Autophagy

Question 1

Interest in autophagy

Answer 1

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

Question 2

What is autophagy

Answer 2

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

Question 3

Yeast model

Answer 3

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

Question 4

Lysosomal degradation pathways

Answer 4

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

Question 5

3 main types of autophagy

Answer 5

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

Question 6

Autophagy - use

Answer 6

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

Question 7

Starvation and stress induced autophagy

Answer 7

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

Question 8

Under nutrient starvation

Answer 8

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

Question 9

How does spontaneous double membrane structure form

Answer 9

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

Question 10

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

Answer 10

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

Question 11

Atg conjugation system and phagophore formation

Answer 11

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)

Question 12

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

Answer 12

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

Question 13

Experimental ways to see LC3 1 & 2

Answer 13

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

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

Question 14

Summary of atg5-atg12, LC3 and lysosome

Answer 14

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

Question 15

Where does the membrane come from?

Answer 15

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

Question 16

Endoplasmic reticulum as a source of membrane

Answer 16

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)

Question 17

Autophagosome maturation and degredation

Answer 17

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

Question 18

Vesicle fusion

Answer 18

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

Question 19

Tethering complex and SNARE mediated vesicle fusion in autophagy

Answer 19

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)

Question 20

Later stage compartments in autophagy pathway

Answer 20

Fusion of lysosome and autophagosome = autolysosome

Phagophore > autophagosome > amphisome > autolydosome

Question 21

Electron microscope of autophagosome stages

Answer 21

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

Question 22

Non selective autophagy

Answer 22

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

Question 23

Selective autophagy

Answer 23

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

Question 24

Selective autophagy - identifying cargo

Answer 24

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

Question 25

Autophagy receptors recognise ubiquitin tagged substrates

Answer 25

Lysine63 (k63) linked ubiquitin changing + e3 ubiquitin ligate = autophagy Lysine48 (K48) linked ubiquitin chains + E3 ubiquitin ligate = proteasome degredation

Question 26

Autophagy receptors

Answer 26

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)

Question 27

Autophagy receptors - xenophagy

Answer 27

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

Question 28

Autophagy dysfunction in disease

Answer 28

Organ specific - CNS eg azhiemers disease, Parkinson’s LUNG - asthma INTESTINE - crohn disease Multisystemic - CANCER - ovarian, sarcoma

Question 29

Human disease associated with autophagy dysfunction

Answer 29

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

Question 30

Innate immune response

Answer 30

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

Question 31

Salmonella infection of intestinal epithelium

Answer 31

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

Question 32

Crohn’s disease (CD)

Answer 32

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

Question 33

Use of autophagy for the benefit of pathogen

Answer 33

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

Question 34

Autophagy and ageing

Answer 34

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

Question 35

Neurodegeneration

Answer 35

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)

Question 36

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

Answer 36

Lofora disease Glycogen like intracellular imvlusions MTOR pathway activated in laforin deficient cells Lysosomal storage disorders Dysfunctional lysosomes (accumulation of cholesterol) effects autophagosome trafficking

Question 37

ESCRT complex and membrane bending

Answer 37

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

Question 38

CHMP2B and frontotemporal dementia

Answer 38

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

Question 39

Mitophagy- autophagy if mitochondria

Answer 39

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)

Question 40

Mitophagy - PINK1, Parkin and Parkinson’s disease

Answer 40

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

Question 41

Therapeutic approach

Answer 41

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

Question 42

Rapamycin ( small molecule inhibitor of mTOR inhibitor)

Answer 42

Inhibits mTORC causing autophagy of small molecules

Question 43

Neuro degeneration- therapeutics

Answer 43

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

Question 44

Huntington’s disease

Answer 44

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

Question 45

Autophagy and hungtintons disease

Answer 45

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

Question 46

Autophagy: good or bad for cancer

Answer 46

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

Question 47

Cancer

Answer 47

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

Question 48

Mechanism of autophagy during cancer

Answer 48

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