ER protein folding and degradation

Question 1

where is the signal sequence for proteins located in the ER

Answer 1

N-term

Question 2

which protein recognises signal sequences on proteins targeted to the ER

Answer 2

cytosolic ribonucleoprotein complex signal recognition particle (SRP)

Question 3

describe the role of SRP and the process of translocation of nascent proteins into the Er

Answer 3

it binds signal sequences and halts translation to prevent folding in the cytosol. it delivers the ribosome and nascent chain to the ER membrane via interactions with the SRPR. SRP is displaced once the ribosome binds the Sec61 translocon and translation resumes. sec61 forms an aqueous pore to translocate the protein. folding and modification occurs as the protein is translocated

Question 4

what is BiP

Answer 4

it is an ER luminal Hsp70 required for translocation, folding and degradation

Question 5

how is BiP involved in protein translocation into the ER

Answer 5

it binds the nascent chain as it is inserted and retains it in the ER

Question 6

how is BiP involved in protein assembly in the ER

Answer 6

energy from ATP hydrolysis enables BiPto bind unfolded regions of proteins. exchange of ATP for ADP causes dissociation and provides an opportuntiy for the protein to fold

Question 7

why does disulphide bond formation occur in the ER

Answer 7

it is an oxidising environment and favours formation

Question 8

at what point can disulphide bond formation occur in the ER on nascent proteins

Answer 8

it can begin when the protein is being translocated

Question 9

how is disulphide bond formation promoted between correct pairs of cysteine

Answer 9

protein disulphide isomerases can oxidise, reduce and rearrange disulphide bonds

Question 10

what occurs in N-linked glycosylation

Answer 10

a preformed oligosacharride is transferred from a dolichol lipid precursor to asparagine (N) by oligo-sacharyltransferase (OST)

Question 11

what is the preformed oligosaccharide chain formed of which is added in N linked glycosylation

Answer 11

2 N-acetylglucosamines, 9 mannose, 3 terminal glucose

Question 12

what is the consensus sequence for N linked glycosylation

Answer 12

Asn-X-Thr/Serwhere x=any amino acid except proline

Question 13

what are the roles of N-linked glycosylation

Answer 13

improves protein solubility, provides binding sites for challenging and calreticulum which facilitates interactions with PDIs and finally it is used to monitor protein folding

Question 14

after addition of the N-linkedoligosacharride what happens to it

Answer 14

it is trimmed to leave a single glucose residue by sequential action of glucosidases I and II. the trimmed oligosacharride is a ligand for calnexin and calreticulum which retain the protein in the ER to prevent aggregation and promote folding by binding PIDs. the final glucose is then trimmed which causes the protein to dissociate from calnexin and calreticulum.

Question 15

what happens to a protein if, after N-linked glycosylation and consequent trimming and folding, the protein is still not correctly folded

Answer 15

a glucose residue is added by UDP-glucose glycoprotein glycosyl-transferase (UGT) and the cycle is repeated. alternatively a slow process of mannose removal catalysed by mannosidases can occur which reduces action of UGT. if sufficient mannose residues are removed the protein is diverted from the folding pathway to the ERAD pathway

Question 16

what proteins recognise misfolded proteins in the ER

Answer 16

adaptor proteins- SEL1L which can also recruit additional adaptors incl. lectins XTP3-B and OS9. also BiP

Question 17

where does ubiqitination of proteins destined for the ERAD pathway occur

Answer 17

the cytosolic face of the ER membrane

Question 18

name an example of a ER membrane bound E3 ubiquitin ligase

Answer 18

HRD1

Question 19

why is SEC61 a candidate for the retro-translocation pore in the ERAD pathway

Answer 19

because mutations can impact on getting proteins out of the ER

Question 20

why is E3 HRD1 a candidate for the retro-translocation pore in the ERAD pathway

Answer 20

it forms multimers in the ER membrane and a simplified system showed that this was sufficient for retrotranslocation

Question 21

what are derlins

Answer 21

membrane bound proteins which promote luminal protein transport across the membrane

Question 22

after ubiquitination how is the protein retrotranslocated

Answer 22

it is recognised by the cytosolic AAA+ ATPase p97/Cdc48 which uses ATP hydrolysis to energise the extraction of the protein from the ER into the cytosol

Question 23

how are proteins in the ERAD pathway degraded

Answer 23

they are delivered to the proteasome in the cytosol

Question 24

how might translocation and degradation in the ERAD pathway be coupled

Answer 24

because there is a population of proteasome at the cytosolic face of the ER membrane and they may act to pull the protein from the membrane via the AAA+ ATPase activity in the 19s cap

Question 25

what triggers the UPR

Answer 25

accumulation of misfolded proteins in the ER

Question 26

what effect do the signals induced by the UPR have overall

Answer 26

they reduce bulk protein synthesis, promote chaperone production, increase ERAD and increase the amount of ER

Question 27

which proteins are unregulated as a result of the cleavage of AFT6 to form the AFT6(N) transcription factor in the UPR

Answer 27

BiP, GRP94 (an Hsp90), PDI

Question 28

what cellular pathways does XBP1 affect as a result of cleavage by IRE1 activation in the UPR

Answer 28

it promotes regulation of lipid biosynthetic enzymes, up regulates ERAD components, induces plasma cell differentiation and degrades mRNAs of secretory proteins thus reducing protein synthesis and producing more ER

Question 29

what is CFTR

Answer 29

an ATP gated plasma membrane channel, expressed in epithelial cells, which transports Cl- ions across the membrane into the mucous to increase water movement and make it less viscous

Question 30

what occurs in the absence of CFTR function in the lungs

Answer 30

there is a build up of viscous mucous resulting in inflammation and infection causing lung damage

Question 31

what is the most common mutation that occurs in CFTR

Answer 31

deletion of F508 (known as DF508)

Question 32

what is the fate of the DF508 mutant

Answer 32

despite retaining some functionality, 100% of it is retained in the ER and regarded via the ERAD pathway

Question 33

describe UPR dependent trafficking of CFTR DF508 to the cell surface

Answer 33

under ER stress, unconventiona protein secretion dependent on GRASP is activated. IRE1 signalling activates GRASP which binds to CFTR DF508 and targets it to the PM.

Question 34

although UPR dependent trafficking of CFTR DF508 to the PM may be used as a therapeutic, what are some of the problems faced by this potential solution

Answer 34

there are QC mechanisms at the PM which may recognise the misfolded protein and recruit chip leading to degradation

Question 35

aside from utilising the UPR dependent trafficking system, what other potential therapies are being researched for CF

Answer 35

small molecules which can stabilise the misfolded protein to increase expression at the cell surface. this is not sufficient by itself but in conjunctio with another drug to cause channel opening may increase function

Question 36

what are the HCMV US2 and US11 proteins

Answer 36

they are ER localised integral membrane proteins which target MHCI for degradation by acting as virally encoded adaptors for the ERAD pathway

Question 37

what is the effect of choler toxin on the body

Answer 37

it promotes electrolyte and water movement into the intestinal lumen resulting in severe diarrhoea

Question 38

what is the molecular mechanism of cholera toxin

Answer 38

it is internalised by cells and trafficked to the ER via the secretory pathway. Here the CTA1 subunit dissociates from CTA2/CTB dependent on PDI. CTA1 is unfolded and retrotranslocated into the cytosol. it escapes proteasomal degradation due to a lack of sine residues for ubiquitination. in the cytosolic binds adenylate cyclase increasing levels of cAMP which activates PKA. PKA phosphorylates CFTR promoting Cl- secretion