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Molecular Cell Biology > Lecture #7 > Flashcards

Lecture #7 Flashcards

1
Q

what is the stress sensor of the inner membrane?

A

OMA1

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2
Q

what happens when stress arises in the organelles?

A

there is the activation of a kinase and the final result is the hyperphosphorylation of eIF2⍺

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3
Q

what is eiF2⍺?

A

an elongation factor of the cytosolic translation

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4
Q

what does the phosphorylation of eIF2⍺ lead to overall?

A

shuts down the cytosolic translation

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5
Q

what is the final aim of the stress response?

A

try to reduce the protein load in order to let the cell recover from damage

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6
Q

what protein is able to escape eIF2⍺?

A

ATF4

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7
Q

what happens to ATF4 upon phosphorylation?

A

it translocates to the nucleus and induced the transcription of several target cytoprotective genes that are involved in the recovery from stress

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8
Q

what can defects in the electron transport chain, ROS, and mitochondrial proteotoxic stress activate depending on the context?

A

activate GCN2, PERK, or HRI

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9
Q

what does the phosphorylation of eIF2⍺ promote?

A

the selective translation of the transcription factor ATF4, which in turn promotes the expression of CEPB homologous protein (CHOP), growth arrest and DNA damage-inducible protein 34 (GADD34), ATF3, and other transcription facts to restore cellular homeostasis

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10
Q

how does OMA1 activate the stress response?

A

OMA1 cleaves a protein in the inner membrane (DELE1), and the cleaved DELE1 is sent back to the cytosol to activate a precise kinase HRI, which in turn phosphorylates eIF2⍺

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11
Q

where was the stress response demonstrated?

A

HeLa cells

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12
Q

what did scientists find in the cerebellum of AFG3L2 ko mice (model of SPAX5)?

A

they found an overreaction of OMA1 - there is a reduced amount of OMA1 → OMA1 gets auto-cleaved after exercising its action

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13
Q

did they discover that OMA1 is involved in inducing the integrated stress response in this mice models?

A

yes, they found an increased level of the phosphorylation state of eIF2⍺, and when they performed qPCR on the ATF4 target genes it all resulted in up regulation in the mutant mice compared to the wild types

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14
Q

did scientists see the same over activation in human patients as they did in the SPAX5 mouse models?

A

yes - in cells from two pediatric SPAX5 patients they saw that there is an increased phosphorylation of eIF2⍺ in the cells coming from patients compared to those coming from controls, meaning that even in this case the integrated stress response is activated

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15
Q

what are the two key parts of protein turnover?

A

both the synthesis and the degradation of proteins

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16
Q

how often is the entire human proteome replaced?

A

every 1-2 months

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17
Q

how are damaged proteins degraded?

A

there are caged organelles in which proteostasis occurs (like in mitochondria) and hydrolase are secreted within the membrane compartments (eg in the lysosomes)

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18
Q

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

A

the presence of a long polyubiquitin chain

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19
Q

what is the function of E1?

A

it is an activating enzyme that, in an ATP-dependent manner, binds ubiquitin to a Cys, activating it

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20
Q

what occurs after ubiquitin has been bound to Cys by E1?

A

the ubiquitin is passed to a conjugate enzyme E2, and finally transferred to another enzyme E3 the gives the specificity of the target (decides which substrate must be degraded)

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21
Q

how many genes exist for E1, E2, and E3?

A

E1: 2
E2: 20
E3: 600 → we need specificity for a lot of substrates

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22
Q

what happens once E3 has bound and polyubiquitinated the target?

A

then the protein is transferred to the proteasome chamber where there are the DUBs enzymes (de-ubiquitinating enzymes) that remove the polyubiquitin chain

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23
Q

what occurs once the polyubiquitin chain has been removed?

A

the protein is then inserted into the proteosomal structure

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24
Q

describe the make-up of the proteosomal structure:

A

4 ring structures - 2 ⍺ subunits at the bottom and periphery and 2 β subunits in the middle → the ⍺ are the regulatory and the β are the catalytic subunits

