autophagy genes
what did identification of the Atg genes allow?
- Disruption of autophagy to investigate its functions, e.g. Atg1 is a kinase, so can find out which target proteins it phosphorylates
- A start on dissecting how the machinery works
- Observation of autophagy in live cells
what is the process of macroautophagy?
- Double membrane vesicle forms
- The vesicle expands into a phagophore
- The vesicle closes into an autophagosome
- Autophagosome fuses with lysosome to form an autolysosome for degradation
what are the molecular mechanisms involved in macroautophagy?
Molecular mechanisms involves coordination of 50-60 proteins which fall into 3 regulatory groups + SNAREs:
- ULK1 is a kinase which is required for initiation of autophagy, and is connected to AMPK which responds to amino acid and sugar levels
- PI3K/Vps34 complex forms specific lipid on the phagophore to define where the autophagosome forms
- 10 final genes translate proteins which controls the addition of new lipid to the autophagosome to allow it to fuse with small vesicles and elongate
- SNAREs regulate fusion with lysosomes to trigger degradation
how is macroautophagy selective?
Ubiquitin tag makes autophagy specific
- Cell recognises damaged organelle for degradation by adding a ubiquitin onto it
- Adaptor proteins inside the cell act as scaffolding proteins with a ubiquitin-binding domain and an Atg8-interacting motif
- Atg8 is a small protein which is incorporated into the autophagosome membrane
- Adaptor proteins therefore bind the ubiquitin-tagged protein to the autophagosome
do all proteins need ubiquitination to be degraded in the autophagosome?
Some proteins already have an Atg8-interacting motif so can directly enter the autophagosome without ubiquitinoylation
what happens if autophagy is defective?
- damaged organelles/proteins accumulate, leading to ageing, muscular dystrophy and neurodegeneration
which cell types are particularly sensitive to autophagy disruption? why?
neurons
- neurons have long lives and travel long distances to spread APs
- neuronal-specific autophagy disruption in mice leads to accumulation of ubiquitinated aggregates and increased apoptosis
give examples of neurodegenerative proteinopathies:
- Huntingtin aggregates in Huntington’s
- Amyloid-beta plaques in Alzheimer’s
- Alpha-synuclein in Parkinson’s
In the brains of patients with these conditions, they all have ubiquitin-protein aggregate accumulation
what mutation causes Huntington’s disease? how is autophagy implicated?
- Caused by polyglutamine (polyQ) expansion in huntingtin protein
- People with Huntington’s have an amplification of the polyglutamine repeats in the first exon
- Q<18 = healthy, Q>35 = disease-causing
- An increased number of Q repeats destabilises the Huntingtin protein, so it misfolds
- with ageing, these misfolded Huntingtin proteins are removed/degraded less and less due to decrease in autophagy
- There is an accumulation of misfolded Huntingtin aggregates due to a decrease in autophagic capacity
what is the mechanism of normal Huntingtin aggregation?
- PolyQ Huntingtin protein is misfolded and aggregates
- Rate of misfolding is proportional to the length of PolyQ repeats - Cells accumulate microaggregates of misfolding Huntingtin
- The misfolded aggregates will be recognised by the cell and ubiquitinated to target for degradation
- The microaggregates will normally be degraded in the proteosome - If the aggregates accumulate over a certain level, the cell forms aggresomes
- these are when adaptor proteins bind to multiple ubiquitin-proteins at once, forming a large package of aggregates called an aggresome - The aggresomes are then targeted for autophagic degradation
which parts of the Huntingtin aggregation process may become faulty in Huntington’s disease?
- It may be due to loss of function in Huntingtin protein due to its misfolding, so there is no wildtype Huntingtin available anymore
- It may be the microaggregates that are toxic which clog up the proteosomes/damage the proteosomes, so that other proteins in the cell cannot be degraded
- It may be the aggresomes which are toxic, as they package damaged aggregates up, meaning the proteins that make the aggresomes are sequestered as they are constantly bound to damaged material and not freely active
- Interacting proteins may be sequestered/degraded in aggresomes, so they cannot perform their normal roles, leading to cellular stress
what is Parkinson’s disease?
- Affects 1-2 per 1000 of the population in the UK
- Loss of dopaminergic neurons
- Main neuropathology is aggregates of α-synuclein (Lewy Bodies)
- Complex genetics - only 5-10% of cases familial
- α-synuclein itself rarely mutated
what rare mutation can cause Parkinson’s?
alpha-synuclein mutations (rare)
how does a mutation in alpha-synuclein lead to Parkinson’s?
- alpha-synuclein normally degraded by chaperone-mediated autophagy
- it is normally sent directly to the lysosome (vesicle fusion isn’t needed) and is recognise by LAMP2 receptor
- mutated version (A53T) of a-syn inhibits the CMA pathway by blocking the LAMP2 receptor
- this leads to accumulation of a-syn aggregates in cytosol
what is the more common cause of Parkinson’s?
mitochondrial dysfunction:
- Damaged mitochondria also accumulate in Parkinson’s
- Mitochondria are the main source of Reactive Oxygen Species (ROS)
- ROS damages cellular components
Parkinson’s may be caused by mitochondrial-derived oxidative damage
which genes regulate mitophagy?
