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Molecular Cell Biology > Lecture VII > Flashcards

Lecture VII Flashcards

1
Q

What is the integrated stress response?

A

response the cell activates in case of proteostatic stress

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2
Q

Where can proteostatic stress rise from?

A

ER or mitochondria

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3
Q

What is an example of a mutatio that induces proteostatic stress?

A

AFG3L2 mutation

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4
Q

What does the absence or mutation of AFG3L2 (m-AAA in the mitochondria) lead to?

A

accumulation of mt-DNA-encoded polypeptides, which leads to proteostatic stress in the mitochondria due to failure of QC of the mitochondria-encoded polypeptides

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5
Q

What does the stress that arises in organelles and relayed back to the cytosol lead to?

A

activation of kinase and hyperphosphorylation of eIF2𝛼 (elongation factor of cytosolic translation

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6
Q

What does the phosphorylation of eIF2𝛼 lead to?

A

inhibition of cytosolic translation and reduction of protein load in order to allow the cell to reover from damage

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7
Q

eIF2𝛼 shuts down translation in general, but what protein escapes this attenuation?

A

ATf4

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8
Q

What is ATF4

A

TF that upon eIF2𝛼 phosphorylation, translocates to the nucleus

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9
Q

What does ATF4’s escape to the nucleus induce?

A

transcription of several target cytoprotective genes that are involved in the recovery from stress

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10
Q

What is the central regulation node of integrated stress response (ISR)?

A

eukaryotic translation initiation factor eIF2𝛼, which when phosphylated, inhibits cytosolic translation

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11
Q

What can activate GCN2, PERK, or HR1?

A

defects in the elctron transport chain (ETC), reactive oxygen species (ROS), and mitochondrial proteotoxic stress

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12
Q

What does the phosphorylation of eIF2𝛼 promote?

A

selective translation of TF ATF4, which promotes the expression of CEBP homologous protein (CHOP), growth arrest and DNA damage-inducible protein 34 (GADD34), ATF3, and other TFs to restore cell homeostasis

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13
Q

How can the mitochondria relay stress to the cytosol?

A

using a pathway that involves the stress sensor of the inner mitochondrial membrane (OMA1)

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14
Q

What is the stress sensor of the IMM?

A

OMA1

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15
Q

What does OMA1 do?

A

cleaves the protein of the inner membrane (DELE1

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16
Q

What happens with the cleaved form of DELE1?

A

it is sent back tot he cytosol and activate a kinase, HRI

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17
Q

What does HRI do?

A

phosphorylates eIF2𝛼, which induces a stress response

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18
Q

Why is protein degradation essential for life?

A

if we did not degrade protein, we would undergo premature cellular senescence

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19
Q

What does the control of protein turnover maintain?

A

homeostasis and opposes aging (proteome plasticity)

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20
Q

What causes proteins to be targeted for degradation?

A

ubiquitination

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21
Q

What are the main pathways in the cytosol that regulate protein degradation?

A

ubiquitin proteosome system and autophagy

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22
Q

In order to be degraded, what must target proteins have?

A

presence of the long polyubiquitin chain

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23
Q

How is ubiquitin attached to target proteins?

A

using a set of enzymes:
E1
E2
E3

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24
Q

What kind of enzyme is E1?

