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ANAT 212 > Lecture 5 > Flashcards

Lecture 5 Flashcards

1
Q

how do cells respond to stress that causes protein misfolding

A

increasing expression of chaperones and other specialized proteins

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2
Q

are all chaperones hsps

A

NOOO
but all hsps are chaperones

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3
Q

describe hsr

A

heat shock response = cytosolic and nuclear proteins, protects against cell death

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4
Q

describe upr

A

unfolded protein response =ER proteins, can promote cell death if stress too severe

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5
Q

what do cells tailor the expression of chaperones to

A

tailor amount of chaperones to need of cell that has to reform misfolded proteins
the level of unfolded and misfolded protein

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6
Q

describe inducible chaperones

A

heat shock proteins

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7
Q

describe constitutive chaperones

A

assisted protein folding = proteins that facilitate folding of others = hold or stabilizing hydrophobic residues and assist folding
universal mechanism of protein homeostasis

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8
Q

describe drosophila and heat

A

heat induces transcription activation of certain genes and expression of specific proteins
discovered by accident
all cells respond to heat the same way = stop producing some proteins and favour production of other proteins

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9
Q

what is heat shock response activated by

A

unfolded cytosolic proteins - heat stress or oxidative damage or proteasome inhibition of alcohol ingestion

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10
Q

what happens during and after stress

A

during = transcription of hsps upregulated and transcription of other genes = down regulated - many hsps are chaperones
response continues after stress removed to help cells recover
expression highest ~12 hrs after = need time to refold proteins

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11
Q

what does HSF1 do

A

activates transcription of hsps

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12
Q

describe HSF1

A

transcription factor mediates hsr
has dna binding domain, regulatory domain, transcription activation domain

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13
Q

describe active and inactive HSF1

A

inactive HSF1 = monomeric
active hsf1 = trimer = recognizes HSE (heat shock element) promoters
trimerizes = quaternary structure

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14
Q

describe regulation of hsf - step 1

A

monomeric hsf1 is folded but mimics unfolded protein and is bound by hsp90
hsp 90 holds as monomer - recognizes patches

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15
Q

describe regulation of hsf - step 2

A

after heat shock unfolded proteins compete with hsf1 for hsp90 binding = more proteins want hsp90 so leaves hsf1 and allows hsf1 to trimerize and bind promoter

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16
Q

describe regulation of hsf - step 3

A

free hsf1 trimerizes and activates transcription

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17
Q

describe regulation of hsf - step 4

A

chaperones including hsp 90 are expressed and help fold or degrade unfolded proteins

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18
Q

describe regulation of hsf - step 5

A

hsf1 is down regulated by binding of excess hsp90 to the monomer form
so hsf1 is now bound again

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19
Q

do all substrates need the same chaperones

A

NOOOO
certain degrees of specificty
some substrates require specific chaperones or combos of chaperones

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20
Q

describe atp dependent chaperones

A

Actively promote folding
substrate binding and release are regulated by atpase cycles

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21
Q

describe atp independent chaperones

A

prevent aggregation and can catalyze some folding steps
good at holding substrate

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22
Q

describe cooperation between chaperones

A

usually work in network
cytosol
endoplasmic reticulum
constitutive and inducible = create network for the cell to have all the proteins folded

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23
Q

describe families of chaperones

A

Different families of chaperone proteins use various biochemical mechanisms – “protein folding toolkit”
3 families of ATP-dependent chaperones, with different structures and ATPase cycles

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24
Q

name atp dependent chaperone families and describe briefly

A

hsp 70 = works as monomer
hsp90 = dimer, nutcracker
chaperonines = hsp60 = provides cage and safe environment for protein to fold by itself

