Eukaryotic Proteostasis (10-13)

Question 1

Where does protein synthesis occur in eukaryotic cells?

Answer 1

Most protein synthesis takes place in the cytosol starting on free cytosolic ribosomes
→ apart from the tin amounts that occur in mitochondria and plastids

Question 2

What are the stages of protein synthesis in a eukaryotic cell?

Answer 2

1. A common pool of ribosomal subunits in the cytosol is used to assemble ribosomes on mRNAs encoding cytosolic proteins: these remain free in the cytosol
2. Starts producing protein → room for docking of another ribosome
   → gangs of ribosomes move along mRNA - polysome, continue making protein until end of translation
3. Newly made proteins are released into the cytosol, ribosomes dismantles re-enters pool in cytosol
   → eukaryotic genes are pseudocircularised making this process very efficient

Question 3

What effect does the overcrowding of macromolecules in cells have?

Answer 3

Promotes rapid biochemistry → overrides diffusion and affinity
Favours aggregation of proteins → many proteins making inappropriate contacts

Question 4

Why are nascent proteins in danger of aggregation?

Answer 4

A single mRNA is translated at the same time by many ribosomes → proteins being made in close proximity
→ exposed hydrophobic patches lead to interactions - aggregation, can misfold easily
Nascent proteins are in a non-native, aggregation-prone, protease-prone conformation
(folds proteins hydrophobic regions generally hidden in core - stable, resistant to proteases and stable)

Question 5

How do nascent proteins find their correct conformation particularly in crowded environments?

Answer 5

Molecular chaperones → ensure the folding of polypeptides occurs correctly, protects vulnerable hydrophobic patches
1. Hydrophobic patches on nascent/unfolded proteins are regained by Heat shock protein 40 family members (Hsp40 co-chaperone)
→ deliver the substrate to ATP-bound (open formation) Heat shock cognate protein 70 (Hsc70 chaperone) and stimulate its ATPase activity
2. ATP molecule on Hsc70 required for it to stay open is degraded to ADP
→ Hsc70 closes around the substrate hydrophobic patches preventing aggregation and allowing time for hydrophilic parts to fold
3. Upon nucleotide exchange Hsc70 adopts its open conformation releasing the substrate with folded soluble structures
→ partly folded protein may now snap into its final conformation

Question 6

What 2 fates can proceed for a protein in an Hsc70-bound state

Answer 6

Hsc70 hides hydrophobic regions of its client reducing aggregation of nascent proteins (holdase - holds client in a soluble condition) → partially folded client can enter 2 routes

Productive → released and find it stable condition - if there’s been enough time for hydrophilic parts to form correctly will snap into final conformation
→ passed onto other chaperones for further folding / assembly into multimeric complexes

Destructive → transported to a lysosome
→ passed to a proteasome for degradation

Question 7

How are protein clients released from Hsc70?

Answer 7

Nucleotide exchange factor (NEF) binds the Hsc70:client complex and removes ADP
→ promotes nucleotide exchange allows entry of ATP into the nucleotide binding site of Hsc70 - adopts open conformation releasing the client protein (can then fold rapdily)
Multiple NEFs: e.g. BAG-1, BAG-2, HSPBP1

Question 8

After being released from Hsc70 where can the client proteins go?

Answer 8

Release of client proteins

Hsp90 → another major chaperone - dimer of 2 arms) provides platform for further protein folding
→ can also take other partially folded substrates and combine

Chaperonin → 14/16/18 x Hsc60 subunits forming a cage - isolates protein from rest of cell allowing specific clients to fold correctly
→ upper chamber opens - makes contact with the client, top ring closes - bottom ring opens, release of fully folded protein

→ there is an interacting competing network of co-chaperones (i.e. Hsc70, Hsp90) that determines the fate of a chaperone client - fate is not pre-determined

Question 9

How can co-chaperones convert Hsc70 into a disaggregase?

Answer 9

Hsc70 in the presence of Hsp40 DNAJB1 and NEF Apg2 can disaggregate Tau, alphaSyn and huntington exon 1 amyloid fibres
1. Hsp40 DNAJB1 binds at end as dimers to alphaSyn fibres → recuites Hsc70
2. Apg2 triggers more Hsc70 binding → starts to stretch fibrils - entropic fulling - can rip the fibrils apart

Question 10

What are the functions of cytosolic chaperones?

Answer 10

1. Prevent aggregation of unfolded proteins
   → e.g. Hsc70 binds hydrophobic regions of a client, delaying folding for correct folding of hydrophilic regions
2. Provide a controlled environment for folding
   → e.g. chaperonins form a cage that encloses the target protein
3. Permit assembly and disassembly of multimeric complexes
   → e.g. histone complexes, clathrin cages, alpha-synuclein fibres
4. Can direct proteins with folding problems for destruction
   → e.g. Hsc70 co-chaperone BAG-1 can engage a Hsc70:client complex with the proteasome and lysosome

Question 11

How are proteosomes structured?

