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Current Topics > Autophagy > Flashcards

Autophagy Flashcards

1
Q

Interest in autophagy

A

Cancer
Neuro degeneration
Energy metabolism and ageing
Liver disease
Innate immunity
Embryogenesis
Cardiomyopathy

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2
Q

What is autophagy

A

Self eating pathway
Maintains cell homeostasis when cell is starved of nutrients or under stress
Protects cell from damage: misfolded aggregated proteins, invading pathogens, damaged organelles (mitochondria)
Auto phagosome: double membrane organelle so unique, fuses with lysosome

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3
Q

Yeast model

A

2016 - Nobel prize
Lysosomal defect yeast- blocked pathway so couldn’t transfer onto vacuole
Looked for a sense of autophagosomes and found essential genes for autophagosome formation

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4
Q

Lysosomal degradation pathways

A

Endocytic pathway (membrane associated cargo, extra cellular molecules)

Autophagy pathway (cytosolic cargo)
Aggregated proteins, damaged organelles, pathogens, long loved proteins, bulk cytosol - double membrane

Both require lysosomal degradation of content

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5
Q

3 main types of autophagy

A

Macroautophagy - substrate encapsulated by an autophagosome that then fuses with lysosome

Microautophay - sequestration of molecules in cytosol directly by lysosomes shown in yeast (unknown in mammals)

Chaperone mediated - protein sequence motif (KFERQ) on substrate targeted by cytosolic chaperone proteins (heat shock proteins) which interact with lysosomal membrane receptor protein (LAMP2A) target directly to lysosome

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6
Q

Autophagy - use

A

Maintains cell homeostasis under times of starvation or stress
Nutrients (AA) withdrawal
Growth factor withdrawal
Mitochondrial damage - oxidative stress, production of reactive oxygen species (ROS)
Infection

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7
Q

Starvation and stress induced autophagy

A

mTORC1 = nutrient sensor
Signals to either protein translation and proliferation or signals toward autophagy
Full of nutrients, complex active, promotes protein translation and inhibits initiation complex of autophagy (ULK1/2 complex) through phosphorylation
Low nutrients so low atp so higher ratio of AMP. Activated AMPKinase which inhibits mTORC1 complex, relieving inhibitory signalling and can activate ULK1/2 by relieving phosphorylation site, autophosphorylation of ULK and other proteins within the complex

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8
Q

Under nutrient starvation

A

Required for random engulfment if cytosol by autophagy and recycling of AA to maintain cellular function

Nutrient rich = mTORC1 > s6k > s6 > cell growth, protein synthesis

Nutrient starvation = ULK1/2 & Atg13 inhibited signals released > vps34 > autophagy

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9
Q

How does spontaneous double membrane structure form

A

Cargo identification and ohagophore biogenesis, closure and maturation, lysosome fusion and degredation/recycling

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10
Q

Autophagosome biogenesis - class III PI3K(Vps34)/ULK complex

A

ULK1-Atg13-FIP200 complex
ULK1 -serine/threonine kinase
Autophosphorylation of ULK1
ULK 1 autophosphorylation/ activation of Atg13 and FIP200
ULK1 phosphorylase’s Beclin 1, Vps34 activated
Autophagy initiation

Then activated VPS34-Beclin1 complex
Vps34 phosphorylates phosphatidylinositol on 3’ position to create PI3P
Triggers recruitment of Atg proteins and initiates phagophore formation
Required for autophagosome biogenesis

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11
Q

Atg conjugation system and phagophore formation

A

2 conjugation systems -covalent attachment

Atg8 (LC3)
Convalent conjugation of Atg8 to phosphatidylethanolamine (PE) lipid
Atg4 (cytosine protease) exposes c terminal glycine on atg8 (reversible process by same enzyme)
Atg7 and 3 mediate ubiquitin like reaction convalentky attaching PE to LC3 producing lipidates LC3-II form

Atg12-atg5-atg16
Convalent conjugation if atg12 and 5, non convalently interacts with atg16
Essential for formation and elongation of autophagosome. (Acts as E3 enzyme, determining the site of Atg8 lipidation)

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12
Q

Microtubule associated protein 1A/1B - light chain 3 (LC3)

