Chaperones and disease Flashcards
(52 cards)
What are chaperones?
proteins that assist folding or unfolding of things in the cell
What do knockout chaperone studies show?
CRT-/- = cardiac problems CNX-/- = demyelination
BiP
Essential for cultured cells-stress
What happens when both CRT and CNX are knocked out?
mild phenotypes- tissue specific effects rather than all cells
ER folding diseases
- cystic fibrosis
- emphysema
- hypothyroidism
How does ER folding affect cystic fibrosis and what happens when you down regulate?
folding of channel- stuck in ER doesn’t reach cell surface
Hsp90 cochaperone Ahal downregulation rescues misfiling of CFTR in CF
Downregulate= allows exit of GFTR from the ER, localisation in the wrong place
How does ER folding affect emphysema
deficiency of secretion of a1-anotrypsin
- old age runs fill up with mucous and oxygen tension goes down
How does ER folding affect hypothyrodidism?
What are the symptoms of hypothyroidism?
- tiredness
- depression
- sustained activation leads to death
ER stress pathway in ALS
Amyotrophic lateral sclerosis
sporadic and familial ALS causes ER stress which activates UPR
- activates= IRE1, PERK, AFT6
What are the different outcomes of the ER stress pathway dependent on ALS
IRE1 goes to either XBP1 (Degeneration- autophagy) or ASK1-JNK (degeneration- aapotosy)
E1F1a- protection (translation)
DIFFERENT OUTCOMES- patients respond differently to ALS
How was COP11 discovered?
identified in sec mutanta- ER tubules
- purified using in vitro assay
- biochemically and ultra structurally distinct from ER
Role of COP11
packaging material destined for golgi
- bud off ER and move to golgi
Different types of COP11 vesicles
- Sar1 (small GTPase)
- Sec23 (adaptor)-link cargo (proteins secreted to the coat
- Sec13/3 (coat)- doesn’t interact directly to the membrane
What happens if KDEL is brought back to the cell?
- cargo molecule is captured by an inner coat layer Sar1, Sec23 and Sec24
- prebudding complexes are concentrated by polymerization of the outer coat scaffold formed by sec13 and sec31- self assemble drives membrane association
- membrane undergoes fission to release a spherical transport carrier from the donor membrane
What was known at the start lf this work about COP11?
found patients with craniotomy- lecticular-sutural dysplasia
- takes time for fontanelle to close, contracts, facial dysplasia
What is Cranio-luticular-sutural dysplasia caused by?
Sec23 mutation leading to abnormal ER to golgi trafficking
What experiment was found the craniotomy condition?
Looked at ER
stained for collagen, sec23 and merge
Sec31 and sec23A mutant not in right place
Em- mutant= more distended
Autosomal recessive disease
What was the underlying defect of cranio dysplasia?
Sar1 GTPase= initiates budding following recruitment by sec12
sec23/sec24= engages cargo
Sec13/31= promotes coat polymerisation, membrane curvature and fission
What is CMRD
Chlylomicron-retention disease, lack of lipoprotein secretion into blood (missence sar1B)
Related disease ion same process Cop11 coat formation
CLSD
Missense in Sec23A
Why does a missense mutation in these paralogues cause tissue specific disease?
Redundancy
related proteins carry out same function
Does the F382L mutation affect Cop11 function in vitro?
Experiments to discover
- budding assay- mammalian cells permeabilised and cytoplasm washed
- prep membrane, add components and look for Cop11 formation
- isolate by centrifuge (ER goes to one place and harvest cop11 coated vesicles)
- Eric 53- used to measure vesicle formation
What is the control in the experiment?
Ribophorin 1- none means its clean
Start of reaction kept bait back and ran 20% input- shows theres enough ribophorin to detect the input
