Protein folding and disease

Question 1

What 2 types of aggregation can occur?

Answer 1

1) . Protein is already folded in a native state and requires an unfolding event to a higher energy level to a partially folded state and from this goes downhill to form a stable aggregate e.g. lysozyme, transthyretin, light chain
2) . Aggregation occurs from intrinsically disordered proteins which are high on the energy landscape and do not fold to the native state e.g. huntingtons, AD and PD

Question 2

2 categories of protein folding diseases

Answer 2

Loss of function, gain of toxic function or both

Question 3

What are the two environments where proteins can fold?

Answer 3

The cytoplasm or the secretory pathway

Question 4

What are amyloid fibrils

Answer 4

Beta strands which run perpendicular to the fiber long axis called a cross-beta structure.

Any protein can form these under the right conditions due to the NH peptide bond so the structure is always the same irrespective of the precursor.

Always 10nm in diameter, long, straight and unbranched, can visualize by EM. Occur by nucleated growth. Diffract x-ray beam and get two bands - 4.7A and 10A (to do with the structure). They bond congo red dye.

Amyloid is very heterogeneous as the protofilaments can differ in number and twist.

Question 5

What are the 3 classes of different diseases

Answer 5

1) . Improper trafficking
2) . Toxic conformer
3) . Degradation

Question 6

Describe the structure of lysozyme

Answer 6

Mixed alpha and beta protein with the active site between the alpha domain and the beta domain

Question 7

Does lysozyme form a cross-beta structure?

Answer 7

Yes

Question 8

What are the two mutations in lysozyme known to cause amyloid?

Answer 8

I56T and D67H - discovered from patients

Question 9

Where is the I56T mutation in the structure of lysozyme?

Answer 9

I56T is in the beta-sheet domain sitting at the bottom in the junction between the alpha and beta in the active site cleft

Question 10

Where is the D67H mutation in the structure of lysozyme?

Answer 10

In the beta-sheet domain at the top

Question 11

Do the I56T and D76H mutants have the same structure as the wild-type? And what does this tell us?

Answer 11

Yes - tells us it is not a folding disease so must be a disease of unfolding

Question 12

Are the I56T and D76H mutants as stable as the WT lysozyme?

Answer 12

No - they are destabilised compared to the WT however they are still stable at 37oC so this is not biologically relevant.

Question 13

Are the I56T and the D76H mutants more aggregation prone than the WT lysozyme?

Answer 13

Yes - this was measured by light scattering

Question 14

Does the reduction in stability of the I56T and the D76H mutants explain why they form amyloid fibrils?

Answer 14

No - other mutants were just as unstable however these did not form amyloid fibrils therefore it is not all about thermostability

Question 15

What did the HX experiments show in the WT and lysozyme mutants?

Answer 15

The mutant HX profiles were different - the beta-sheet domain exchanges D for H all at the same time so shows there is transient unfolding of the entire beta-sheet domain.

So the native state is in dynamic equilibrium with a partially unfolded state where the b sheet domain is unfolded but the alpha helix domain is folded. This partial unfolding is critical for the formation of the AFs as the beta sheets NH’s start to H-bond intermolecularly which is the nucleation event.

Question 16

How should we target lysozyme amyloid disease?

Answer 16

Stabilize the native state to prevent partial unfolding

Question 17

What molecule did they use to stabilize the lysozyme native state?

Answer 17

Found a nanobody (heavy chain only) which binds the active site of lysozyme which stabilized it. It decrease the rate of aggregation in vivo and EM showed almost no amyloid presence. They retested the HX and the rate of exchange was slower in the presence of the camelid.

Question 18

What is the similarity between lysozyme and transthyretin?

Answer 18

They both unfold from a native state

Question 19

Is TTR a loss of function or gain of toxic function disease?

Answer 19

Gain of toxic function

Question 20

What does TTR destroy?

Answer 20

Post-mitotic tissue

Question 21

Where is TTR made and where does it deposit?

Answer 21

Made in the liver but deposits in either the heart causing myopathy or the nerve systems causing neurological problems

Question 22

What are the 2 types of TTR amyloidosis classes?

