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BIOC212 > MVU4 PROTEIN FOLDING IN THE CELL -3 > Flashcards

MVU4 PROTEIN FOLDING IN THE CELL -3 Flashcards

1
Q

what is the nature of many chaperones?

A

they are heat shock proteins
all HSP are chaperones but not all chaperones are HSP

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2
Q

how do cells respond to stress that causes protein misfolding?

A

increase the expression of chaperones and other specialized proteins

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3
Q

where does the heat shock response happen and what does it do?

A

happens in cytosolic and nuclear proteins
protects against cell death

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4
Q

where does the unfolded protein response (UPR) happens and what does it do?

A

happens in ER proteins
can promote cell death if the stress is too severe

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5
Q

what are inducible chaperones?

A

the heat shock proteins
heat induces the transcriptional activation of specific genes, which induces the transcription of certain proteins

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6
Q

what are constitutive chaperones?

A

assist in protein folding
proteins that facilitate the folding of others
hold or stabilise the hydrophobic residues
universal mechanism of protein homeostasis

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7
Q

what can cause unfolded cytosolic proteins?

A

heat stress
oxidative damage
proteasome inhibition

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8
Q

how long after the stress is removed does the stress response continue?

A

only after 24 hours does the cell return to normal

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9
Q

what transcription factor mediates the heat shock response?

A

HSF1

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10
Q

what domains does HSF1 have?

A

DNA binding domain
regulatory domain
transcription activation domain

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11
Q

what are the active and inactive forms of HSF1?

A

inactive = monomeric
active = trimer

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12
Q

what sequence in the DNA does active HSF1 recognise?

A

recognises heat shock element promoters (HSE)

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13
Q

what does the regulatory domain of HSF1 do?

A

allows it to become a trimer

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14
Q

how is HSF1 regulated? (before, during and after the heat shock)

A

before the heat shock: monomeric HSF1 is folded, but mimics unfolded proteins and is bound by Hsp90 (has exposed hydrophobic patches)
after: unfolded proteins compete with HSF1 for Hsp90 binding
free HSF1, liberated from Hsp90, trimerises and activates transcription
chaperones, included Hsp90 are expressed and help fold or degrade unfolded protein
after: HSF1 is down regulated by binding excess Hsp90 to the monomer form

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15
Q

what do some substrates require?

A

some require specific chaperones or combination of chaperones

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16
Q

what do ATP dependent chaperones do?

A

actively promote folding
substrate binding and release is regulate by ATPase cycles

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17
Q

what do ATP independent chaperones do?

A

prevent aggregation and can catalyse some folding steps

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18
Q

how do chaperones work?

A

in networks, cooperate

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19
Q

what do different families of chaperone proteins use?

A

different biochemical mechanisms

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20
Q

what are the 3 different families of ATP dependent chaperones?

A

Hsp70 family
monomers
locking pliers

Hsp90 family
dimers
nutcrackers

chaperonins (Hsp60)
trimer
cage

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21
Q

which two chaperones from the HSP70 family are present in the cytosol?

A

HSC70 (constitutive)
HSP70 (inducible)

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22
Q

which two chaperones from the HSP90 family are present in the cytosol?

A

HSP90 alpha and beta

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23
Q

which chaperonin (HSP60) is present in the cytosol?

A

TRiC

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24
Q

which protein from the HSP70 family is present in the ER?