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25
describe hsp70 - table
cytosol = hsc70 (active) and hsp70 (inducible) ER = BiP others = mitochondria, ribosomes
26
describe hsp90 - table
cytosol = hsp90, alpha and beta er = GRP94 others = mitochondria
27
describe hsp60- table
chaperonin cytosol = TRic er = - others = hsp60 in mitochondria
28
what does hsp60 function like
E coli GroEL
29
which chaperones are constitutively expressed
all are always expressed except HSP70
30
which chaperones are induced by heat shock response
hsp70 hsp90 hsp60
31
which are induced by ER unfolded protein response
Bip GRP94
32
describe hsp70 family
70kda monomers atpase domain controls substrate binding domain of hsp70 atp bound = no substrate binding adp bound = substrate binding domain is closed tightly on peptide binds short hydrophobic sequences
33
how does hsp70 function
with help of cochaperones = proteins which contact chaperones to regulate activity some can bind to polypeptide substrates themselves and are both chaperones and cochaperones
34
name hsp70 co-chaperones
DNAJ (HSP40) family promote hsp70 substrate binding nucleotide exchange factors (NEFS) promote substrate release
35
describe hsp70 functional cycle - step 1
hsp40 recognzies misfolded protein Hsp40-mediated delivery of substrate to ATP-bound Hsp70 induces atpase activity
36
describe hsp70 functional cycle - step 2
Hydrolysis of ATP to ADP mediated by Hsp40 results in closing of the α-helical lid and tight binding of substrate by Hsp70 conformational change = allows hsp70 to bind substrate
37
describe hsp70 functional cycle - step 3
NEF catalyzes exchange of ADP for ATP adp liberates and binds to hsp70
38
describe hsp70 functional cycle - step 4
Opening of the α- helical lid, induced by ATP binding, results in substrate release
39
describe hsp70 functional cycle - step 5
Released substrate either folds to native state or given to another hsp
40
describe DNAJ co-chaperones
regulate hsp70 function many = at least 53 genes in humans
41
describe domains of DNAJ co-chaperones
all have conserved j domains = bind transiently to hsp70, activate it to hydrolyze atp (induces atpase activity) and bind polypeptide, DOES NOT bind substrate other domains determine their specific biological function = could bind substrate or for trimerization
42
which DNAJs are the most highly conserved
substrate binding dnajs
43
describe substrate binding dnajs
j domain, substrate binding, dimerization homodimers = 2 subunits of 40-50kda (originally identified as hsp40) bind short hydrophobic sequences transfer substrate to hsp70 during atp hydrolysis
44
describe dnajs that bind substrate
some bind substrate through specific domains and act as atp independent chaperones like HSP40 can hold and help fold
45
describe dnajs that do not bind substrate
specific domains attach dnaj to protein complex or intracellular membrane these recruit hsp70s to complex or membrane - bring to complex that has substrate
46
describe NEFs
Nucleotide Exchange Factors (NEF) remove ADP from HSP70 and allow ATP to bind NEF binding opens up HSP70 ATPase domain and weakens interactions with nucleotide ATP binds when NEF dissociates ATP-bound HSP70 to releases polypeptide (transition) Several NEF families in humans
47
how does hsp70 help folding
HSP70 binds hydrophobic regions of folding intermediates and prevents incorrect contacts from forming Release of polypeptide from HSP70 provides chances for it to fold Balance between DNAJs and NEFs supports an optimal rate of HSP70 substrate binding and release Substrate-binding DNAJs may provide additional assistance Can form multi-chaperone complex with HSP90
48
describe hsp90 family
homodimers with 2 identical subunits joined at c terminus - human - hsp 90 = 2x90kda=180kda open and close like nutcracker atp controls opening and closing cochaperone p23 = stabilizes closed form thought to bind polypeptides at late stages of folding --> stabilizes intermediate folded states (specific for hsp90) substrate bound along sides of subunit diff substrates can bind to diff sites on sides (unlike hsp70s and chaperonins)
49
describe hsp90 functional cycle - informal
n terminus = atp binding domain c terminus = dimerization domain dimer Recognizes client HOP = cochaperone recognoizes hsp70 and 90 p23 stabilized binding and now closed atp hydrolyzed FKBP52 open and cannot bind client
50
describe hsp90 functional cycle - 3 steps
1 - Substrate is bound weakly in the open nucleotide-free state 2 - ATP binding allows dimer to close and bind substrate tightly 3 - ATP hydrolysis to ADP compacts the dimer and releases substrate
51
describe hsp70-hsp90 co-chaperone system
Cytosolic HSP70 and HSP90 form a multi- chaperone system cooperate to assist substrates Substrate is released from HSP70 and bound by HSP90 in coordination HOP assists complex formation HSP70 dissociates when HSP90 binds ATP
52
describe the co-chaperones that regulate hsp 70 and hsp90
many cochaperones regulate = sometimes active on substrates, provide flexibility, folding and non folding functions
53
what is similar between hsp70 and hsp90
similar eevd motif cytosolic hsp70 and 90 = not homologous but have similar c terminal sequence motids 4 aas = motif (4-5 recognized by domain)