Answer 22

1) . Senile systematic amyloidosis (SSA) - cardiomyopathy, WT TTR and occurs i older years >60 years old where 15% of people above 80 years old are effected
2) . Familial amyloid polyneuropathy (FAP) - peripheral neuropathy and cardiomyopathy, mutant TTR and WT TTR, heterozygotes with hybrid tetramers (get mutant and WT coassembly), 15-60 years old as it is variant dependent.

Question 23

Where is TTR found? and what does it do?

Answer 23

In the plasma and is retinol binding protein carrier

Question 24

Is TTR beta sheet or alpha helix rich?

Answer 24

beta sheet rich

Question 25

Oligerisation state of TTR?

Answer 25

Homotetramer

Question 26

Does TTR bind thyroxine?

Answer 26

Yes but this is not its main function

Question 27

Which protein carries thyroxine?

Answer 27

Thyroxine-binding protein - has a much higher affinity than TTR

Question 28

Where does thyroxine bind TTR?

Answer 28

In the 2-fold axis of symmetry between the 2 dimers of TTR with negative cooperativity

Question 29

How does TTR form amyloidosis?

Answer 29

1) . Tetramer dissociates into native-like monomers
2) . Monomers then misfold
3) . Misfolded monomers form a cross-beta structure

Native monomer cannot form cross-beta without misfolding first so this is the rate limiting step

Question 30

Which axis does the tetramer dissociate from?

Answer 30

2-fold axis where Thyroxine binds is the weak axis

Question 31

How was TTR targetted to prevent disease? and what is the benefit of this?

Answer 31

Stabilising the tetramer to prevent dissociation into monomers than therefore misfolding and amyloid formation.

Benefit is that you do not need to know that the toxic species is as you are preventing all of them

Question 32

What was the evidence that stabilizing the TTR tetramer prevented the disease? and how did Jeff Jelly prove this?

Answer 32

In Portugal there was a family which had the V30M mutation which meant they should have got FAP however they were heterozygous for the T119M mutant too which is a protective mutation.

Jeff Kelly tagged each mutant and looked at amyloid formation of the different ratios - see diagram - where homo T119M had the least and homo V30M had the most fibrils. In terms of kinetics homo-T119M has a higher barrier than homo-WT showing it has slower amyloid formation and by stabilizing the tetramer.

Question 33

Did Jeff Kelly look at TS analogues?

Answer 33

No as this is hard to do

Question 34

How did Jeff Kelly test if one or two binding sites were needed?

Answer 34

TTR contains no cysteines therefore he introduced one near the 2-fold axis and attached Thyroxine by different linker lengths so he had a uniform population of 0, 1 or 2 ligands bound. Showed 1 ligand was enough to prevent aggregation and this meant that he didn’t have to deal with the negative cooperativity.

Question 35

Describe how Jeff Kelly designed TTR small molecules to stabilise TTR

Answer 35

Already knew Thyroxine binds therefore want an analogue which binds tightly to TTR and binds selectively to TTR in the plasma.

Split the design into 3 partsX, Y and Z where he optimised each part and switched them together. Used information from the crystal structure to help design and then screened a range looking at the efficacy score and rate of amyloid formation. Took some forward and looked at selectivity in the blood plasma where if it still bound 1:1 it was binding specifically to TTR. The molecule they took forward was called Tafamidis and they solved the crystal structure in complex with this.

Question 36

How well does Tafamidis work in patients

Answer 36

Looked at nerve function and after 18 months everyone got the drug as no other treatment (liver transplant) and everyone showed improvement.

Saw improvements in small and large nerve fiber function, body mass index and lower extremity neurological exams.

Question 37

Are there other drugs to target TTR?

Answer 37

Not on the market but are siRNA phase 1 drug targeting TTR gene and an antisense agent also targeting TTR in phase 1.

Question 38

What is the proteostasis network?

