Proteostasis

Question 1

what is gaucheries disease

Answer 1

it is a lysosomal storage disease in which lipid accumulates in cells

Question 2

what causes gaucheries disease

Answer 2

hereditary deficiency in glucosylceramidase resulting in accumulation of glucosylceramide mostly in macrophages

Question 3

which signalling pathways are involved in regulation of the proteostasis network

Answer 3

Heat shock response, dietary restriction, calcium signalling, HDACs, chaperones

Question 4

at what point does gaucheries occur

Answer 4

when activity levels of GC fall below 10%

Question 5

why must GC be able to fold in a number of different conditions

Answer 5

because it is synthesised in the ER (pH7.4), trafficked to the golgi in vesicles (pH6), then buds from the golgi in vesicles to the lysosome (pH5)

Question 6

under what conditions is the L444P mutant of GC folded and trafficked and what does this say about it

Answer 6

it only occurs at 30 degrees, not 37 showing its only stable enough in the ER at low temps

Question 7

what is the result of overzealous monitoring of protein folding in relation to GC

Answer 7

mutated proteins are able to fold but they are still recognised as mutated and so they’re sent to the ERAD pathway

Question 8

what are proteostasis regulators

Answer 8

small biological molecules that control concentration, conformation, quaternary structure and location of proteins by manipulating the proteostasis network through influencing signalling pathways

Question 9

how can Endo-H digestion analysis be used to determine whether GC has been properly folded and left the ER

Answer 9

in the ER mannose rich glycans are added to GC which can be cleaved by Endo-H however in the golgi more complex carbs are added and these are not able to be cleaved by endo-H. therefore a protein stuck in the ER will be cleaved and this can be visualised on a gel

Question 10

what is the overall effect of adding celastrol or MG-132 on GC trafficking

Answer 10

they increase the active concentration of GC in the lysosome

Question 11

what is the mechanism of celastrol and MG-132 in increasing active GC conc in the lysosome

Answer 11

they up regulate PERK, IRE-1 and ATF6 to up regulate transcription of genes involved in chaperones and folding machinery in the ER lumen thus destabalising misfolded state and increasing the flux of moleculaes to a folded state in the ER lumen over lower energy barriers

Question 12

what are AFT6, PERK and IRE1

Answer 12

3 branches of the UPR

Question 13

what happens which HSP70/BIP binds AFT6, PERK or IRE1

Answer 13

inactivates them

Question 14

how are the IRE1, AFT6 and PERK pathways initiated

Answer 14

a misfolded protein in the ER lumen causes Bip to dissociate from AFT6, PERK and IRE1 to act as a chaperone. in doing so those 3 proteins are activated, initiating the signalling pathways

Question 15

describe the mechanisms of action of the IRE1 signalling pathway and how does addition of celastrol and MG-132 affect this

Answer 15

BIP dissociates and IRE1 dimerises then transphosphorylates another subunit to activate it. this activates mRNA splicing in the cytoplasm, acting specifically on XBP1 mRNA to remove an intron. the eons are joined together by ligase activity to create the active form which is then translated. the mRNA codes for the XBP1s transcription factor which results in transcription of chaperones, lipid synthesis and ERAD proteins. addition of small molecules (celastrol and MG-132) results in more spliced XBP1 compared to un-spliced

Question 16

describe the mechanisms of action of the AFT6 signalling pathway and how does addition of celastrol and MG-132 affect this

Answer 16

bip is released causing aft6 to be transported to the golgi where it is cleaved. the soluble domain becomes a transcription factor for genes involved in production of ER resident chaperones and UPR. celastrol and mg132 increase cleavage

Question 17

describe the mechanisms of action of the PERK signalling pathway and how does addition of celastrol and MG-132 affect this

Answer 17

bip dissociates causing perk dimerisation. it then phosphorylates EIF2 which is involved in translation initiation of mRNA by ribosomes. phosphorylation activates EIF2 which slows down protein synthesis to allow ER to recover from misfolding and selectively translates ATF4. this leads to production of CHOP which is a transcription factor which up regulates genes of the UPR. addition of celastrol and mg-132 up regulates levels of CHOP

Question 18

how could a pharmacological chaperone be used to treat lysosomal storage diseases and give an example of this

Answer 18

it enters the ER and stabalises gaucheries protein which would allow it to be trafficked to the lysosome. it would then need to dissociate in the lysosome. NN-DNJ is an inhibitor of GC which stabilises it and increases activity of the N370S mutant in the lysosome. it binds tightly in the ER for trafficking but once in the lysosome substrate displaces the inhibitor as it has a much higher affinity