- PARKIN
- PINK1
what is the role of PARKIN in mitophagy?
PARKIN gene encodes a cytosolic E3 ubiquitin ligase
- the ubiquitin ligase adds ubiquitin to proteins to target them for degradation
- the ligase does this in response to mitochondrial damage
what is the implication of PARKIN in Parkinson’s?
PARKIN is mutated in 50% of autosomal recessive and 10-15% of sporadic early-onset Parkinson’s
what is the role of PINK1 in mitophogy?
PINK1 gene encodes a mitochondrial kinase which responds to mitochondrial damage
what is the implication of PINK1 in Parkinson’s?
PINK1 has a loss of function mutation in 5-10% sporadic early-onset Parkinson’s
how may mutations in PARKIN and PINK1 lead to the neurodegenerative disease Parkinson’s?
- Kinase and ubiquitin ligase recognise depolarised mito and tag them with ubiquitin
- This allows mito to be targeted to autophagosomes
If there are mutations in the genes making these enzymes, damaged mito accumulates, meaning more ROS is produced, leading to cell damage and misfolding of proteins
- This damage feeds-back to damage more mitochondria –> degeneration and neuronal death
how is autophagy implicated in neurodegeneration?
Autophagy defects leads to cells being more susceptible to accumulation of damage, and this damage leads to neuronal death
what causes cancer?
Cancer is caused by the accumulation of DNA damage
- Cells then grow in unregulated manner
how is autophagy linked to cancer? is it oncogenic or tumour-suppressive?
autophagy is tumour suppressive:
- Autophagy decreases damaged organelles, protein toxicity and ROS in cells, so inhibits oxidative stress, DNA damage and tumourigenesis
how is Beclin 1/Bec1 (Atg6) implicated in cancer?
Bec1/Atg6 is monoallelically deleted in 40-75% of ovarian, breast and prostate carinomas
- Atg6 protein is decreased in tumours, so tumours undergo less autophagy and damage can accumulate
how may autophagy be oncogenic?
Cancers can have upregulated autophagy to survive starvation:
- Autophagy is unregulated in hypoxic, nutrient-poor tumour regions
- Autophagy helps cancer cells survive starvation
- Blocking autophagy causes necrosis or apoptosis of cancer cells
how is autophagy a difficult mechanism in treating cancer?
autophagy can both inhibit and enhance cancers (reduces DNA damage but helps them survive nutrient starvation)
how may autophagy drive tumour survival and chemotherapy resistance?
autophagy inhibits apoptosis:
- autophagosome formation is pro-cell survival as it removes damage so that cells can continue to function
- Apoptosis is a last resort and orderly cell death
- autophagy prevents apoptosis from occurring, leading to cancer survival
what are the roles of Beclin 1 and Beclin 2 in autophagy and apoptosis?
- Beclin 1 is involved in making lipid PI3P and autophagosome formation
- Beclin 2 is an anti-apoptotic protein
- Apoptosis is triggered by polarisation of mito and release of caspases into the cytosol, and this is regulated by proteins which are bound to the mitochondria
- When Bcl2 is bound to mito, it prevents mito releasing caspases, so prevents apoptosis
how are autophagy and apoptosis linked?
Beclin 1 and Beclin 2 are linked - co-regulation:
- Bcl1 and Bcl2 directly interact, so when autophagy activity is low, Bcl1 sequesters Bcl2 away from the mito
- This means that there is less autophagy, and the anti-apoptotic Bcl2 protein is no longer bound to mito, meaning caspases can be released to trigger apoptosis
how is the co-regulation of autophagy and apoptosis disrupted in cancer?
- If there is upregulation of autophagy in tumours, the apoptotic pathway is blocked
- This means that the cancers ability to survive is increased via autophagy, and the cancers ability to die is decreased as apoptosis is inhibited
- Leads to uncontrolled tumour growth
- This drives tumour survival and chemotherapy resistance
how can autophagy be anti-oncogenic?
- Cell Homeostasis
- Damage removal
- Reduced ROS/genotoxicity
- Reducing inflammation
how can autophagy be pro-oncogenic?
- Survival during oxygen or nutrient shortage
- Prevention of apoptosis
- Survival during chemotherapy
what is key in using autophagy to treat cancer or prevent cancer formation?
it is key to balance activation and inhibition of autophagy to ensure that cells do not accumulate damage, and if they do, to ensure that they undergo apoptosis
what strategies may be used for autophagy therapeutics against cancer?
- Blocking survival to metabolic stress with autophagy inhibitors
- Stop tumour from feeding on itself and surviving - Inhibit autophagy to increase apoptosis in chemotherapy
- Dissociate Beclin 2 from mito to increase apoptosis - Elevate autophagy to remove damage and prevent cancer
- Preventative method by removing damage accumulation and DNA mutation
