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Molecular Cell Biology > Lecture VI > Flashcards

Lecture VI Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What 3 compartments does protein synthesis and folding occur in eukaryotic cells?

A

cytosol

ER

mitochondria

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2
Q

What is the native structure?

A

correct #d structure of a protein

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3
Q

What is RNAse like in its native structure?

A

contains 4 disulfite bonds

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4
Q

What experiment first explained how proteins fold?

A

RNase folding studied by Anfinsen

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5
Q

Where is folding information found?

A

inside the primary structure (aminoacidic sequence of the protein)

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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)

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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)

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8
Q

What kind of process is protein folding?

A

thermodynamic process

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9
Q

When are proteins most stable?

A

in their native conformation (lowest energy)

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10
Q

What is the misfoldeing confirmation?

A

when proteins have problems folding the correct way

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11
Q

What is more stable: misfolded or unfolded proteins?

A

misfolded proteins

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12
Q

Review the diagram and observe the energy levels with respect to the protein’s conformation:

A
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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)

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14
Q

What helps proteins fold?

A

chaperone proteins

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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

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16
Q

What is the primary site of the secretory pathway?

A

ER

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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

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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

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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)

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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

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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

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22
Q

What are “private chaperones”?

A

chaperones that are used by specific proteins that are more difficult to fold

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23
Q

What is Hsp47 a private chaperone for?

A

collagen

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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

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25
Q

What is BiP always bound to?

A

heavy chains of immunoglobulins

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26
Q

Describe an immunoglobulin:

A

ATPase domain (needs ATP to fold protein)

substrate-binding domain (binds the exposed hydrophobic regions of proteins that are trying to fold)

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27
Q

What can BiP do?

A

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

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28
Q

What is the most up-regulated chaperone under stress conditions?

A

BiP (Hsp70)

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29
Q

How are ER proteins stabilized?

A

via different covalent modifications

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30
Q

List the different modification ER proteins go through to be stabilized:

A

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)

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31
Q

In terms of google, what is N-Glycosylation?

A

modification of appropriate asparagine residues with oligosaccharide (sugar) structures

*this happens on proteins that need to be folded

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32
Q

What kind of sugar is an oligosaccharide?

A

polymeric sugar

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33
Q

What is the oligosaccharide (polymeric sugar) composed of?

A

3 glucoses

9 mannoses

2 n-acetyl-glucoaminases

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34
Q

Why is the composition of the oligosaccharide important?

A

the “remodeling” of the sugar is important for the folding of the protein itself

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35
Q

Where is the sugar (oligosaccharide) synthesized?

A

synthesis starts in the cytosol

flippases insert the structure into the lumen of the ER

synthesis continues on the ER membrane

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36
Q

What happens after the sugar (oligosaccharide) has been completely synthesized?

A

an enzyme (oligosaccaril-transferase) binds the sugar to the Asparagine in the consensus sequence of the nascent protein

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37
Q

What happens after the sugar has been bound to the consensus sequence of the nascent protein?

A

glucosidase I cuts the 1st glucose

glucosidase II cuts the second glucose

*protein is left with only 1 glucose in its branch glycosylation

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38
Q

What is the protein with 1 glucose recognized by?

A

2 chaperone (calreticulin and calnexin)

*both are lectins that specifically recognize a sugar

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39
Q

What do calreticulin and calnexin work with?

A

enzyme Erp57

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40
Q

What is the goal of calnexin and calreticulin working with enzyme Erp57?

A

fold the protein

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41
Q

What cuts the last glucose off the protein?

A

glucosidase II

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42
Q

What recognizes a misfolded proteins?

A

enzyme, UGGT

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43
Q

hat does UGGT do?

A

gives back a glucose so that the protein can be bound to calnexin and calreticulin again to have another chance to fold

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44
Q

At a certain point, if the protein continues to be misfolded, what happens?

A

Mannosidase I cuts the mannoses

UGGT is not able to re-glutosidate the protein

**mannosidase I is the TIMER

45
Q

What happens after mannosidase I cuts the mannoses?

A

protein will be recognized by the degradation system of the ER

46
Q

Why do proteins need to form disulfide bonds (SS)?

A

it is a characteristic of a protein that has to be secreted

47
Q

What proteins usually do not have disulfide bonds?

A

cytosolic proteins (unless they are regulatory disulfide bonds)

48
Q

What does the formation of a disufide bonds in secretory proteins do?

A

increases its stability

49
Q

What is special about the extracellular environment and the secretory pathway?

A

they are both oxidizing

50
Q

What is more oxidizing than the cytosol?

A

the ER

51
Q

How can we measure the fact that the ER is more oxidizing than the cytosol?

A

using a redox ratio of glutathione/oxidized glutathione

52
Q

What is the formation of SS bonds needed for?

A

protein stability

compactness

polymerization

QC

53
Q

Why are SS bonds needed for polymerization?

A

link 2 different polypeptides

54
Q

What do oxidoreductases do?

A

catalyze the formation and the disruption or isomerization of disulfide bonds in secretory proteins

55
Q

What are the most famous protein disulfide-isomerase (PDI)?

A

oxidoreductases

56
Q

What is Erp57?

A

oxidoreductase

*catalyze an oxidation of a secretory protein

57
Q

What is the donation of an SS bond called?

A

oxidation

58
Q

If a protein is not able to fold and has to be degraded but has already ormed some SS bonds, what needs to happen?

A

it needs to be reduced

59
Q

How can a protein be reduced?

A

using an enzyme known as oxidase, which forms a covalent intermediate so that the protein can be reduced and the enzyme oxidized

60
Q

What is isomerization?