Answer 11

20S core particle → 4 rings of 7 subunits, 2 beta rings each flanked by an alpha ring
→ catalytic activities arranged around the core: chymotrypsin-like, trypsin-like, caspase-like (3 different proteolytic activities within core of proteasome)
Each 20S core has 19S caps, regulatory particles (RP) at one or both ends

Question 12

How is a protein targeted to the proteasome?

Answer 12

Targeting required ubiquitin → multiple in a row for degradation signal (4 or more) - allows them to be bound by the 19S RP of the proteasome
→ tetra-Ub is a degradation signal usually on a lysine residue (Ub is a conserved 76aa protein found in all eukaryotic cells)

e.g. a protein that has failed Hsc70-mediated folding
1. Ub is activated by an E1 ubiquitin-activating enzyme (E1 kept in a reduced state, following oxidation you get addition of ubiquitin)
2. Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme
3. E2-Ub conjugate associates with an E3 ubiquitin ligase - binds the target protein and transfers Ub to the target marking for destructive pathway

Question 13

How does the specificity of E1s, E2s and E3s vary?

Answer 13

E1s → ~9 in mammalian cells - vital enzymes (can’t knock them out)
E2s → >30, each select their own E3s, can provide some substrate specificity
E3s → 100s, each type selects its target protein by recognising some specific feature: extended residence in a chaperone system, their N-terminus, misfiled regions, exposure of a degradation signal
→ control the stability of proteins involved in key cellular processes: timing the key transitions in the cell cycle circadian rhythms, development, signalling, immunity q

Question 14

How are polyubiquitylated proteins degraded?

Answer 14

1. Polyubiquitylated proteins bind the 19s regulatory particle of the proteasome
2. The RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core
   → deubiquitylases removed Ub molecules and return them to a common pool for recycling
3. 3 proteolytic activities are encoded by the beta subunits of the 20S core
4. Target protein degraded into small peptides which are ejected from the proteasome

Question 15

What function, other than destruction, do proteasomes have?

Answer 15

Not just a destructive machine → fail-safe mechanism
One of the RP units acts as a chaperone that directs some clients for destruction and allows re-folding of others back to their native conformation

Question 16

What happens when the ubiquitin-proteasome system fails?

Answer 16

The UPS is relevant for: proteins that fail to fold correctly, normal turnover of cytosolic proteins, proteins whose conc. changes rapidly, viral proteins, misfiled proteins ejected from the ER

When proteasomes or E3s fail:
→ proteins that normally be destroyed accumulate instead - can lead to formation of aggregates e.g. in neurones of people with Parkinson’s and Alzheimer’s
→ if cell cycle proteins not degraded properly can lead to cell proliferation (e.g. cancer)

Overactive proteasomes:
→ implicated in autoimmune diseases e.g. systemic lupus erthematosus and rheumatoid arthritis

Question 17

What is the cytosolic post-translational modification of proteolytic cleavage?

Answer 17

Targeted specific proteolytic cleave e.g. to activate a protein
→ the effector proteases of apoptosis are stored in an inactive condition - activation by proteolytic cleave and subunit rearrangement

Question 18

What is the cytosolic post-translational modification of addition of lipids?

Answer 18

Permits membrane targeting → man regulatory proteins are modifies by the addition of lipids, e.g. Rabs that regulate membrane traffic
→ when the prenyl groups are masked by GDI Rab-GDP is cytolsolic - following nucleotide exchange GDI dissociates and the prenyl groups enter the target membrane
→ 2 lipid groups - long can embed in membranes, Rab can co-ordinate movement of vesicle along cytoskeleton

Question 19

What is the cytosolic post-translational modification of phosphorylation?

Answer 19

Additional and removal of phosphates → large -ve charge - can alter charge, shape thus protein function
→ e.g. control of CDK activation

Can be highly complex → phosphorylated p53 protein tetramerises and binds DNA where it acts as a trans-activator of a huge number of genes
→ has 24 phosphorylation sites (not all activated at once) different stressors activate different kinases with different substrate specificities - changes in phos. pattern depending on the stressors

Question 20

What is co-translational targeting?

Answer 20

An evolutionarily-conserved mechanism to target proteins into the secretory pathway
→ protein synthesis in eukaryotes is cytosolic - on free ribosomes that cluster to form polysomes
→ if the protein is destined for secretion the n-terminus is a signal peptide - a targeting device that allows engagement with a translocon on the ER membrane
→ directs nascent protein into ER lumen where the other parts of the secretory pathway can be accessed

Question 21

How are proteins secreted in the secretory pathway?