A

Specific autophagosome associated protein
2 forms:
LC3-I: cystolic, not bound to membrane (autophagy inactive)
LC3-II: lipidated, bound to autopgahomal membrane (autophagy active)
Multiple different similar genes I’m mammals

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13
Q

Experimental ways to see LC3 1 & 2

A

Western blot
LC31 - above and lighter
LC32 - below and darker

LC3 in green imagining
Growth - dull green
Starved - bright spots

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14
Q

Summary of atg5-atg12, LC3 and lysosome

A

LC3-II inserted into inner and outer leaflets of autophagosome membrane
Atg5-Atg12 complex on outer membrane and lost as autophagosome matures
LC3 and associated cargo degraded upon fusion of autophagosome with lysosome

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15
Q

Where does the membrane come from?

A

Can be anything but…
ER membrane takes up a large proportion of total intracellular membrane - good source during starvation induced autophagy (non selective)

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16
Q

Endoplasmic reticulum as a source of membrane

A

In response to starvation
Atg14 always on er
Omegasome - subdomain of er where autophagosome forms
Occurs at double FYVE-containing protein 1 (DFCP1) positive subdomains of ER- binds phosphatidylinositol 3 phosphate (PI3P)
WD-repeat domain phosphoinositide-interacting 2 (WIPI2) (essential)

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17
Q

Autophagosome maturation and degredation

A

Requires aquisition of proteins (tethering complexes and SNARE proteins) for fusion with different vesicular compartments
Gradual decrease in interns pH
Intersection of endolysosomal and autophagy pathways - essential machinery - fusion of multi vesicular bodies and endosomal with autophagosome (amphisome)
Fusion with lysosome and delivery of lysosomal proteases

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18
Q

Vesicle fusion

A

Vesicle tethering complex and motor proteins required for bringing membranes into close contact
SNARE protein complex required for vesicle and membrane fusion

Approach > tethering > SNARE assemble > fusion

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19
Q

Tethering complex and SNARE mediated vesicle fusion in autophagy

A

HOPS (honotypic fusion and protein sorting) tethering complex - multi subunit complex required for tethering of autophagosome and lysosome to promote fusion

Syntaxin 17 - SNARE present on autophagosomes required for fusion of autophagosome with lysosome (vamp8)

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20
Q

Later stage compartments in autophagy pathway

A

Fusion of lysosome and autophagosome = autolysosome

Phagophore > autophagosome > amphisome > autolydosome

21
Q

Electron microscope of autophagosome stages

A

Autophagosome - inside looks same as outside
Amphisome/autolysosome - more electron dense so appears darker (indicating degraded material)

22
Q

Non selective autophagy

A

Result of nutrient starvation, random digestion of cell cytosol (response to increase AMT/ATP levels)
ER major source of membrane

23
Q

Selective autophagy

A

Targeted again specific substrate (eg aggregated protein, damaged mitochondria, bacteria), ubiquitin-tagged for recognition
Requires specialised proteins for identification of cargo
May obtain membrane to form autophagosome from many different intracellular sources

24
Q

Selective autophagy - identifying cargo

A

Different substrates targeted for degredation by autophagy require unique recognition receptors (autophagy receptors)
Linked to LC3 on membrane

25
Q

Autophagy receptors recognise ubiquitin tagged substrates

A

Lysine63 (k63) linked ubiquitin changing + e3 ubiquitin ligate = autophagy

Lysine48 (K48) linked ubiquitin chains + E3 ubiquitin ligate = proteasome degredation

26
Q

Autophagy receptors

A

1) Function: link between autophagosome and cargo
2) Ability to interact with LC3 on autophagosome membrane and ubiquitin in substrate to be degraded
3) Specificity for different substrates based on types of ubiquitin chains on cargo or different LC3 isoforms on autophagosome membrane
4) LC3 interactions potentially regulated by phosphorylation (optineurin)

27
Q

Autophagy receptors - xenophagy

A

TAX1BP1 - green salmonella - red
Contains LC3 interaction region and ubiquitin binding domain
Recognises ubiquinated salmonella in cytosol of infected cells and targets them for degredation by autophagy
Limits hyperproliferation of salmonella so suppressing infection