Answer 38

It is a compilation of integrated biological pathways that influence the proteosome and its function from birth to death. Includes protein synthesis, folding, trafficking and degradation to maintain the proteosome (~600 genes). Everything is in balance so everything can happen at the right time.

The network is highly conserved across organisms from yeast to man where the only thing which is different is the amount of TRPs increase as the complexity increases (repeat involved in protein:protein interactions). Lots of different molecular chaperones and regulators work together to maintain the quality of the proteosome.

Question 39

What do chaperones do?

Answer 39

Help the protein fold both the 1st time and through thermal kicks Also can interact with misfolded proteins and target for degradation

Question 40

Is Gaucher’s a genetic disease?

Answer 40

Yes

Question 41

Name a lysosomal storage disease

Answer 41

Gaucher’s

Question 42

What are the symptoms of Gaucher’s disease?

Answer 42

Bruising, fatigue, anemia, low blood platelets and enlargement of the liver

Question 43

What causes Gaucher’s disease?

Answer 43

Hereditary deficiency in the enzyme Glycosylceramidase (GC) which converts glycosylceramidase to glucose and ceramide. In Gaucher’s get a build up of the fatty acid in white blood cells and macrophages, which can collect in the spleen, liver, kidneys, bone marrow, brain and lungs.

It is to do with the secretory pathway.

Question 44

What is the threshold for developing Gaucher’s

Answer 44

~10% if it drops below this get Gaucher’s disease. We produce more enyzme than we need for health but if it drops below this you get disease.

Question 45

Where is GC found?

Answer 45

Lysosome

Question 46

Where is GC made?

Answer 46

RER and it folds in the ER therefore has to be released by vesicle budding/fusion and transported to golgi and then to the lysosome.

Question 47

Describe the differences in pH in GC trafficking

Answer 47

ER pH 7.4, Golgi pH 6.2 and lysosome pH 5

pH decreases, becomes more acidic as you traffick towards lysosome

Question 48

What are the mutants which cause Gaucher’s?

Answer 48

N370S and L444P

Question 49

Describe the differences in stability of N370S and WT and therefore what happens to GC?

Answer 49

WT is stable at both pH 7 (ER) and pH 5.3 (lysosome)
N370S is stable at pH 5.3 (lysosome) but NOT stable at pH 7 (ER) where it has to fold. This means GC is secreted from the ER and gets Ub for degradation.

Question 50

What does the L444P GC mutant tell us in Gaucher’s disease?

Answer 50

WT and lysosomes co-localise at both 37 and 30oC
L444P only co-localises with lysosomes at 30oC not 37oC. This shows that it is a disease of the QC machinery of the ER being overzealous, so if could turn this off a little bit the GC would be trafficked to the lysosome where it is stable.

(Lower temps are better for folding)

Question 51

What are proteostasis regulators?

Answer 51

Small molecules or biologicals that control the concentration, conformation, quaternanry structure and location of proteins comprising the proteome by manipulating the proteostatasis network, otfen by influencing the signalling pathways that control the proteostatasis network

Question 52

What are the 2 small molecules called which increase L444P GC activity in cells? and where do they work?

Answer 52

Celastrol and MG-132

Already know they work in the cytosol but also have a function in the UPS

Question 53

How can you measure where GC is in the secretory pathway?

Answer 53

Proteins in the ER are mannose richwhere as proteins in the golgi have more complex sugars which are resistant to EndoH cleavage.

Question 54

What happens to GC trafficking when Celastrol and MG-132 are added?

Answer 54

It makes protein endoH resistant therefore they are leaving the ER and entering the golgi. lso immunofluroscnece experiments showed lots of proteins in the lysosomes and it’s active

Question 55

Describe the unfolded protein response

Answer 55

IRE1 - homodimerizes and becomes P when Bip dissociates which swithces on signalling pathway resulting in splicing of Xbp-1 which allows the transcription of an active TF which switches on genes for chaperones, lipid synthesis and ERAD.

