Lecture VI Flashcards
(108 cards)
1
Q
What 3 compartments does protein synthesis and folding occur in eukaryotic cells?
A
cytosol
ER
mitochondria
2
Q
What is the native structure?
A
correct #d structure of a protein
3
Q
What is RNAse like in its native structure?
A
contains 4 disulfite bonds
4
Q
What experiment first explained how proteins fold?
A
RNase folding studied by Anfinsen
5
Q
Where is folding information found?
A
inside the primary structure (aminoacidic sequence of the protein)
6
Q
What is the Levinthal Paradox?
A
mathematical calculation to try all the possible random conformation that can occur for 1000 amino acids (10²⁷)
7
Q
Does protein folding occur by chance?
A
no, it is driven by forces that are able to overcome Levinthal Paradox (information inside the primary structure)
8
Q
What kind of process is protein folding?
A
thermodynamic process
9
Q
When are proteins most stable?
A
in their native conformation (lowest energy)
10
Q
What is the misfoldeing confirmation?
A
when proteins have problems folding the correct way
11
Q
What is more stable: misfolded or unfolded proteins?
A
misfolded proteins
12
Q
Review the diagram and observe the energy levels with respect to the protein’s conformation:
A
13
Q
What are the forces that drive protein folding?
A
hydrophobic interactions
electrostatic interactions
hydrogen bonds (formed on the lateral residues of different amino acids, that are a part of the protein, and among the atoms of the peptide backbone of the protein)
14
Q
What helps proteins fold?
A
chaperone proteins
15
Q
What are heatshock proteins (Hsp)?
A
chaperone proteins discovered in the late 70’s by Kelley and Schlesinger who were studying protein folding when cells were subjected to heatshock
16
Q
What is the primary site of the secretory pathway?
A
ER
17
Q
What happens at the ER?
A
proteins that have to be secreted are exposed to the plasma membrane
enzymes that are involved in the secretory pathway are synthesized
18
Q
Why is protein folding in the ER difficult?
A
there is a high probability of being trapped in “energy traps”, so misfolded conformation is high due to different covalent modifications in the ER
19
Q
What are the different covalent modifications in the ER?
A
cleavage of the leader peptide
N-Glycosylation
GPI anchors
SS bond formation
Hydroxylation (Pro and Lys)
20
Q
Why is it so hard for proteins to fold in the ER?
A
it is a crowded environment
high protein concentrations
specific enzymes or chaperones are needed to help proteins fold in the ER
21
Q
What are the different families of chaperones and enzymes that assist protein folding in the ER?
A
chaperones
lectins
redox enzymes
proline isomerases
sugar processing
22
Q
What are “private chaperones”?
A
chaperones that are used by specific proteins that are more difficult to fold
23
Q
What is Hsp47 a private chaperone for?
A
collagen
24
Q
What is BiP (immunoglobulin binding protein)?
A
most famous chaperone in the ER (aka Hsp70) that is the most abundant chaperone in all cells
25
What is BiP always bound to?
heavy chains of immunoglobulins
26
Describe an immunoglobulin:
ATPase domain (needs ATP to fold protein)
substrate-binding domain (binds the exposed hydrophobic regions of proteins that are trying to fold)
27
What can BiP do?
recognize the hydrophobic regions of the immunoglobulin and bind to them to help them fold using ATP hydrolysis and this prevents the aggregation between unfolded proteins
28
What is the most up-regulated chaperone under stress conditions?
BiP (Hsp70)
29
How are ER proteins stabilized?
via different covalent modifications
30
List the different modification ER proteins go through to be stabilized:
cleavage of the leader peptide by singal peptidase when the protein enters the ER
some proteins then undergo N-Glycosylation (addition of sugar on the lateral residue of Asparagin (-NH2) *not all asparagine: only the ones with Asn-X-Ser/Thr sequence (X is every aa except proline)
31
In terms of google, what is N-Glycosylation?
modification of appropriate asparagine residues with oligosaccharide (sugar) structures
*this happens on proteins that need to be folded
32
What kind of sugar is an oligosaccharide?
polymeric sugar
33
What is the oligosaccharide (polymeric sugar) composed of?
3 glucoses
9 mannoses
2 n-acetyl-glucoaminases
34
Why is the composition of the oligosaccharide important?
the "remodeling" of the sugar is important for the folding of the protein itself
35
Where is the sugar (oligosaccharide) synthesized?
synthesis starts in the cytosol
flippases insert the structure into the lumen of the ER
synthesis continues on the ER membrane
36
What happens after the sugar (oligosaccharide) has been completely synthesized?
an enzyme (oligosaccaril-transferase) binds the sugar to the Asparagine in the consensus sequence of the nascent protein
37
What happens after the sugar has been bound to the consensus sequence of the nascent protein?
glucosidase I cuts the 1st glucose
glucosidase II cuts the second glucose
*protein is left with only 1 glucose in its branch glycosylation
38
What is the protein with 1 glucose recognized by?