28
Q

Autophagy dysfunction in disease

A

Organ specific - CNS eg azhiemers disease, Parkinson’s LUNG - asthma
INTESTINE - crohn disease

Multisystemic - CANCER - ovarian, sarcoma

29
Q

Human disease associated with autophagy dysfunction

A

Atg16L1 - autophagosome formation - T300A mutation - Crohn’s disease

Beclin 1 - autophagosome formation - monoallelix deletion - ovarian, colorectal cancers

PARK2/parkin & PARK6/PINK1- selective autophagy mitophagy induction - mitophagy induction - recessive or apprising early onset Parkinson’s

SQSTM1/p62 - selective autophagy, autophagy receptor - mutations, pagers disease of bind and amyotrophic lateral sclerosis (ALS)

UVRAG - autophagosome maturation/degredation - deletion mutation eg colorectal cancer

30
Q

Innate immune response

A

Protects against invading pathogens -
Always present, not antigen specific, no memory

Autophagy - target and degraded pathogens in cytoplasm if a cell (selective autophagy xenophagy) eg salmonella, TB,
Regulates signalling involved in innate immune response, receptors interact with partners in NF-kB pathway and regulate pro inflammatory signalling

Some pathogens have adapted mechanisms to evade some of these responses

31
Q

Salmonella infection of intestinal epithelium

A

Invasion > proliferation within epithelium (salmonella containing vacuole) > escape and infection of other cell types eg macrophages (immune response - cytokine secretion and inflammation)

NDP52, TAX1BP1, p62, optineurin recognise ubiquitin tagged substrates and recruit LC3 positive autophagosomal membrane

32
Q

Crohn’s disease (CD)

A

Chronic inflammatory bowel disease from defective innate immune response to bacteria in gut
Excessive cytokine release (IL 1b) and inflammation
Polymorphisms in immunity related GTPase family M proyein (IRGM) associated with CD risk (regulates autophagosome formation in response to cellular pathogen infection)
Threonine 300 to alanine polymorphisms in Atg16L1 (essential autophagy regulator, defective autophagy disease progression)

Mice model: Atg16L1 deficient. Fewer lyric granules in intestines that help control gut bacterium - alter host cells with bacterium

33
Q

Use of autophagy for the benefit of pathogen

A

Listeria monocytogenes bacteria
Express pore firing toxin listeriolysin O (LLO) required for life cycle (makes holes in membrane)
Listeriolysin O decouples pH gradient across membrane (low growth phase), disrupting fusion of vesicles with lysosomes allowing bacteria to replicate in autophagosome like vesicle within macrophages

34
Q

Autophagy and ageing

A

When autophagy is active:
increase cytoprotection, decreased cell attrition, reduced oncogenic transformation, decreased dysfunctional mitochondria, decreases Intracytoplasmic aggregate- prone proteins

Dysfunctional autophagy in long lived cells may lead to intracellular damage

35
Q

Neurodegeneration

A

Neuronal function is susceptible to protein toxicity due to limited self renewal
Disease characterised by accumulation of intracellular protein aggregates in the brain eg Parkinson’s - alpha synclein (A53T), Alzheimer’s disease - Tau (hyper phosphorylation)

Numerous aggregate prone proteins have roles in neuronal trafficking
Huntingtin- interacts with dyein-synaptic complex regulating transport
Tau - interacts and stabilised Microtubules
Alpha synuclei - synaptic vesicle trafficking (fusion or recycling)

36
Q

Neurodegeneration and other conditions can be caused by mutations in autophagy regulators

A

Lofora disease
Glycogen like intracellular imvlusions
MTOR pathway activated in laforin deficient cells

Lysosomal storage disorders
Dysfunctional lysosomes (accumulation of cholesterol) effects autophagosome trafficking

37
Q

ESCRT complex and membrane bending

A

Multi subunit complex of proteins required to perform unique type of membrane bending and scission
Functions during cytokinesis, inward vesicle formation in multivesicular bodies and vital budding from cells
Implicated in autophagosome closure or fusion events between autophagosome and lysosomes/endosomes

38
Q

CHMP2B and frontotemporal dementia

A

CHMP2B - escort protein part of ESCRT complex
Rare mutations in CHMP2B associated with autosomal dominant frontotemporal dementia and Neurodegeneration