A

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
Q

Oxidation

A

enzyme at the beginning is oxidized then reduced

62
Q

Reduction of the Cargo Protein

A

enzyme is reduced then oxidized

63
Q

Isomerization

A

enzyme is reduced at the beginning and end

64
Q

What is able to oxidize a protein?

A

PDI

*at the end it is reduced

65
Q

What can recharge PDI after it has oxidized a protein and become reduced?

A

Ero1 (it is oxidized and donates its SS bonds to PDI, which leads it to be reduced)

66
Q

How can Ero1 be re-oxidized?

A

giving its reducing equivalent to molecular oxygen to have oxidized Ero1 and one molecule of H₂O₂

67
Q

Since H₂O₂ is dangerous in high amounts, what is able to detoxify it to water?

A

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
Q

How can we ensure that only native proteins are secreted?

A

using quality control systems of eukaryotic cells

69
Q

What is the 1st QC system?

A

calreticulin/calnexin cycle

70
Q

What does the calreticulin/calnexin cycle ensure?

A

oly correctly folded proteins can proceed to the Golgi compartment

71
Q

What is the 2nd form of QC system?

A

formation of the quaternary structure

72
Q

What is the 1st response we produce against pathogens?

A

IgM polymeric protein

*it is a pentameric IgM composed of 21 polypeptides, 51 glycans and 100 SS bonds

73
Q

List the steps in IgM formaation and assembly:

A

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
Q

Can B cells produce IgM and polymerize them?

A

no

75
Q

Can plasma cells produce IgM and polymerize them?

A

yes

76
Q

If IgM are not assembled, are they secreted?

A

no

77
Q

Why is the heavy chain always retained?

A

because it bonds to BiP, and BiP stays in the ER

78
Q

Why is the monomer retained?

A

it contains a cysteine at the C-terminal that is not involved in the SS bond

79
Q

What are the 2 forms of QC in the secretory pathway?

A

in the ER, it is mediated by calnexin/careticulin and BiP (proximal QC)

in the Gogli

80
Q

What is proximal QC ensuring?

A

monomeric proteins are folded correctly and dimers are linked with no covalent interactions

81
Q

What is golgi QC ensuring?

A

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
Q

What is TCR?

A

polymeric transmembrane protein composed of different transmembrane molecules that travel together from the ER to the plasma membrane

83
Q

Why are the enzymes (PDI, calreticulin, BiP, and Ero1) not normally secreted by cells?

A

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
Q

What is the KDEL sequence?

A

an aminoacidic sequence (lysin, aspartic acid, glutamic acid, and leucine)

85
Q

What are RDEL and KEEL?

A

aminoacidic sequence (positive charge, 2 negative charges, and a hydrophobic)

86
Q

What happens if the enzymes concentrated in vesicles leave the ER?

A

KDEL receptor waits for them i the golgi, will catch them from the ER and bring them back

87
Q

What are 2 enzymes that do NOT have a KDEL sequence?

A

Ero1 and peroxiredoxin

88
Q

What do Ero1 and peroxiredoxin use since they do not have a KDEL sequence?

A

ERGIC, which is a compartment between the golgi and ER

89
Q

What happens when Ero1 and peroxiredoxin reach ERGIC?

A

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
Q

What do Ero1 and PDI have a role in?

A

blood coagulation

*Ero1 also plays a role in control of blood pressure

91
Q

What maintains the QC of oligomeric proteins linking via disulfide bonds?

A

ERp44 (44 is the MW and ER because when it was first isolate, it contained reticular staining)

92
Q

Describe the composition of the protein, ERp44:

A

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
Q

Describe the composition of the protein, PDI:

A

4 thioredoxin-like domain

94
Q

Describe ERp44 in the ER:

A

it is in closed conformation and it binds to ERGIC-53 (a cargo receptor) to move from the ER to the golgi

95
Q

At a steady state, where is ERp44?

A

not in the ER, it is in the middle

96
Q

Describe the golgi:

A

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
Q

What happens when ERp44 arrives in the golgi compartment?

A

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
Q

What is the cycle of ERp44 able to do?

A

keep Ero1 inside the ER and act on immunoglobulins

99
Q

What is ERp44 capable of doing?

A

recognize unassembled structures, isomerize the disulfide bonds, and help correct polymerization

100
Q

What was a problem when generating a KO ERp44 mouse model?

A

they died very early, their skull did not completely close, and they had less bone density, and more spine curvature

101
Q

What did the phenotype of the KO ERp44 mice resemble?

A

Ehlers’ Danlos mouse model, which is a syndrome that is characterized by problems in the EC matrix formation

102
Q

What do patients with Ehlers’ Danlos syndrome suffer from?

A

vascular problems or frequent dislocation due to malformed tendons

103
Q

What is Ehlers’ Danlos syndrome linked with?

A

zinc

104
Q

What was hypothesized in the zebrafish lab in regards to the lack of ERp44?

A

lack of ERp44 could affect vertebrate formation and mineralization

it for sure affects collagen deposition and fibrils assembly

105
Q

A hypothesis is that the presence or absence of ERp44 affects ______.

A

the presence or absence of ERp44 affects zinc homeostasis in the secretory pathway and collagen assembly

106
Q

Why is zinc important?

A

zinc affects collagen folding for the alteration of signaling cascades necessary for bone development

107
Q

What kind of relationship do ERp44 and Zinc have?

A

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
Q

Where does the formation of collagen trimers start?

A

C-terminal and it is mediated by the formation of specific intermonomer disulfide bonds

Molecular Cell Biology (10 decks)

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