Answer 21

1. From the cytosol proteins translocate the ER membrane and exit in vesicles
2. Vesicles fuse with the Golgi and proteins are transported through into secretory vesicles
3. After fusion of the last vesicles with the plasma membrane the protein content (cargo) is secreted to the outside of the cell

→ the entry point for the secretory pathway is the ER - requires a signal peptide

Question 22

Where is the signal peptide (SP) encoded on mRNAs?

Answer 22

mRNAs that encode secretory pathway proteins → encode a 5’ signal sequence in frame to the sequence encoding the mature secreoty protein
→ the primary product following translation is a protein with an N’terminal signal peptide and the rest of the protein
→ during the process of ER entry the SP is removed - processed by proteolytic cleavage

Question 23

What do ER signal peptides look like?

Answer 23

There is a huge variation in what they look like - length variable ~15-30aa

They have broad similarities for recognition by the ER, tend to have:
→ +ve aa (Lys, Arg) towards the N’ terminus
→ a hydrophobic stretch thats recognised by signal recognition particles
→ cleavage site usually follows a small amino acid

Even highly related proteins can have different signal peptides
→ seems they are free to evolve rapidly as long as they maintain their overall features - have limited constraints

Question 24

How does a signal peptide (SP) ensure that the translating ribosome binds the ER membrane?

Answer 24

The SP is recognised by signal recognition particles in the cytosol → targets the SP to the translocon
→ SPs targets proteins to the ER but don’t directly bind the ER membrane

Question 25

How does a protein enter the ER with a signal peptide (SP)?

Answer 25

1. An emerging SP (from a ribosome) captures SRP (recognition step)
2. SP:SRP binds the alpha subunit of an SRP receptor (targeting step) recruiting a closed translocon
   → SRP receptor is dimeric - alpha/beta subunits - beta is transmembrane and associated with a closed translocon
3. Transolocon opens allowing entry of at STP into the channel
   → translation is stopped until translocon opens and hook shape inserted then SP:SRP complex disengages and SRP recycled
4. Signal peptidase (SPase) cleaves the SP from the protein
   → significance of the fold - cleavage site presented to ER lumen available for SPase
5. SP leaves translocon through a lateral gate - signal peptide peptidase cleaves the SP into two
   → cuts in the middle of the hydrophobic patch making it easier to extract
6. Translation continues until termination
   → protein passed into the ER lumen

Question 26

What does the specificity of ER targeting depend upon?

Answer 26

Broad substrates specificity in the recognition step
The folding of the SP into a hook shape by SRP
→ which allows the signal peptidase to cleave the signal within the translocon releasing the SP into the ER membrane
SPs disrupt the ER membrane which can cause problems downstream - they require chopping with signal peptide peptidase

Question 27

Why are ER targets nascent proteins prone to aggregation?

Answer 27

They are formed in locally crowed conditions → all made close together by membrane-bound polyribosome
→ so ER chaperones are required for efficient folding

Question 28

What are the 2 major ER modifications?

Answer 28

ER lumen is the major site of protein folding - which requires chaperones - ER modifications depend on whether they have the right aa sequence motifs
1. N-glycosation → covalent attachment of trees of sugars to asparagine residues on target proteins
2. Disulphide-bonded → forms between 2 adjacent cysteines in oxidising conditions

Question 29

What is retro-translation (dislocation)?

Answer 29

When misfiled proteins are ejected from the ER and then degraded in the cytosol with proteosomes
→ if a protein doesn’t fold properly it will fail a quality control check carried out by ER chaperones - acts as a stress sensor
→ when stress levels are high misfiled proteins ejected via secretory pathway rapidly

Question 30

What is N-glycosylation of proteins?

Answer 30

The covalent addition of an oligosaccharide tree of sugars (core N-glycan) from a lipid carrier to a target protein by the membrane-integral OST (oligosaccharyltransferase) as its being extruded into the ER from ribosome
→ as the protein moves through the secretory pathway the core N-glycans are modified to make complex N-glycans

Question 31

What does a core N-glycan consist of?

Answer 31

Core N-glycan: Glc3 Mann9 GlcNac2 (starting at the terminal glucose)
3 glucose on the long branch, tree of mannose, 2 N-acetly glucosamine → attached to N atom on the side chain of an asparagine (N) residue in a specific context - the N-glycosylation signal N X S/T
→ N = asparagine, X = residue of any aa except proline, S/T = serine or threonine

Question 32

What are the functions of N-glycosylation?