Experimental: expression of CHP2B mutants cause accumulation of autophagosomes and Neurodegenerative phenotypes

39
Q

Mitophagy- autophagy if mitochondria

A

Mitochondria required for electron transport chain and ATP production
Oxidative stress, mitochondria become damaged and depolarise mitochondrial membrane triggering autophagy (defective in Parkinson’s)
Fission machinery gets rid of bad mitochondria - PINK1 recruits Parkin1 (ubiquitin E3 ligase) - mutations = early onset Parkinson’s

Parkin ubiquitylates substrates on outer membrane of mitochondria leading to recruitment of autophagy machinery (normally)

40
Q

Mitophagy - PINK1, Parkin and Parkinson’s disease

A

Normal steady state conditions: PINK1 rapidly degraded by proteasomes
Depolarisation/damage to mitochondria- PINK1 accumulates on OM activating mitophagy
phosphorylates ubiquitin on mitochondria so platform for Parkin (also phosphorylated so fully activated) more ubiquitin and Parkin key driver of recruitment of autophagy receptors, ubiquitin binding domains and LC3 interaction region recruiting autophagosomal membrane & damage signal

Mutations in PINK1 and Parkin = recessive early onset Parkinson’s disease

PINK1 = phosphate and tendon homolog induced putative kinase 1 - serine threonine kinase

41
Q

Therapeutic approach

A

Eg Tau aggregates
Induction by autophagy enhancers so take up aggregate proteins, degredation of mutant aggregate prone proteins in autolysosomes, reduction in aggregates, block neuro degeneration

42
Q

Rapamycin ( small molecule inhibitor of mTOR inhibitor)

A

Inhibits mTORC causing autophagy of small molecules

43
Q

Neuro degeneration- therapeutics

A

Eg Alzheimer’s, Parkinson’s

Direct targets of mTOR pathway
Rapamycin - enhanced clearance of aggregate prone proteins, FFA approved for transplant rejection but lots of side effects

Torin1 - more potent induced if autophagy, inhibits mTORC1 and mTORC2 complexes

Indirect targeting - potential target: transcription factor EB, master regulator of autophagy and lysosome genes, indices autophagy, decrease Intra cellular aggregates in mice models

44
Q

Huntington’s disease

A

Autosomal dominant neurodegenerative disease
Caused by nucleotide (CAG)n expansion mutant = long polyglutamine (poly q) tract at the n-terminus of huntingtin protein
Polyglutamine expanded huntingtin proteins aggregates and forms inclusions in neurones
Suggests aggregates are deleterious and lead to gain of function mechanisms to disease progression

45
Q

Autophagy and hungtintons disease

A

No rapamycin, mTOR active, mTOR inhibits autophagy, huntingtin aggregates accumulate in neurones

With Rapamycin, mTOR inactive, mTOR, activates autophagy, huntingtin aggregates broken down by neurone. Showed improved behaviour

Non selective though

46
Q

Autophagy: good or bad for cancer

A

Autophagy activation in cancer
Cell survival under nutrient and oxygen shortage
Cell survival during chemotherapy treatment
Prevention of apoptosis
Escape from stress and protection from cell damage (oxidative stress and damage to mitochondria)

Autophagy function in normal cells
Mitochondrial quality cintrol under oxidative stress
Cell growth control
Autophagosome cell death
Inhibition of inflammation
Inhibition of chromosomal instability

47
Q

Cancer

A

Autophagy is upregulated in oncogenic RAS- driven cancers and in hypoxia regions of solid tumours

Autophagy supports tu at growth and promote cancer progression
Inhibition of autophagy may lead to decreased tumour mass

48
Q

Mechanism of autophagy during cancer

A

Autophagy can act both +ve and -ve in cancer cell survival
Autophagy functions to prevent cancer initially but once tumour develops cancer cells utilise autophagy for own protection
High lvls of autophagy within tumour core protects from cell death (apoptosis and necrosis) limiting metabolic damage
Cells normally undergo cell death after detachment from extra cellular matrix (anoikis) - Autophagy activation after detachment protects them and promotes cancer metastasis

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