Chaperones and disease Flashcards
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
Cells incubated under different conditions
- cells with nothing added- no budding
- cytosol but no ATP added back- no budding
- cytosol and ATP- budding
- reduce cytosol conc- reduce budding
- reduced cytosol + coat + Sar1= some budding
- Reduced cytosol + purify components- budding
Sar1A + mutant Sec23A
some budding
Sar1B + mutant Sec23A
No budding
F382L - sec23A
is not competent for budding of cargo containing vesicles in vitro when paired with Sar1B
What is the underlying cause of the in vitro budding defect?
GTPase of SAR1- locked in GTP form
intrinsic ability to hydrolyse by GAP (GTP hydrolyse protein)
Experiment to show SAR1Bs effect hydrolysis?
Meausure intrinsic fluorescence
- way less hydrolysis with SAR1B (F382L +13/31)
What initiates Cop11 binding
Sar1 activation
What is the role of Sec23/24 and Sec13/31
23/24- recognises cargo, weak GAP activity
13/31- enhances the GAP activity
Highly homologous but some non overlapping functions are cargo specific
What is the cargo transported by Cop11 vesicles
- secreted molecules
- transmembrane proteins destined for intracellular organelles
- recycling components- SNAREs and putative cargo
- p23/24
ERGIC-53
receptor for luminal cargo
mutations= combined deficiency of coagulations factors V and V11
Why do you put pro collagen into a cop11 vesicle?
To visualise cargo inside is difficult
Model for Cop11
- Large vesicular carriers (protein become incases in vesicle)
- non vesicular intermediate- between golgi and ER
What do the microscopes show?
- show collagen
- see colocalisation with Sec31
Only see to the refraction limit of life
What happens when you have abnormal vacuolisation of notochord cells in the BBF2H7-KO medaka
- don’t have appropriate lining up
- for lining you need the UPR= activation for ER stress
How do Cop1 move transport vesicles
- move materials retrograde cargo- KDEL and Dilysina motifs
- anterograde cargo (VSUG) through secretory pathway
ARF1
GTP exchange activates myristroyl switch
What is KDEL in yeast?
ERD2
What inhibits retrograde transport?
Anti-B-COP antibodies
What is the TGN is the major cargo sorting station for
- sorts newly synthesised lysosomal proteins
- sorting between constitutive and regulated secretory pathways
- apical and basolateral membranes
- Establish planar cell polarity
Signal for lysosomal proteins
M-6-M
What does the lysosomal proteins allow for
- It to be packaged
- addition of phosphate, binding to M6P receptor
- receptor dependent transportation
- remove phosphate, dissociate at Acidic PH
- receptor recyling
Sorting of lysosomal enzymes in steps
- N lined oligosaccharide, binds signal patch to recognition
- Transfer of GLcNAc-p to manage catalytic site
- release
- lysosomal hydrolyase with GlcNAc-p attached to mannose in oligosaccharide
What is important for cargo sorting at the TGN
AP1, AP3 and GGAs
What is I cell disease?
Inclusion cell
- fibroblasts contain no lysosomal hydrolyses (found in blood)
- recessive gene
What are the motifs recognised by AP1 and GGA
- tyrosine based
- dileucine based
- inner membrane contact sites
What is tyrosine based motif?
AP4 mediated transport of APP
- lysosomes, basolateral, somatodendritic
Dileucine based motif
AP1 signal recognise compartments by u subunit
- Eddo-Lysosomal, basolateral endosomal
Inner membrane contact sites
proteins tethered visualised by EM, MCD protein components are often extended to allow bridges to form