Answer 32

1. N-glycans act as flags for folding and ER quality control → removal of 2 Glc residues (leaving gluc-1 residue) in the ER allows interactions with ER chaperones (e.g. calreticulin) required for efficient folding - trimming of core glycans allows interactions with folding environment provided by calretuclin and calnexin
   → as the proteins proceeds through the folding steps the final Glc removed - correct folding, can leave folding environment and a Man residue is removed - can leave ER to Golgi
   (in Golgi further additions to N-glycans occur, giving complex N-glycans, allowing the cell to track progress of a protein through secretory pathway)
2. Increase protein stability → a core N-glycan is very large and made of hydrophilic sugars thus can increase protein stability and reduce aggregation problems during protein folding in ER
3. Influence folding rates and final protein conformation → N-glycans are bulky and thus constrain the alpha-carbon backbone of the polypeptide
   → if the final conformation of the protein is constrained may influence the activity of a protein and its interaction with other molecules

Question 33

How do disulphide bonds form in the ER?

Answer 33

Disulphide bonds require oxidising conditions to form (the ER in eukaryotes or the bacterial periplasm)
They form where 2 cystine residues are brought close together during protein folding
The biological catalyst are protein disulphide isomerase (PDI) and its relatives
→ PDIs may make, break and shuffle disulphide bonds
Covalent S-S bonds contribute to the stability of protein tertiary structures (of secreted proteins, not cytosolic proteins), many are essential for the activity of proteins

Question 34

What are the functions of protein disulphide isomerase (PDI)?

Answer 34

Can shuffle disulphide bonds
1. PDI recognises unstable proteins
→ PDI is reduced has 2 sulfhydryl groups
2. PDI binds as a chaperone forming PDI:client complex with mixed disulphide
3. Isomerisation of the disulphide bonds continues until the client protein reaches a stable conformation when its released from PDI

Can make/break disulphide bonds
Oxidised PDI, with its own disulphide bond, can bind reduced client
→ forming PDI:complex with mixed disulphide
PDI changes shape of disulphide resulting oxidised client and reduced PDI → can do it reversibly until protein is stable

Can join and separate 2 proteins
2 reduced clients bind oxidised PDI forming PDI:client protein with mixed disulphide → oxidised clients joined by disulphide bond and reduced PDI

PDI only binds unstable proteins, once reached stability it will let go
PDI can assemble and dismantle protein complexes as well as aid folding and stability of individual proteins

Question 35

How does the ER protein folding process work for MHC Class I assembly?

Answer 35

ER protein folding processes don’t work in isolation - they are co-ordinated
1. Bip maintaines solubility of MHC I HC until beta2 micro globulin binds → holds MHC in inactive, soluble condition
2. N-glycosylation allows entry into a folding environment → once trimmed can interact with calreticulin and its PDI-related partner ERp57 - which is disulphide linked to transmembrane tapasin, forming cage of chaperones
3. Assembly and folding of Class I molecules providing a groove for insertion of a peptide
→ fed in from the cytosol by TAP transporter that is recruited by tapasin - peptides derived from proteosomal degradation
→ allows contents of cytosol to be displayed as peptides on membrane

Question 36

What happens to an ER protein that fails quality control (fails to fold correctly)?

Answer 36

ERAD (ER-assocaited protein degradation) → misfiled ER proteins are destroyed in the cytosol
e.g. an MHC Class I HC that failed for find beta2m
1. If the MHC Class I has a long residence time with ER chaperones e.g. Bip (HSc70 molecules can direct down productive or destructive pathways - long residence time flags destruction)
2. Allows access of a mannosidase that removes one particular mannose - removing the protein from the folding environment
3. Protein is unfolded and fed through a multisubunit dislocon and is ubiquitylated during dislocation
→ which targets it to the proteasome where it is deubquitylated and degraded

Question 37

What does the secretory system modification of O-glycosylation (attachment of sugars to O) do?

Answer 37

Occurs in the ER/Golgi apparatus in eukaryotes (also occurs in archaea and bacteria)
O-N-acetylgalactosamine → may be attached to serine or threonine residues - interacts with water the high conc of carbohydrates gives mucus its slimy feel
O-mannose → can be attached to serine and threonine residues in secretory pathway proteins, common in both prokaryotes and eukaryotes
O-frucose and O-glucose → can be added to cysteine residues

Question 38

What is the secretory system modification of proteolytic cleavage?