ATF6 - Translocated into the golgi where it is cleaved by S1P/S2P and this allows it to enter into the nucleus where it can upregulate proteins related to UPS

PERK - dimerises and P eIF2alpha which switches off translation so you only get selective translation (GCN4/ATF4 –> CHOP on UPR gene)

Question 56

How do MG-132 and Celastrol effect the UPR?

Answer 56

Induce splicing of Xbp-1 indicating activation of IRE1
Induce cleavage of ATF-6
Induce expression of CHOP indicating activation of PERK

Question 57

Do MG-132 and Celastrol effect all 3 arms of the UPR?

Answer 57

Yes

Question 58

Describe how GC is targetted by small molecule

Answer 58

Stabilizing the native state in the ER with NN-DNJ (inhibitor similar to substate) allows it to leave the ER and then it dissociates when it gets to the lysosome as the substrate binds tighter. Increases trafficking of GC to lysosomes

Question 59

What is the best strategy for treating Gaucher’s

Answer 59

Combining small molecule stabilizers of the native state with small molecule proteostasis regulators

Question 60

What are the other ways to regulate proteostasis

Answer 60

Ca2+ blockers

Question 61

What are the cellular roles of degradation?

Answer 61

Turnover of protein, response to starvation, removal of misfolded proteins, regulation of cellular pathways on/off and antigen presentation

Question 62

Why is degradation so important?

Answer 62

Many newly synthesised proteins fail to fold due to lots of reasons, cellular stresses and other factors can result in misfolding and need to remove misfolds as they are inactive and to prevent pathological aggregates e.g. amyloid

Question 63

What are the two principle sites for protein degradation?

Answer 63

Cytosolic proteosomes and lysosomes

Question 64

Describe the Ub proteasome system (UPS)

Answer 64

Short-lived proteins are mainly degraded by the UPS

Up to 5% of the human genome is dedicated to the UPS which many of these genes encoding E3 ligases

Ub is a 76aa protein which is highly conserved

Ub is cross linked to proteins via a Gly76 of Ub and a Lys of the substrate protein. Poly Ub chains are formed by Lys48-Gly76 isopeptide bonds and chains of 4 or more tag for degradation.

1) . E1 forms a thioester with the carboxyl group of Gly 76 on Ub
2) . E2 transiently carries Ub as a thioester
3) E3 promotes the transfer of Ub to the substrate protein or Ub

Question 65

What is the proteosome

Answer 65

It is a large 26MDa complex which is present in the cytol, it is made up of 2 subcomplexes - 19S cap and the 20S proteosome core

Question 66

Are E1, E2 and E3 proteins conserved?

Answer 66

Yes from yeast to man

Question 67

Describe the proteosome 20S core

Answer 67

Site of the substrate cleavage where proteolysis occurs in the central chamber of the 20S core
Entry into the channel is via a narrow channel therefore proteins must be unfolded to enter

Question 68

Can be proteosome degrade folded proteins?

Answer 68

No

Question 69

What are the 3 enzymes in the 20S proteosome core?

Answer 69

1) . Tryptic activity - after basic
2) . Chymotryptic activity - after hydrophobic
3) . Peptidylglutamyl peptidase activity - after acidic

Therefore lots of different proteins can be broken down
Peptides range from 3-44aa in length and these can be further broken down in the cytosol by amino-peptidases

Question 70

Describe the 19S proteosome cap

Answer 70

Each 20S core has 2x 19S caps on either end
The cap recognises the Ub-protein and cleaves off the Ub so that it can be recycled.The unfolding of the substrate is mediated by AAA ATPase activities in the 19S cap which enables the protein to enter the core.

Question 71

What proportion of newly synthesised proteins are degraded immediately after synthesis? and what are they called?

Answer 71

1/3 due to defects - they are called defective ribosomal initiation products (DRiPs)

Question 72

Why do DRiPs occur?

Answer 72

Maybe because of inaccurate transcription/translation and have errors for example the wrong stop codon or initiation codon and these need to be removed quickly to prevent a build up

Question 73

What happens to DRiPs which lack a stop codon?