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25
about how long are the proteins fragments after degradation?
about 25 aa → then other proteases are engaged in order to recycle each single aa
26
what are the three types of autophagy?
1. macroautophagy 2. chaperone-mediated autophagy 3. microautophagy
27
what is the purpose of macroautophagy?
toe grade big dimensional materials - organelles, bacteria, or aggregates of proteins
28
what is the purpose of chaperone-mediated autophagy?
used when the cells need to degrade quickly or in the case of small aggregates / proteins
29
what must proteins have to be targetted by the chaperone-mediated autophagy?
a KFERQ-specific pentapeptide
30
what is the KFERQ pentapeptide motif recognized by (chaperone-mediated autophagy)
chaperone protein HSPA8 that delivers the cargo directly to the lysosome
31
what occurs in microautophagy?
occurs in stress conditions in order to get rid of damaged material in a fast way - least studied
32
where do the membranes needed for macroautophagy come from?
the ER, Golgi, ERGIC, and endosomes
33
what kinase complex triggers the formation of the autophagosome, and what occurs for this to happen?
de-phosphorylation of ULK 1/2
34
what occurs once ULK 1/2 is de-phosphorylated?
it recruits a lot of components of the ATG system and engages with LC3 and these complexes help with th formation of the autophagosome and the engulfment of the cargo
35
how does LC3 bind the cargo inside of the autophagosome?
LC3-1 (starting molecule) becomes LC3-II, the de-lipidated form which then is able to interact with the cargo
36
once LC3 is in its de-lipidated how does it interact with the cargo, and what occurs?
the autophagosome is formed which can fuse directly with the lysosome for degradation, or there can be an intermediate step in which it fuses with the endosome to form the amphisome, which then goes on to fuse with the lysosome
37
what receptor is essential in order to bind the de-lipidated form of LC3-II in a specific interaction?
p62
38
describe p62:
part of the family of sequestosome like receptors (SLRs) that serve as a bridge between the ubiquitinated cargo and the LC3-II
39
what are the three domains in p62?
PB1: polymerization domain UBA: binds the ubiquitin residues LIR: interacts with LC3
40
what happens to LC3 and p62 when fusion with the lysosome occurs?
they are engulfed and degraded as well
41
it is known that there is crosstalk between the UPS and autophagy, what does a dysfunction in either cause?
a dysfunction in proteosomal activity cells can burst autophagy and vice versa
42
what must we do to measure the autophagic flux in cells?
we need to measure the steady state condition and the condition in which we block the lysosomal degradation → in particular we must measure the fusion between the lysosome and the autophagosome
43
what drug is used to measure the autophagic flux in cells?
Chloroquine or Bafilomycin → alter the pH of the lysosomes hampering the fusion of the autophagosome with the lysosome
44
if cells are given Chloroquine treatment blocking autophagosome degradation, what do we see?
an increase in autophagic structures, or puncta, as well as a great increase in the conversion from LC3-1 to LC3-II
45
what does it mean if chroloquine is given and we see an accumulation of p62?
if both LC3-II and p62 accumulate it means the autophagy is working fine
46
what other drug can be used to stimulate autophagy, and how does it work?
Rapamycin → works on MTORC1 and activates the ULK complex triggering the autophagic flux
47
what is used if we want to measure proteosome activity?
MG132 → directly binds to the proteasome impeding protein degradation
48
what are the two ways that proteins can be ubiquitinated?
ribosomal quality control pathway (RQC) or co-translational quality control
49
what does ubiquitination do?
marks the nascent chain to the proteosome and marks it for degradation
50
when is ribosomal quality control activated?
triggered when there are defects in the translation machinery, especially in the mRNA
51
when do newly synthesized polypeptide chains get ubiquitinated?
when they are still being translated and are bound to ribosomes
52
in the ribosomal quality control pathway, what occurs once the mRNA is damaged?
there is the formation of secondary structures, the mRNA that caused the ribosomes to stall is sensed by the machinery and causes the splitting of the ribosome and the ubiquitination of the nascent polypeptide chain which is then targeted to the proteasome
53
what also importantly happens in the ribosomal quality control process?
the degradation of associated mRNA takes place
54
what occurs in the co-translational quality control pathway?
the nascent chains get ubiquitinated while they are still being translated → but there is no issue with the translational machinery and the ribosome translated normally
55
what happens to the polypeptide chain in co-translational quality control?
it is ubiquitinated and targeted to the proteasome → there is evidence that this is coupled with the folding of the nascent chain, meaning that if there are translational issues there is a problem when it comes to the folding of the chain that is sense by the cellular machinery which causes ubiquitination
56
when does folding occur?
while the nascent chain is being translated, and it can fold while the next domain is being translated
57
describe the stalled translational complex (quality control):
translational arrest is the trigger, and it is associated with the splitting and degradation of the associated mRNA
58
describe the active translational complex:
ubiquitination is caused by misfolding - translation is active so the ribosome is translated
59