Answer 38

Proteolytic cleavage can e.g. activate a protein such as albumin
→ albumin is activated in the secretory pathway be an endosomal/lysosomal enzyme called furin
preproalbumin → pro albumin → albumin

Proteolytic activation occurs for: active digetsive enzymes, hormones, neurotransmitters and proteins that would otherwise aggregate

Question 39

What is amyloid precursor protein (APP)

Answer 39

(function in the brain is not well understood)
If beta secretes cuts APP then y secretes will cleave auto release an alphabeta fragment that aggregates to form plaques on the brain - Alzheimers

If alpha secretase cuts first then it cleaves within alphabeta preventing release of the toxic fragment - major effort underway to study the regulation of the secretases

Question 40

What is the secretory system modification of addition of lipid to permit/maintain membrane targeting?

Answer 40

GCPRs are polytopic membrane proteins that have a myristic acid lipid anchor → allows it to float in the lipid environment of membrane

GPI-linked proteins have a glychosphoatidylinositol membrane anchor linked via sugar to the protein

Question 41

What are the different types of intracellular trafficking?

Answer 41

Gated transport → nucleus to cytosol
Transmembrane transport → cytosol to peroxisomes/ER/mitochondria/plastids, ER to cytosol
Vesicular transport → ER to Golgi, Golgi to secretory vesicles/plasma membrane/early endoscopes/late endosomes, ER to peroxisomes, Golgi to ER, etc

These can be anterograde (forward ER to surface) or retrograde

Question 42

What are the 5 major interdependent strategies for ER selective transport - to maintain ER homeostasis and reduce cellular stress levels?

Answer 42

Cargo capture → receptor-mediated export of proteins from the ER to the Golgi in costumer protein II (COPII) vesicles
Bulk flow → some proteins and lipids are included in COPII vesicles by default
Retention → prevents proteins from entering the transport vesicles
Retrieval → retrograde transport from there ER-Golgi intermediate compartment (ERGIC)/early Golgi back to the ER
ERAD → cytosolic elimination of ER proteins that fail quality control

Question 43

What is the process of cargo capture?

Answer 43

1. In the ER secretory cargo is loaded into COPII (costumer protein II) transport vesicles at ER exit sites (ERES)
   → requires cargo receptor proteins in the vesicle membrane and export signals in fully folded client proteins
2. COPII vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC)
   → when they are close to the cis-Golgi membrane they shed their coats - COPII components are recycled
3. ERGIC vesicles fuse with the cis-Golgi - change in pH allows receptors to let go of their cargo and deliver it
   → receptors usable return to the ER by retrieval pathways

Question 44

What is the process of bulk flow?

Answer 44

Transport of proteins through anterograde transport without need of receptors or export signals
→ some soluble and membrane proteins (and membrane lipids) enter COPII vesicles by default - transported with forward flow of the membranes
→ biotech benefit - lack species specific recognition signals

Question 45

What is the process of retention?

Answer 45

Some proteins are selectively excluded from COPII vesicles → maintained in the ER

Question 46

What is the process of retrieval?

Answer 46

COPI coated vesicles retrieve transport machinery, cargo receptors, lipid membrane and escaped ER-resident proteins from ERGIC and the cis-Golgi
→ e.g. cargo receptors need to be returned to the ER to be reused

Retrieved membrane proteins typically possess a C-terminal dilysine motif (KKXX) or a close variant e.g. OST (human) …EKEKSD
Retrieved soluble proteins typically have a C-terminal ‘KDEL” motif (HDEL in yeast, K/HDEL in plants)
→ e.g. Bip has strong KDEL sequence so is highly retrieved, ERp57 QDEL and CRT KDEL - proteins required for ER folding are retrieved

Question 47

What is Rab6 retrograde transport?

Answer 47

A retrograde route governed by the small GTPase Rab6 protein - lipid retrieval
→ if sending vesicles in anterograde way causes depletion of ER membrane lipids - they need to be returned
→ Rab6 organises return via long tubular elements - independent of COPI

Question 48

What are some stresses that increase misfiling/stimulate unfolding?

Answer 48

Abiotic → heat stress (plants, fungi, homeothermic animals), osmotic stress (plants - drought), high light intensity (plants)
Biotic → infection (plants, animals, fungi), stress related hormones (salicylic acid secreted by competitor plant)

Question 49

What is the unfolded proteins response (UPR)?

Answer 49

ER as a stress sensor → the protein folding capacity of the ER is tightly regulated by a network of signalling pathways
→ UPR sensors monitor the ER folding status of proteins in the ER
→ once UPR triggered - many physiological events that ultimately reduce the stress and adjusts the folding capacity of the ER according to need

Question 50

What are the sensing mechanisms for ER stress mediated by?

Answer 50

Ire1 (Inositol-requiring Enzyme 1) → animals, plants, fungi
PERK (PRKR-like endoplasmic reticulum kinase) → animals and plants
ATF6 (activating transcription factor 6) → animals and fungi, (bZIP28 and bZIP17 in plants)
→ these systems monitor ER stress by counting misfiled proteins and adjusting protein folding capacity

Question 51

What is the process of stress sensing with Ire1?