Answer 73

The mRNA lacks a stop codon and therefore the ribosome stalls when it reaches the poly A tail where in yeast Ltn1 E3 ligase Ub them for degradation. Ltn1 is part of the quality control complex which associates with stalled ribosomes via the 60S subunit. It is thought the mammalian homologue plays a similar role and if K.O. it is embryonically lethal

Question 74

Descibe cytosolic chaperones and the UPS

Answer 74

Cytosolic chaperones promote folding and stabilize proteins that cannot fold to prevent aggregation. Misfolded proteins are degraded so likely to find misfolded proteins bound by chaperones so there is a relationship between the UPS and chaperones.
Chaperones binds to surface exposed hydrophobic patches where are present in misfolded proteins not folded proteins therefore chaperones recognize proteins for degradation. A number of E3 ligases interact with chaperones, for example E3 Ub ligase constitutive Hsc70 interacting protein (CHIP) binds to the chaperones Hsc70, Hsp70 and Hsp90 and hence associates with misfolded proteins. CHIP can interact with the 26S proteosome via the co-chaperone BAG-1

Question 75

What is autophagy?

Answer 75

‘Self-eating’ a process which cytoplasmic components are degraded by delivery to the lysosome

Question 76

What are the 3 classes of autophagy?

Answer 76

1) . Macroautophagy
2) . Chaperone-mediated autophagy
3) . Microautophagy

Question 77

Describe lysosomes

Answer 77

Found in all mammalian cells except RBCs
Typically 200-400nm in diameter
Membrane-bound organelle
pH 4.5-5.0 - distinct environment maintained by vacuolar proton pump
Contain a range of hydrolases and proteases including:
1) cathepsin D - asprtate endopeptidase
2) cathepsin L - cysteine endopeptidase
3). cathepsin B - cysteine protease with both endo/carboxypeptidase activity
Membrane transporter to transport the free aa into the cytosol

Question 78

Describe macroautophagy

Answer 78

Involves the removal of cytoplasmic components (organelles and proteins) for degradation by lysosome
Long-lived cytosolic proteins and protein aggregates can be broken down
It is enhanced by cell starvation, hence aa recycling
It is a multistep process involving the formation of an autophagosome (cytoplasmic components are surrounded by a characterised double-membrane - a number of organelles have been proposed to donate the membrane including ER, golgi, mitochondria and PM) then this fuses with a lysosome (autophagasomes are transported along MT to the MTOC where the concentration of lysosomes is high), fusion is SNARE-dependent.

Question 79

Macroautophagy and misfolded proteins

Answer 79

Macroautophagy compliments the proteosome and CMA by degrading proteins which resist unfolding and aggregates. It can encapsulate large volumes of cytoplasmic material therefore can sometimes be non-specific but aggregates can be selectivity targeting via recognition with specific adaptor proteins such as p62 which link the aggregates to the assembling autophagosome

Question 80

Describe chaperone-mediated autophagy (CMA)

Answer 80

Involves direct delivery of proteins to the lysosome and doesn’t involved the formation of membrane-bound autophagosomes
Recognizes KFERQ-like motifs that are present in 1/3 of all cytosolic proteins and is only exposed if the proteins are unfolded/misfolded or a complex disassembled. The cytosolic chaperone Hsc70 recognises FKERQ-like motif and this binds to the cytosolic portion of LAMP-2A (receptor on lysosome) and get translocation into the lysosome. Substrate binding promotes LAMP-2A multimerisation, unfolding of substrate and translocation.

Question 81

Can the CMA pathway degrade folding proteins or aggregates?

Answer 81

No

Question 82

Describe alpha-synuclein

Answer 82

140aa protein found in the nerve terminals with a role in synaptic vesicle recylcing
Forms amyloid fibrils whicha re a majoy component of Lewy bodies
Alpha-syn amyloid can be transmitted between cells i.e. seeding further aggregation in previously healthy neurones

Question 83

What is Parkinson’s disease?

Answer 83

Neurodegenative disorder (2nd most common)
Movement disorder associated with the death of dopaminergic neurones in SN
Hallmark is the formation of Lewy bodiesies in the cytoplasm of effected neurones
Majority of cases are sporadic which increases with age but subset due to mutations in a number of genes including alpha-syncuclein

Question 84

How are proteostasis and alpha-syn linked?