A

activating enzyme that binds ubiquitin onto Cys in an ATP-dependent manner

*ubiquitin is activated this way

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25
What kind of enzyme is E2?
conjugating enzyme
26
What kind of enzyme is E3?
free ligase that gives specificity of the target
27
In our genome we have 2 genes for E1, 20 genes for E2, and 600 genes for E3, what does this suggest?
we need high specificity for a lot of substrates
28
What happens after E3 binds and polyubiquitinates the target?
the protein is transferred to the proteosome chamber where there are DUBs enzymes (deubiquitinating enzymes) that remove the polyubiquitin chain
29
What happens after DUBs remove the polyubiquitinated chain?
protein is inserted into the proteosomal structure that is composed of 4 rings
30
What are the 4 ring structures in the proteosome?
2 𝛼-subunits (regulatory) and the bottom and periphery 2 β subunits (active catalytic) in the middle
31
What happens after the protein is inserted into the proteosomal ring structure?
the protein is degraded in proteolytic fragments (peptides) that are about 25 aa long *this infers other proteases are further engaged in order to recycle each single aa
32
What are the 3 types of autophagy in mammalian cells?
macroautophagy chaperone-mediated autophagy microautophagy
33
What is macroautophagy?
degradation os large dimension material (organelles, bacteria, or large protein aggregates
34
What is chaperone-mediated autophagy?
used when the cells need to degrade in a fast manner small proteins or small aggregates
35
What is needed for chaperone-mediated autophagy to take place?
proteins must contain a KFERQ specific pentapeptide to be targeted for this mechanism, and the pentapeptide motif is recognized by the chaperone protein HSPA8, that delivers the cargo directly to the lysosome
36
What are tau and 𝛼-sinuclein proteins?
proteins that accumulate in Alzheimer's disease and when they are unfolded or misfolded, they are targeted for chaperone-mediated autophagy
37
What is microphagy?
occurs in stress condition in order to get rid of damaged material quickly *cargo directly enters the lysosome
38
What is needed to trigger autophagy?
membranes to engulf cargoes machineries (kinases)
39
What are the membranes involved in autophagy derived from?
ER Golgi ERGIC endosomes
40
What is the kinase complex involved in autophagy?
ULK1/2 complex
41
What does the ULK1/2 complex do?
triggers the formation of the autophagosome when it is dephosphorylated
42
What does ULK1/2 recruit?
components of the ATG system
43
What does the ULK1/2 complex engage?
LC3
44
What do the ATG system and LC3 help ULK1/2 do?
form the autophagosome and engulf the cargo
45
Where and what does LC3 bind?
cargo inside the autophagosome *LC3-I becomes LC3-II (de-lipated form)
46
Describe the autophagy mechanism:
LC3-I binds phospholipids (phosphoethanolamine) and becomes the de-lipidated form of LC3-II (the one interacting with the cargo)
47
What can the autophagosome directly fuse with?
the lysosome for degredation or endosome to form an "amphisome", which will later fuse with the lysosome
48
What is the receptor, p62, essential for?
bind the de-lipated form of LC3-II
49
What is p62?
prototype of a family of receptors that are called Sequestrome Like Receptors (SLRs) they bridge between ubiquitinated cargo and LC3-II
50
Describe p62:
contains: polymerization domain (PB1) ubiquitin residues binding domain (UBA) LC3 interaction domain (LIR)
51
Where are LC3 and p62 located? What happens to them/
they are inside autophagosomes, and they are degraded when they fuse with the lysosome
52
How can we measure the autophagic flux in cells?
we have to measure the steady state condition and the condition in which we block the lysosomal degradation *in particular, the fusion between the autophagosome and the lysosomes
53
What drugs can we use to measure the autophagic flux in cells?
Chloroquine or Bafilomycin (both alter the pH of the lysosomes hampering the fusion of the autophagosome with the lysosome
54
If we use chloroquine treatment to block autophagosome degradation, what happens?
there is an increase in the number of puncta (autophagic structures) *this is how we should measure autophagy
55
What happens to LC3-I and LC3-II when Chloroquine is given?
there is a great increase in the conversion of LC3-I to LC3-II (seen in WB)
56
Using choroquine treatment, what does the accumulation of p62 and LC3-II infer?
autophagy is working fine
57
What other drug, apart from Chloroquine can be used to stimulate autophagy?
Rapamycin, which works on mTORC1 and activates the ULK complex and triggers the autophagic flux
58
What happened when Prof. Maltecca and her team gave Chloroquine to patients?
LC3-II band was big (implying autophagy was increased)
59
What happened when Prof. Maltecca and her team analyzed the basal state??