Answer 51

1. Ire (transmembrane protein) ER luminal domain is captured by Bip
   → maintains it in a soluble state and keeps it inactive
2. Bip recognises unfolded or misfiled clients and leaves
   → Ire dimerises and also binds unfolded proteins
3. As stress increases there’s an increasing number of misfolded proteins
   → lots of Ire activated, multimerises - maintains solubility
   → the cytosolic domains of Ire1 transautophosphorylate activating Ire1 RNAase activity - levels of stress correlated with level of activated Ire
4. The RNAase activity of Ire1 recognises a highly conserved unusual cytosolic RNA (stem loop structures) - cuts exons then spliced together in association with specific ligase leaving unusual intron
5. The splicing exons together creates an open reading frame thats translated to make a transcription factor
   → transported into nucleus - turns on response pathways leading to reduction in ER stress (increases ERAD, [chaperones] - folding and trafficking)

Question 52

Why is the Ire1 splicing mechanism unusual?

Answer 52

Most eukaryotic splicing requires two transesterifcations coordinated by snRPs and occurs in the nucleus

Ire1 has RNAse activity which recognises cytolic RNA and which removes a highly conserved intron
→ the exons are fused using RNA ligase activity

Question 53

How can you stimulate ER stress experimentally?

Answer 53

DTT (dithiothritol) → a reducing agents: breaks disulphide bonds
→ if cells are treated with DTT their ER proteins become destabilised - destabilised proteins more likely to unfold
TG (thapsigargin) → blocks sarco/endoplasmic reticulum Ca ATPase (SERCA) pumps
→ depletion of ER calcium stores leads to ER stress
Heat shock (e.g. 42C) → destabilises/unfolds some proteins

measure with spliced and unspliced mRNA

Question 54

What is regulated Ire1 dependant decay of mRNA (RIDD)?

Answer 54

Degrades mRNA in complex with ER-associated ribosomes → reducing ER import and thus reduces stress
→ only mRNA close to ER membrane which is being used to translate protein into the ER - degraded non-specifically
→ cytosolic translation continues normally

Question 55

How can Ire1 trigger apoptosis?

Answer 55

If the ER stress is too great and Ire responses don’t control it
→ then it can stimulate signalling via c-Jun N-terminal kinase (JNK) triggering apoptosis

Question 56

What are the similarities and differences between PERK and Ire1 stress sensors?

Answer 56

Similarities → both transmembrane, ER domains share sequence and structural similarity, cytosolic portion both possess kinase domains that transautophosphorylate, both interact with Bip
Differences → Ire1 activation leads to specific splicing and production of transactivators of transcription
→ PERK activation leads to interference with global translation - reduces stress in every compartment

Question 57

What is the mechanism of PERK stress sensing?

Answer 57

No ER stress Bip is bound to PERK

Unfolded or misfiled clients → Bip detects, PERK dimerises, autotransphosphorylates, activated PERK phosphorylated elF2alpha which is required for translation
→ reduces translation globally - reducing protein expresión and relieves stress
However, a few genes are preferentially expressed that don’t require elF2alpha - transcription factors for apoptosis

Question 58

What is the mechanism of ATF6 ER stress sensing?

Answer 58

No ER stress - Bip is bound to ATF6

Unfolded or misfolded proteins → activated ATF6 - transported to the Golgi
→ ATF6 is cleaved
→ ATF6f (fragment) is a transcription factor - exported to nucleus and Bip and other ER chaperones increased - unregulated ERAD
→ chain of events that reduce ER stress

Question 59

What are the roles of autophagy?

Answer 59

Removal of protein aggregates, old and damaged organelles, and invading microbes
& developmental modelling
& providing amino acids, nucleotides, lipids, sugars under low nutrients conditions

Question 60

What are the types of autophagy?

Answer 60

Macroautophagy → the most well-characterised form, bulk capture non-specific
→ beginning point: phagophore - isolation membrane forms an capture cargo, isolation membrane fuses to form a vesicle called an autophagosome, this fuses with a late endosome forming an amphisome, this fuses with a lysosome forming an autolysosme - end point: lysosome

Microautophagy → direct targeting into a lysosome
Chaperone-mediated autophagy (CMA) → entry into lysosome via a membrane channel, delivers specific protein to be degraded

Question 61

How are lysosomal enzymes delivered?

Answer 61

Entry into ER as a preproenzyme, signal peptide cleave to generate proenzyme, N-glycosylation and folding
cis-Golgi mannose phosphorylation and TGN sorting to the LE
LE fuse with lysosome, delivering the cache-sins to a hybrid EL - activation by controlled hydrolysis in an acidic compartment
Regeneration of lysosomes

Question 62

Why do lysosomes need low pH?