Answer 84

The aggregation of alpha-syn into amyloid is a failure in proteostasis as it fails to prevent and remove misfolded alpha-syn and mutant forms of alpha-syn and its aggregates can distrupt the degradation machinary - nicous cycle
Trisomy (extra copy of the gene) is associated with an increase risk of PD as increased levels of alpha-syn is a major risk factor
alpha-syn is degraded by both UPS and autophagy although CMA may be the dominant pathway
Neurons from PD patients have reduced levels of capthepsin D, LAMP-2A and Hsc70 - degradation machinary
alpha-syn has a KFERQ-like motif for degradation by CMA

Question 85

What is the dominant pathway for the removal of alpha-syn?

Answer 85

CMA pathway

Question 86

What are the mutants in alpha-syn associated with PD? and how do these interfere with CMA?

Answer 86

A53T and A30P - Get bound by Hsc70 to the KFERQ-like motif and delivered the lysosome membrane but not the lumen and this impairs other substrates degradation including neuronal survival factor MEF2D

Question 87

Are alpha-syn aggregates broken down by macroautophagy?

Answer 87

Yes is thought to play a role however Lewy bodies may not get broken down and may inhibit macroautophagy

Question 88

Does mTOR regulate macroautophagy?

Answer 88

Yes - active mTOR suppresses macroautophagy and starvation causes mTOR inhibition promoting macroautopahgy and the recycling of aa’s.

Question 89

Does rapamycin inhibit mTOR? Can it be used as a PD drug?

Answer 89

Yes and this means it promotes macroautophagy and therefore can promote alpha-syn degradation. Shown to reduce neuronal death in animal models

Question 90

What is TFEB?

Answer 90

TFEB is a TF which is a positive regulator of macroautophagy and lysosome biogenesis therefore overexpression or chemical activation promotes clearance of alpha-syn and prevents neuronal death in animal models

Question 91

How much of the proteosome is transcribed on the ER?

Answer 91

1/3 - major site for protein folding

Question 92

How do proteins get into the ER?

Answer 92

Signal recognition particle (SRP) sequence is at the N-term of proteins and targets proteins for the ER as it is recognised by cytosolic ribonucleoprotein complex signal recognition particle (SPR) which pauses translation to prevent folding in the cytosol. SRP delivers the ribosome/nascent chain to the ER membrane and interacts with the SRP receptor which displaces it once it binds to SEC61 translocon. This is a pore across the membrane which resumes protein translation into the ER lumen. Folding nd modifications can occur as the protein is being translocated into the ER.

Question 93

Protein folding in the ER

Answer 93

Multi-faceted process - chaperones promote protein folding and assembly, DS formation formation and rearrgement and proteins are glycosylated (can be used to monitor protein folding.

Question 94

What is BiP? and how does it work?

Answer 94

Binding immunoglobulin protein
ER lumenal Hsp70 chaperone with roles in translocation, folding and degradation. It is required for translocation into the ER and retains the proteins in the ER
ATP hydrolysis enables BiP o bind to unfolded regions of proteins and exchange of ADP for ATP cause BiP to dissociate and provides a chance for the protein to fold. Facilitates protein folding but if still attached can be a signal for degradation

Question 95

Is the ER an oxidizing or reducing environment?

Answer 95

Oxidizing so favors the formation of DS bonds. PDI’s isomerise the DS bonds to the correct cysteines

Question 96

Describe N-linked glycosylation and the advantages of it.