2 chaperone (calreticulin and calnexin)
*both are lectins that specifically recognize a sugar
39
What do calreticulin and calnexin work with?
enzyme Erp57
40
What is the goal of calnexin and calreticulin working with enzyme Erp57?
fold the protein
41
What cuts the last glucose off the protein?
glucosidase II
42
What recognizes a misfolded proteins?
enzyme, UGGT
43
hat does UGGT do?
gives back a glucose so that the protein can be bound to calnexin and calreticulin again to have another chance to fold
44
At a certain point, if the protein continues to be misfolded, what happens?
Mannosidase I cuts the mannoses
UGGT is not able to re-glutosidate the protein
**mannosidase I is the TIMER
45
What happens after mannosidase I cuts the mannoses?
protein will be recognized by the degradation system of the ER
46
Why do proteins need to form disulfide bonds (SS)?
it is a characteristic of a protein that has to be secreted
47
What proteins usually do not have disulfide bonds?
cytosolic proteins (unless they are regulatory disulfide bonds)
48
What does the formation of a disufide bonds in secretory proteins do?
increases its stability
49
What is special about the extracellular environment and the secretory pathway?
they are both oxidizing
50
What is more oxidizing than the cytosol?
the ER
51
How can we measure the fact that the ER is more oxidizing than the cytosol?
using a redox ratio of glutathione/oxidized glutathione
52
What is the formation of SS bonds needed for?
protein stability
compactness
polymerization
QC
53
Why are SS bonds needed for polymerization?
link 2 different polypeptides
54
What do oxidoreductases do?
catalyze the formation and the disruption or isomerization of disulfide bonds in secretory proteins
55
What are the most famous protein disulfide-isomerase (PDI)?
oxidoreductases
56
What is Erp57?
oxidoreductase
*catalyze an oxidation of a secretory protein
57
What is the donation of an SS bond called?
oxidation
58
If a protein is not able to fold and has to be degraded but has already ormed some SS bonds, what needs to happen?
it needs to be reduced
59
How can a protein be reduced?
using an enzyme known as oxidase, which forms a covalent intermediate so that the protein can be reduced and the enzyme oxidized
60
What is isomerization?
wrong SS bonds are formed
oxidoreductase is reduced
oxidoreductase binds to the protein to favor the exchange of the disulfide bonds to get to the correct disulfate bonds in the protein
oxidoreductase is still reduced
61
Oxidation
enzyme at the beginning is oxidized then reduced
62
Reduction of the Cargo Protein
enzyme is reduced then oxidized
63
Isomerization
enzyme is reduced at the beginning and end
64
What is able to oxidize a protein?
PDI
*at the end it is reduced
65
What can recharge PDI after it has oxidized a protein and become reduced?
Ero1 (it is oxidized and donates its SS bonds to PDI, which leads it to be reduced)
66
How can Ero1 be re-oxidized?
giving its reducing equivalent to molecular oxygen to have oxidized Ero1 and one molecule of H₂O₂
67
Since H₂O₂ is dangerous in high amounts, what is able to detoxify it to water?
GPX7 and GPX8
or it can be used by PRX4 to obtain an oxidized PRX4 form that can donate an SS bond to PDI
68
How can we ensure that only native proteins are secreted?
using quality control systems of eukaryotic cells
69
What is the 1st QC system?
calreticulin/calnexin cycle
70
What does the calreticulin/calnexin cycle ensure?
oly correctly folded proteins can proceed to the Golgi compartment
71
What is the 2nd form of QC system?
formation of the quaternary structure
72
What is the 1st response we produce against pathogens?