Answer 62

Lysosomal enzymes include proteases, nucleases, glycosidases, ligases, phospholipases and phosphatases
→ these hydrolytic enzymes work best at ~pH 4.5-5

Question 63

How is low lysosomal pH maintained?

Answer 63

Proton-pumping V-type ATPases → convert energy from ATP hydrolysis to pump protons H+ into the lysosome lumen
→ this generates a transmembrane voltage
→ as [H+] increases pH falls - can’t be maintained as forcing up gradient being made thus a counterion is needed to maintain ability to pump H+ (e.g. pump out Ca2+ or pump in Cl-)
→ lysosome is under dynamic pH control, low pH is what finally activates protein to degrade captured cargo

Question 64

What is chaperone-mediated autophagy (CMA)?

Answer 64

Selective lysosomal degradation of proteins bearing Hsc70-binding KFERQ motifs via a membrane channel formed from lysosome-associated membrane protein type 2A (LAMP-2A)
KFERQ motifs → up to 2 +ve residues (K, R), up to 2 hydrophobic residues (I, F, L, V), a single negatively charged residue (E, D), a single Q that can be N- or- C terminus
→ KFER can be in any order
→ up to 40% of mammalian proteins contain a KFERQ motif
→ also post translational modification can generate more active motifs e.g. a motif lacking a -ve charge can be converted to an active motif by phosphorylation of S, T or Y

KFERQ-driven CMA is used for the disposal of a very wide range of substrates

Question 65

What are LAMP proteins?

Answer 65

LAMP proteins → C-terminus: receptor for cargo binding, trans-membrane domain, N-terminus in lysosomal lumen: form channel for entry of target protein
LAMP-2A → receptor and channel for chaperone-mediated autophagy
LAMP-2B → required for fusion of autophagosomes with late endoscopes/lysosomes
LAMP-2C → autophagy of nucleic acids

Question 66

What is the process of chaperone-mediated autophagy (CMA)?

Answer 66

1. Recognition of KFERQ motif (on a damaged, misfolded protein needs to be cleared) by Hsc70 and delivery to LAMP-2A
2. Multiermisation of LAMP-2A aided by Lys-Hsp90, substrates unfolding, translocation mediated by Lys,Hsc70 and hydrolysis
   → forms a channel
3. Hsc70-mediated dismantling of the CMA translocation complex, transport to cholesterol-containing lipid domain and degradation of LAMP-2A by cathy-sin A and metalloproteinase

Lipid microdomain → sites of proteolysis of LAMP 2 receptor sub channel

Question 67

How is chaperone-mediated autophagy (CMA) down regulated?

Answer 67

1. mTORC2 complex activates Akt1 by phosphorylation
2. Activated Akt1 phosphorylates GFAP (glial fibrillary acidic protein)
   → keeps LAMP-2A inactive - resulting reduced substrate docking

Nutrient rich diets, high fat content diets and increasing age stimulate mTORC2 activity → inhibit CMA

Question 68

How is chaperone-mediated autophagy (CMA) up-regulated?

Answer 68

Inhibition of mTORC2 and Akt1 reduce phosphorylation of GFAP → non-phosphorylated GFAP stabilises multimerised LAMP-2A
→ results in increased substrate docking

Withdrawal of growth factors, starvation, oxidative stress, DNA damage and hypoxia → stimulate CMA

Question 69

How does chaperone-mediated autophagy (CMA) link with health and disease?

Answer 69

Cancer cells → degrades tutor suppressors and pro-apoptotic proteins, protects against radiation and hypoxia, favours growth and metastasis

Neurodegenerative patients → CMA dysfunction: mRNA maturation, MAP2 promoter variants, Tau accumulates
→ CMA toxicity: mutant alpha-synuclein, Tau variants

Question 70

What is the process of microautophagy?

Answer 70

Autophagic cargo is taken up directly by late endosomes and lysosomes → direct delivery, target protein inside lumen of lysosome
→ some microautophagy substrates bear KFERQ motifs and are delivered by Hsc70 by direct binding of the chaperone to phosphatidylserine in the LE membrane
→ the autophagic cargos are then degraded on the end-lysosomal or lysosomal lumen

Question 71

What are digestive lysosomes?

Answer 71

Fusions of lysosomes with: phagosomes - forming phagolysosomes, autosomes - forming autolysosomes
→ degrade material

Lysosomes are regenerated by tubulation from digestive lysosomes, scission and maturation

Question 72

How are autophagosomes formed?