why should a cell target a polypeptide to be degraded if the polypeptide has not been given the chance to be completely synthesized and folded?
in the case of RCQ this means that translation is arrested, and the ribosome cannot go on so the polypeptide product is truncated and a protein is truncated, and the cell will process to get rid of the product
60
how is RQC similar to nonsense mediated decay?
they both have a defect in the mRNA because of a premature stop codon that cause the premature arrest of RNA translation, which is associated with the degradation of the mRNA there is also evidence that in NMD that the truncated protein gets ubiquitinated
61
what occurs in NMD?
there is a genetic mutation that causes a premature stop codon, like a substitution or frameshift constitutively triggering the decay of mRNA causing the molecular pathology of genetic disease
62
what can an abundance of misfolded proteins cause?
toxic aggregation
63
what is ARSACS?
autosomal recessive spastic ataxia of Charlevoix-Saguenay
64
what is ARSACS characterized by?
cerebellar atrophy accompanied by a progressive degeneration of cerebellar purkinje cells
65
what type of genetic disorder is ARSACS?
autosomal recessive - second most common
66
what are purkinje cells?
the main neurons of the cerebellar cortex responsible for the processing of the information in the cerebellum and ensure proper delivery to the CNS
67
what is ARSACS caused by?
a LOF mutation in a gene known as Sacs that encodes for the sacsin protein
68
what makes the sacs gene unique?
it is the one of the biggest proteins in the human genome - highest expression is in purkinje cells
69
what is the function of sacsin?
regulation of the cytoskeleton, in particular neurofilaments and intermediate filaments of the cytoskeleton of neurons in purkinje cells
70
name the two domains of sacsin related to ubiquitination and the ubiquitisome domain?
DNAJ and HEPN
71
describe the DNAJ domain:
a Hsp40 protein which is a core chaperone of Hsp70
72
describe HEPN:
higher eukaryote and protakyotes nucleotide-binding domain - poorly characterized
73
what are the main types of mutations causing ARSACS?
full deletion of an allele and single point mutations (frameshift due to insertions or deletions / substitution)
74
when studying the molecular pathogenesis of ARSACS, what type of cells were used?
patient derived cells from skin biopsies (fibroblasts)
75
why did they have to generate a novel antibody to detect the possible truncated sacsin protein?
there was only one antibody available but it attached to the c-terminis, so they have to produce an antibody to recognize all possible truncated protein products that use the n-terminal
76
what did the addition of the novel antibody with the n-terminal attachment reveal?
there is a loss of different proteins, and that there are some products that are truncated and therefore not stable
77
what was discovered in patient mRNA who had two truncating mutations?
their mRNA is significantly reduced compared to the control
78
what was hypothesized to happen in patents with two truncating mutations to cause the loss of mRNA?
due to a nonsense mediated decay due to a premature stop codon that differentiates the analysis substitution
79
what was seen in patents with one or two missense mutations in PN5 and PN9?
the delivery of mRNA was identical to the control so the mRNA was not degraded and appeared normal
80
what was observed in patients carrying a missense mutation in regards to the protein formed?
the protein is never fully synthesized
81
what can be used to inhibit autophagy?
chloroquine
82
what was concluded to occur when there is a missense mutation?
the protein is never fully synthesized - when there are frameshift mutations the mRNA is degraded
83
what did they do to see if sacsin mRNA with the mutation is being translated?
polysome profiling
84
what occurs in polysome profiling?
sacs is treated with an antibiotic that targets ribosomes and blocks the translation freeing everything in that moment (ribosomes remain bound to the mRNAs but translation stops) then the lysed cells are taken and a sucrose granite density fractionation is performed and it allows for the separation of all of the ribosomal monosomes
85
what was found after polysome profiling occurred?
in patents with one or two missense mutations the SACS mRNA was present at the same level as the control cells → the protein with the missense mutation could undergo co-translational degradation
86
what method of degradation was hypothesized?
thought that the sacsin protein was not able to fold, which was sensed and drove the ubiquitination of the nascent chain therefore causing it to be targeted to the proteasome before the full length this synthesized
87
how were the nascent chains detected?
through immunoprecipitation and the validated n-terminal antibodies
88
what was found when the the cells were treated with a ubiqitin chain to increase ubiquitination?
the nascent chains were able to be detected in the patient cells - it was demonstrated that the were ubiquitinated and were degraded by the proteasome because it was possible to inhibit the proteasome
89
what was determined to be the final model when a mutation occurs in the SACS gene?
whether the mutation is a nonsense or a frameshift, the cells go through nonsense mediated decay → the SACS mRNA is degraded and the protein is absent In the case of missense mutations the protein is absent due to co-translational quality control
90
what potential method could be used to diagnose ARSACS quickly?
it has been demonstrated that sacsin is present in small but detectable amounts in the blood and hypothesized that this mechanism can also occur in blood lymphocyte cells

Molecular Cell Biology (10 decks)

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