54
what recognizes EEVD motifs
TPR domains recognize eevd motifs can be specific for hsc70, hsp90 or both
55
describe TPR cochaperones
tpr domains are adaptors to hsp70/90 often have other domains which interact with substrates directly
56
name tpr cochaperones
HOP FKBP52 CHIP
57
describe HOP
has domains which bind hsp70 and hsp90 specifically 2 tpr domains
58
describe FKBP52
has an hsp90 binding tpr domain and PPlase domains (cochaperone and chaperone) PPlase = peptidyl-prolyl isomerase = chaperone specific to prolines (P52) = proline is cis and trans so domain helps transition of proline from cis to trans
59
describe CHIP
binds either hsp70 or 90 has ubiquitin ligase domain that helps degrade proteins = if no way to fold = degrade
60
what are many hsp substrates
signal transduction proteins = kinases, receptors, transcription factors many also require hsc70
61
what can hsp90 bind without needing HSC70
hsp90 can bind kinases without needing hsc70
62
what do mutations in signalling proteins cause
cancer `
63
what are drug targets for cancer treatments
HSP90 and HSC70
64
describe c-src and v-src
c-src = cellular, normal kinase involved in signalling cell growth, cells have this, kinase not always active, autoregulated v-src = viral, mutant kinase that causes cancer, modified so not regulated region = always active and always cell division
65
descibe v-src - drug therapy
must inhibit hsp90 = viral kinase won't achieve native conformation - folded functional state 1 - v-src expressed in epithelial cells causes them to become cancerous 2 - treat cells with hsp90 inhibitors 3 - hsp90 cannot chaperone v-src anymore 4 - cells revert from cancer to normal growth
66
describe hsp60 family
large oligomeric complexes with typical double ring structure 2 cages = one up and down, have alternate cycles depends on what binds = can be a bigger or smaller cage - accommodates substrate differently
67
describe groel cavity
rings are identical and work in alternating cycles
68
describe groel cavity - down position
no nucleotide subunits around ring bind to hydrophobic polypeptide smaller wall of hydrophobic residues - interact with other residues = cavity will attract misfolded proteins
69
describe groel cavity - up position
atp bound subunits bind to groes cap instead of substrate large cavity with polar surface formed Substrate released inside cavity = enclosed but no longer bound provides space to fold
70
describe groel subunits
Each GroEL subunit has an ATPase domain and a substrate- binding domain The ATPase domain is the interface with the opposite ring (upside down) Movement of the substrate binding domain is controlled by the ATPase in both rings atp bound = conformation change
71
describe groel functional cycle - one ring
smaller conformation -- bring substrate conformational change and promotes binding of cap ~7 secs = atp hydrolyzes and proteins folds by itself then liberate groes, folded protein and adp
72
how does groel help folding
Substrate enclosed inside polar cavity provides chance to fold confinement promotes folding by favouring more compact conformations atp hydrolysis acts as a timer for substrate release
73
what functions like groel
human mitochondrial HSP60
74
describe human chaperonin in cytosol
TRiC (TCP1 ring complex) does not have cap co-chaperone long substrate binding domains form the cavity themselves
75
what causes heat shock response - summary
stress conditions that trigger protein misfolding induce hsr
76
what does heat shock response refer to - summary
activation of expression of certain constitutive and inducible chaperones known as hsps
77
how are hsps identified - summary
by molecular weight and atp dependent or independent
78
what do hsps do - summary
fold proteins in cytosol and nucleus and are pro-survival
79
describe hsp families - summary
3 main atp dependent = hsp90, hsp70, hsp60 use diff approaches and co-chaperones to fold proteins
80
do misfolded proteins usually only need one chaperone - summary
many client misfolded proteins need cooperation of several co-chaperones
81
describe groel cycle - step 1
top ring binds substrate in down position - no nucleotide
82
describe groel cycle - step 2
binds atp and moves up to position = conformational change
83
describe groel cycle - step 3
binds groes and encloses substrate inside cavity
84
describe groel cycle - step 4
hydrolyzes atp Substrate stays inside
85
describe groel cycle - step 5
release adp, moves down to position, substrate released native substrate will not be bound again
86
what is a consequence of inhibiting hsp90 for cancer therapies
hsp90 keeps hsf1 in monomer = so will lead to many more chaperones (since hsf1 will be in active trimer state ) = more proteins and more heat shock response
87
describe GroEL - hsp60 family
2 rings x 7 identical subunits x 60kda = 840kda
88
describe GroES - hsp60 family
cap co-chaperone 7 subunits x 10kda = 70kda
89
what are homologs of human mitochondrial HSP60 and HSP10
GroEL GroES
90
what does substrate binding domain of groel bind
substrate-binding domain either binds substrate (down, no nucleotide) or GroES (up, ATP-bound)

ANAT 212 (34 decks)

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