Answer 72

Initiation: formation of the phagophore
→ inhibition of mTORC complexes, activation of ULK1 complexes, capture of Atg9 vesicles, activation of PI3K complex, phosphorylation of membrane lipids
Nucleation → recruitments of WIPIs to phosphorylated membrane lipids, recruitment of ATG16 complex and lipidation of LC3, recruitment of ATG2
Growth of the phagophore → ATG9 vesicle fusion, transfer of membrane lipids from the omegasome
Closure of the phagophore → role of ESCRT complexes

Question 73

What activates autophagosome formation?

Answer 73

mTORC complexes at the lysosomal membrane inhibited

Activation of mTORCs reduces both chaperone-mediated and macro autophagy
→ mTORC2 phosphorylates Akt1, which phosphorylates GFAP - inhibits LAMP-2A and delivery via CMA
→ mTORC 2 phosphorylates Akt1, which phosphorylates mTORC1 - phosphorylates two parts of the ULK complex (ATG13 is a regulator of ULK1 kinase) - inhibits macroautophagy

Starvation stimulates autophagy → withdrawal of insulin, growth factors and nutrients inhibits mTORC complexes
→ autophagosome is formed that captures cytoplasmic contents non-specifically
→ provides amino acids, nucleotides, lipids and sugars under low nutrient conditions

Question 74

What is involved in autophagosome formation initiation?

Answer 74

1. mTORC complexes activate ULK complexes which associate on the ER membrane and capture ATG9 vesicles
   → ATG13 regulates ULK complex - captures vesicles containing ATG9 associated with PI3k complexes
2. ULK1 kinase phosphorylates and activates Beclin (in the autophagyspecific PI3K complex)
3. Beclin activates the PI3K activity (Vps34)
4. Vps34 is a lipid kinase → phosphorylates membrane lipids converting membrane phosphatidylinositol to Ptdlns(3)P

Question 75

What is the function of ATG9?

Answer 75

Protein required for maintenance of membranes → ensures there is an equal amount of lipids on each side
→ ATG9 cycles between the plasma membrane, endosome and trans-Golgi network - ATG9 vesicles are recruited from these endosomal and TGN compartments

Question 76

What is involved in autophagosome formation nucleation?

Answer 76

Recruitment of other factors → vesicles captures and decorated with autophagy associated proteins
→ once vesicles captured phosphorylated lipids capture WIPI proteins
→ recruitment of cytosolic LC3-1 and its lipidation by ATG16L1
→ ATG2 binds the omega some and ER_domain that might be the same as an ER-exit site (ERES)

Question 77

What is involved in autophagosome formation vesicle fusion?

Answer 77

Formation of the isolation membrane → once vesicles captured and decorated with autophagy associated proteins they fuse together
→ a phagophore (isolation membrane) is formed: captured by ATg13, connected by ATG2 to the omega some and bearing multiple molecular markers from multiple cellular locations - the phagophore is now expanded

Expansion of phagophore involves ATG2 and ATG9 → ATG2 is a lipid transporter, ATG9 is a lipid ‘flippase’ so both inner and outer leaflets are expanded with ER-derived lipids (maintains membrane homeostasis)

Question 78

What is involved in phagosome formation closure?

Answer 78

Expansion leads to almost closed autophagosome → ESCRT (endosomal sorting complex required for transport) 1 bridges the gap and ESCRT-III subunits are recruited by FIP200, a member of the ULK complex - forming a plug
→ the membranes seal and the ESCRT proteins removed
→ the autophagosome is now competent for lysosomal function

Question 79

How is LC3 processed?

Answer 79

(LC3 is part of bulk autophagy and can be used for selective processes)
By proteolytic cleavage and lipidation
→ 3 isoforms of LC3 are processed by proteolytic cleavage after a gly residue (LC3A, LC3B, LC3C) go from proLC3 to LC3-I mature products
On demand a proportion of LC3-I is conjugated to phosphatidylethanolamine (PE) in membrane to generate LC3-II → LC3 becomes associated with membrane but choices are made - LC3A-II and LC3B-II associate with different vesicles (some specificity)

Question 80

How can LC3-I proteins capture cargo receptors?

Answer 80

e.g. ubiquitylated cargo can be captured by cargo receptors such as p62, which can be collected by cytosolic LC3-I, stimulating conjugation to membrane PE by ATG16L1
→ thus availability of cargo can stimulate selective autophagy

There are various cargo receptors - proteins that bind autophagy cargo e.g.
→ intracellular pathogens: NDP52
→ damaged organelles: NDP52, OPTN
→ proteins aggregatesL p62, TAX1BP1

Question 81

How can LC3-II capture cargo receptors?

Answer 81

LC3-II captures cargo receptors that contain an LC3-interacting region (LIR) motif
→ the cargo receptor recruits cargo