A

BiP

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25
which protein from the HSP90 family is present in the ER?
GRP94
26
what chaperone in E.coli works like HSP60?
GroEL
27
which is the one chaperone that is not expressed constitutively?
HSP70
28
how do HSP70 chaperones work?
70kDa monmers the ATPase domain controls substrate binding domain ATP bound: no substrate peptide binding ADP bound: the substrate binding domain is closed tightly on the peptide binds short hydrophobic sequences
29
what are co chaperones?
co chaperones are proteins which contact chaperones to regulate their activity some can bind to polypeptide themselves and are both chaperones and co chaperones
30
what are some HSP70 co chaperones?
DNAJ (HSP40) promoter HSP70 substrate binding nucleotide exchange factors (NEFs) promote substrate release
31
what is the HSP70 functional cycle?
1. HSP40 mediated delivery of substrate to ATP bound HSP70 2. hydrolysis of ATP to ADP mediated by HSP40 results in closing of the alpha helical lid and tight binding of substrate by HSP70 3. NEF catalyses exchange of ADP for ATP 4. opening of the alpha helical lid induced by ATP binding, results in substrate release 5. released substrate either folds to native state or given to another HSP
32
what do DNAJs do?
regulate HSP70 function
33
how many genes code for DNAJs?
at least 53
34
what are the different domains of DNAJs?
all have a conserved J domain bind transiently to HSP70, activate it to hydrolyse ATP and bind polypeptide do not bind substrate other domains determine their specific biological function substrate binding (only some have this, bind to hydrophobic sequences) dimerization domain
35
what do DNAJ co chaperones do?
homodimers: 2 subunits of 40-50kDa bind short hydrophobic sequences transfer substrate to HSP70 during ATP hydrolysis
36
what do DNAJs that bind substrates act as?
bind through specific domains act as ATP independent chaperones
37
what do DNAJs that do not bind substrates do?
specific domains attach DNAJ to a protein complex or intracellular membrane recruit HSP70 to the complex or the membrane
38
what do NEFs do?
remove ADP from HSP70 and allow ATP to bind NEF binding opens up HSP70 ATPase domain and weakens interaction with the nucleotide ATP binds when NEF dissociates ATP bound HSP70 releases the polypeptide there are several NEF families in humans
39
how does HSP70 help folding?
HSP70 binds hydrophobic regions of folding intermediates and prevents incorrect proteins from forming release of the polypeptide from the HSP70 provides a chance for it to fold
40
the balance between which two things provides an optimal rate for HSP70?
DNAJs and NEFs
41
what is the structure of HSP90 chaperones?
homodimers, 2 identical subunits joined at the C termini human HSP90= 2x90kDa= 180kDa
42
what is the analogy of how the dimer works
can open and close like a nutcracker
43
what controls the opening and closing of the HSP90 dimer?
ATP
44
what stabilises the closed form of the HSP90 dimer?
co chaperone p23
45
at what stage of folding does HSP90 bind? and what does it bind to?
late stage folding binds to hydrophobic and polar surfaces, stabilises intermediate folded states substrate is bound along the sides of the subunit different substrates can bind to different sites on the sides, unlike HSP70 and chaperonins
46
what do HSP70 and HSP90 form?
a multi chaperone system they cooperate to assist substrates released from HSP70 and bound by HSP90 HSP70 dissociates when HSP90 binds ATP
47
what assists in the formation of the HSP70 and HSP90 complex?
HOP co chaperones provide flexibility, have folding and non folding functions
48
what is the similarity between HSP70 and HSP90?
not homologous but have similar C terminal sequence motif EEVD-COO-
49
what domain recognises these EEVD motifs?
TPR domains in HOP proteins can be specific for HSC70, HSP90 or both
50
what are TPR domains?
adaptors to HSP70 and HSP90
51
what do TPR co chaperones often have?
other domains which interact with substrate directly
52
what specific domains does HOP have?
domains that bind HSP70 and HSP90
53
what TPR domains does FKBP52 have?
HSP90 binding domain and PPIase domains peptidyl-propyl isomerase: chaperone specific to prolines
54
what does CHIP bind?
either HSP70 or HSP90 and has a ubiquitin ligase domain that helps degrade proteins
55
what types of proteins are many HSP90 substrates?
signal transduction proteins kinases, receptors, transcription factors
56
what do those signal transduction proteins also require and what is the exception?
many require HSC70 as well except kinases, HSP90 binds to them without needing HSC70
57
what do mutations in signaling proteins cause?
cancer
58
what chaperones are often drug targets for cancer treatment?
HSP90 and HSC70
59
what is the difference btween c-src and v-src
c-src (cellular) is the normal kinase involved in signaling cell growth it is auto regulated and not always active v-src (viral) is a mutant kinase that causes cancer HSP90 helps it stay active and make tumors
60
how can you treat cancer caused by v-src?
v-src expressed in fibroblast epithelial cells causes them to become cancerous treat cells with HSP90 inhibitor HSP90 cannot chaperone v-src anymore cells revert from cancer to normal growth
61
how does the HPS90 functional cycle work? (steps)
1. the C terminus of the proteins is the dimerisation domain, the middle region recognises the client, and the N terminus is the ATP binding domain 2. the client binds to HSP90, accompanied by HSP40 and HSP70, with the help of the co chaperone HOP 4. ATP binds to the N terminus of the protein, and the p23 stabilises that binding and closes the conformation 5. FKBP52 also stabilises the dimer 5. ATP is hydrolysed to ADP, and the client and other proteins are liberated 6. ADP is now bound to the protein, but ADP needs to be liberated so that HSP90 can bind to the next client
62
what is the structure of the E.coli GroEL?
2 rings x 7 identical subunits x 60kDa = 840kDa 2 cages, one up and one down with alternative cycles
63
what is the structure of the E.coli GroES cap co chaperone?
7 subunits x 10 kDA= 70kDa
64
what are the different domains of the HSP60?
substrate binding domain ATPase domain substrate binding domain +GroES cap
65
how does ATP change the conformation of HSP60?
ATP binding changes the size of the cage
66
what is the conformation of the down position of the HSP60?
no nucleotide smaller hydrophobic subunits around the ring bind to the hydrophobic polypeptide
67
what is the conformation of the up position of the HSP60?
ATP bound subunits bind to GroES cap instead of the substrate large cavity with polar surface is formed doesn't interact with polypeptide, gives it space to fold released into the cavity
68
what are the domains in each of the GroEL subunits and their functions during down and up positions?
ATPase domain (hydrolyses ATP) linker region substrate binding region in the down conformation the substrate binding region has a hydrophobic residue that points towards the center the movement of the substrate binding domain is controlled by the ATPase domain in the up conformation the subunit is taller, the hydrophobic residue binds to the GroES
69
what is the GroEL functional cycle (for one ring)
the protein is in its down conformation ATP binds to it switches to up conformation, gets bigger binds the cap releases the protein inside the cavity, gives it space to fold ATP is hydrolysed to ADP releases the protein
70

BIOC212 (32 decks)

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