IgM polymeric protein
*it is a pentameric IgM composed of 21 polypeptides, 51 glycans and 100 SS bonds
73
List the steps in IgM formaation and assembly:
formation of the base (aka "monomers": 2 heavy chains and 2 light chains)
heavy chain of Ig is bound by BiP
SS bonds between the cysteines form, which is called polymerization (they are present at the C-terminal of the heavy chain of IgM
74
Can B cells produce IgM and polymerize them?
no
75
Can plasma cells produce IgM and polymerize them?
yes
76
If IgM are not assembled, are they secreted?
no
77
Why is the heavy chain always retained?
because it bonds to BiP, and BiP stays in the ER
78
Why is the monomer retained?
it contains a cysteine at the C-terminal that is not involved in the SS bond
79
What are the 2 forms of QC in the secretory pathway?
in the ER, it is mediated by calnexin/careticulin and BiP (proximal QC)
in the Gogli
80
What is proximal QC ensuring?
monomeric proteins are folded correctly and dimers are linked with no covalent interactions
81
What is golgi QC ensuring?
monomers compose polymeric structures linked by SS bonds
*if the monomers are folded correctly, they leave to be polymerized and if theyare correctly assembled when they arrive at the golgi, they will be released
82
What is TCR?
polymeric transmembrane protein composed of different transmembrane molecules that travel together from the ER to the plasma membrane
83
Why are the enzymes (PDI, calreticulin, BiP, and Ero1) not normally secreted by cells?
they are part of the matrix of the column, which is inserted into vesicles that exit the ER
cargoes that exit the matrix are also transported out of the ER
84
What is the KDEL sequence?
an aminoacidic sequence (lysin, aspartic acid, glutamic acid, and leucine)
85
What are RDEL and KEEL?
aminoacidic sequence (positive charge, 2 negative charges, and a hydrophobic)
86
What happens if the enzymes concentrated in vesicles leave the ER?
KDEL receptor waits for them i the golgi, will catch them from the ER and bring them back
87
What are 2 enzymes that do NOT have a KDEL sequence?
Ero1 and peroxiredoxin
88
What do Ero1 and peroxiredoxin use since they do not have a KDEL sequence?
ERGIC, which is a compartment between the golgi and ER
89
What happens when Ero1 and peroxiredoxin reach ERGIC?
they bind the protein ERp44, which has a KDEL sequence at the C-terminal and with the ERp44 it can be brought back so that the cell maintains homeostasis of the secretory pathway
90
What do Ero1 and PDI have a role in?
blood coagulation
*Ero1 also plays a role in control of blood pressure
91
What maintains the QC of oligomeric proteins linking via disulfide bonds?
ERp44 (44 is the MW and ER because when it was first isolate, it contained reticular staining)
92
Describe the composition of the protein, ERp44:
3 thioredoxin-like domains (domain typical of the oxidoreductase in the ER)
tail that covers the active site
* only 1 cysteine, so it cannot act as a oxidase or reductase (could be an isomerase)
93
Describe the composition of the protein, PDI:
4 thioredoxin-like domain
94
Describe ERp44 in the ER:
it is in closed conformation and it binds to ERGIC-53 (a cargo receptor) to move from the ER to the golgi
95
At a steady state, where is ERp44?
not in the ER, it is in the middle
96
Describe the golgi:
more acidic than ER due to lysosome exchange and GPHR which pumps proteins into the golgi
high zinc concentration pumped into the golgi by 4 zinc transporters: ZnTs 4, 5, 6, and 7
97
What happens when ERp44 arrives in the golgi compartment?
the pH change and presence of zinc induces a conformational change
protein opens its tail and the histidines bind zinc
protein can now interact with substrate covalently
*protein also exposes C-terminal RDEL sequence
98
What is the cycle of ERp44 able to do?
keep Ero1 inside the ER and act on immunoglobulins
99
What is ERp44 capable of doing?
recognize unassembled structures, isomerize the disulfide bonds, and help correct polymerization
100
What was a problem when generating a KO ERp44 mouse model?
they died very early, their skull did not completely close, and they had less bone density, and more spine curvature
101
What did the phenotype of the KO ERp44 mice resemble?
Ehlers' Danlos mouse model, which is a syndrome that is characterized by problems in the EC matrix formation
102
What do patients with Ehlers' Danlos syndrome suffer from?
vascular problems or frequent dislocation due to malformed tendons
103
What is Ehlers' Danlos syndrome linked with?
zinc
104
What was hypothesized in the zebrafish lab in regards to the lack of ERp44?
lack of ERp44 could affect vertebrate formation and mineralization
it for sure affects collagen deposition and fibrils assembly
105
A hypothesis is that the presence or absence of ERp44 affects ______.
the presence or absence of ERp44 affects zinc homeostasis in the secretory pathway and collagen assembly
106
Why is zinc important?
zinc affects collagen folding for the alteration of signaling cascades necessary for bone development
107
What kind of relationship do ERp44 and Zinc have?
bidirectional relationship
ERp44 binds to zinc
in thigh zinc concentration, ERp44 can form a dimer in which 5 zinc atoms are inserted and function as a zinc buffer for the secretory pathway
108
Where does the formation of collagen trimers start?
C-terminal and it is mediated by the formation of specific intermonomer disulfide bonds