Answer 96

Majority of proteins in the ER undergo N-linked glycosylation. A pre-formed oligosaccharide chain is co-translationally transferred from a dolichol lipid precursor onto an Asparagine (N) by OST enzymes.
Consists of 2x GlcNac’s, 9x Mannose’s and 3x Glucose’s.
OST enzyme associates with the translocon and scans for consensus sequence Asn-X-Thr/Ser.
Addition improves protein stablity. provides binding site for calnexin and calreticulin (faciliates DS bond isomerases) and it is used to monitor protein folding and identify for degradation

Question 97

Describe N-linked glycosylation and how it is used to monitor protein folding

Answer 97

After addition the oligosaccharide is trimmed leaving 1x glucose by the enzyme glucosidases. This trimming makes it a ligand for calnexin (mem associated) or calreticulin which retain proteins in the ER, prevent aggregation and promote binding of DS isomerase ERp7. When the final glucose is trimmed it is no longer a substrate for calnexin and calreticulin. If folding is complete protein can leave the ER but if not complete a Glucose can be added on by UGT enzyme and the cycle gets repeated - multiple rounds of folding. Removal of mannose residues over time as acts as a molecular clock that monitors protein folding, enzyme is called mannosidases. This reduces the addition of glucose by UGT and if sufficient mannoses are removed the protein is diverted from the folding pathway to the degradation pathway ERAD.

Question 98

Describe ERAD pathway

Answer 98

95% of nascent proteins correctly fold in the ER but misfolded proteins are recognized for removal by ERAD.
Adaptors recognize misfolded proteins and they get retro-translocated into the cytosol and poly-Ub for degradation by the proteosome.
Adaptor e.g. SEL1 is an ER membrane protein which can recruit additional adaptors e.g. lectins, XTP-B, OS9 which will recognize mannose trimmed substrates. BiP can also act as an adaptor and deliver them to E3 ligases.
HRD1 is a E3 ligase and is membrane bound - Ub occurs on the cytosolic face of the ER membrane.
Candidates for the translocation pore are SEC61 in reverse, E3 HRD1 forming a multimer or Derlins.
Ub-substrate is recognized by cytosolic AAA ATPase p97/cdc48 which uses ATP hydrolysis to energize the extraction of the protein from the ER. Protein is delivered to the proteosome where a population is present at the cytosolic face of the ER - the 19S cap may also help extract the protein.

Question 99

What do the differential of B cells into plasma cells trigger?

Answer 99

Unfolded protein response (UPR) as plasma cells make antibodies

Question 100

Which system does HCMV hijack and why?

Answer 100

ERAD? - to degrade MHC1 molecules

Question 101

How does HCMV target MHC1 for degradation?

Answer 101

By ERAD - US2/11 hijack ERAD t cause correctly folded MHC1 proteins to be delivered to E3 ligases causing Ub, translocation back into the cytosol and degradation. US2 is dependent on signal peptidase for MHC1 degradation whereas US11 is dependent on Derlin-1 and SEL1L

Question 102

Describe CF disease

Answer 102

CFTR is a large protein complex found on the PM of epithelial cells and is an ATP-gated channel which transport Cl1 ions into mucous to increase water movement and therefore make it more viscous. Only ~25% of WT folds correctly as it is very complex.dom

1/27 Caucasians carry mutant form of CFTR and CF affects 1/2000 - severe life-limiting disease.

F508 mutant is the most common - autosomal recessive mutation resulting in loss of F508 at the interface of the membrane spanning domain and the nucleotide binding domain 1. Can still transport some Cl- ions but all is retained in the ER and degraded by ERAD (multiple E3 ligases - RNF5, RNF185 and CHIP). CHIP is recruited to Hsc70 bound CFTR, RNF recruited via Hsc70-associated Hsp40 DnajB12 and associates with Derlin-1. RNF5/185 can detect defects as CFTR is being synthesized.

Question 103

Pharmacological targets of CFTR

Answer 103

Lumacaftor increase the ER exit of F508 and increase Cl- transport by stabilizing the NBD1 and membrane spanning domain interfaces. Limited clinical benefit alone but best it combination with ivacaftor which increases channel opening.

ER stress response can also activate unconventional pathway of protein secretion on GRASP. IRE1 mediated signalling activates GRASP dependent secretion via P of this protein. GRASP can bind F508 and target it to the cell surface where it can function as an ion channel. Transgenic mice can recuse F508 phenotype using this pathway so might provide a new therapy.