they had a very faint band that could be interpreted in 2 ways: LC3-I is not converted to LC3-II (autophagy is impaired) LC3-I is converted to LC3-II, but it is rapidly degraded by the autophagic process (autophagy is increased)
60
How can the basal state results be interpreted correctly?
using blockers or inducers
61
If we want to measure proteasome activity, we need to block it with some drugs. What is the drug used?
MG132, which directly binds to the proteasome impeding proteins' degradation
62
What can compensate for the impairment of the proteasome?
autophagy
63
Why does the ubiquitination of proteins increase when we block autophagy?
when we block autophagy, there is a burst in the proteasome degradation (crosstalk between 2 systems)
64
What is ubiquitination?
mark that targets the nascent chains to the proteasome and marks it for degredation
65
What does it mean if ubiquitination happens co-translationally?
nascent polypeptide chains get ubiquitinated as they are being translated
66
What are 2 main pathways in co-translational ubiquitination?
ribosomal quality control (RQC) cotranslational quality control
67
What is RQC?
triggered when there are defects in the translation machinery (mRNA) fats damaged in the mRNA acutely lead to the prolonged stalling (arrest) of the translation the mRNA that caused the ribosome to stall is then sensed by the machinery and causes the splitting of the ribosome and the uniquitination of the nascent polypeptide chain nascent chain is targeted to the proteasome degradation of the associated mRNA takes place
68
What is cotranslational quality control?
nascent chain get ubiquitinated while they are being translated (active translation: NO problem in translation machinery) ribosome is translated normally the nascent polypeptide chain is ubiquitinated and targeted to the proteasome
69
When does protein folding begin?
while the nascent chain is still being translated in a domain-wise fashion *once a single domain has been fully translated, it can begin to fold while the next domain is being translated **if the domain cannot be folded properly, this is sensed and causes ubiquitination
70
WHat kind of translational complexes can co-translational ubiquitination be associated with?
stalled translational complex (RQC) active translational complex
71
What is stalled translational complex (RQC)?
translational arrest (stalling of translation) is the trigger and it is associated with the splitting and degradation of the associated mRNA
72
What is active translational complex?
misfolding causes ubiquitination translation is active and the ribosome is translated
73
How is nonsense mediated decay (NMD) similar to RQC?
both have a defect in the mRNA because of a premature stop codon that causes premature arrest of RNA translation, which is associated with the degradation of the mRNA *there is evidence that in NMD the truncated protein gets ubiquitinated
74
What does ARSACS stand for?
Autosomal Recessive Spastic Ataxia of Chatlevoix-Saguenay
75
What is ARSACS characterized by?
cerebellar atrophy by a progressive degeneration of cerebellar Purkinje cells autosomal recessive disorder
76
What does the atrophy of the cerebllum lead to?
death and degeneration of Purkinje cells
77
What are Purkinje cells?
main neurons in the cerebellar cortex, and they are only on efferent neurons of the cerebellar cortex they are responsible for processing information and relaying it to the CNS
78
When is ARSACS diagnosed? What causes it?
between 1 & 5 caused by a loss of function mutation if the Sacs gene that encodes for the sacsin protein
79
What is special about sacsin?
it is the 2nd largest human protein and highly expressed in nervous tissue and the CNS
80
What is the determined function of sacsin?
regulate cytoskeleton, neurofilaments, and intermediate filaments of the cytoskeleton of neurons in Purkinje cells
81
What happens when the ARSACS patients have a missense mutation?
the protein is never fully synthesized
82
What happens when the ARSACS patients have a frameshift mutation?
mRNA is degraded
83
What happens in ARSACS patients if there is a missense mutation in the protein?
mRNA is present but the protein is not fully synthesized since the full length is never rescued
84
What does active translation mean?
polysome
85
What was the hypothesis in terms of the sacsin protein with the missense mutation?
the sacsin protein with the missense mutation is not able to folde and it is sensed this drives the ubiquitination of the nascent chain nascent chain is driven to the proteasome befor the full length is synthesized the proteasome was inhibited with the mg132 in order to stabilize the nascent chains
86
How are nascent chains detected?
using immunoprecipitation and N-terminal antibody
87
What happens when there is a mutation in the Sacs gene?
cells go through nonsense mediated decay (NMD)

Molecular Cell Biology (10 decks)

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