Lecture VII Flashcards
What is the integrated stress response?
response the cell activates in case of proteostatic stress
Where can proteostatic stress rise from?
ER or mitochondria
What is an example of a mutatio that induces proteostatic stress?
AFG3L2 mutation
What does the absence or mutation of AFG3L2 (m-AAA in the mitochondria) lead to?
accumulation of mt-DNA-encoded polypeptides, which leads to proteostatic stress in the mitochondria due to failure of QC of the mitochondria-encoded polypeptides
What does the stress that arises in organelles and relayed back to the cytosol lead to?
activation of kinase and hyperphosphorylation of eIF2๐ผ (elongation factor of cytosolic translation
What does the phosphorylation of eIF2๐ผ lead to?
inhibition of cytosolic translation and reduction of protein load in order to allow the cell to reover from damage
eIF2๐ผ shuts down translation in general, but what protein escapes this attenuation?
ATf4
What is ATF4
TF that upon eIF2๐ผ phosphorylation, translocates to the nucleus
What does ATF4โs escape to the nucleus induce?
transcription of several target cytoprotective genes that are involved in the recovery from stress
What is the central regulation node of integrated stress response (ISR)?
eukaryotic translation initiation factor eIF2๐ผ, which when phosphylated, inhibits cytosolic translation
What can activate GCN2, PERK, or HR1?
defects in the elctron transport chain (ETC), reactive oxygen species (ROS), and mitochondrial proteotoxic stress
What does the phosphorylation of eIF2๐ผ promote?
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
How can the mitochondria relay stress to the cytosol?
using a pathway that involves the stress sensor of the inner mitochondrial membrane (OMA1)
What is the stress sensor of the IMM?
OMA1
What does OMA1 do?
cleaves the protein of the inner membrane (DELE1
What happens with the cleaved form of DELE1?
it is sent back tot he cytosol and activate a kinase, HRI
What does HRI do?
phosphorylates eIF2๐ผ, which induces a stress response
Why is protein degradation essential for life?
if we did not degrade protein, we would undergo premature cellular senescence
What does the control of protein turnover maintain?
homeostasis and opposes aging (proteome plasticity)
What causes proteins to be targeted for degradation?
ubiquitination
What are the main pathways in the cytosol that regulate protein degradation?
ubiquitin proteosome system and autophagy
In order to be degraded, what must target proteins have?
presence of the long polyubiquitin chain
How is ubiquitin attached to target proteins?
using a set of enzymes:
E1
E2
E3
What kind of enzyme is E1?
activating enzyme that binds ubiquitin onto Cys in an ATP-dependent manner
*ubiquitin is activated this way
What kind of enzyme is E2?
conjugating enzyme
What kind of enzyme is E3?
free ligase that gives specificity of the target
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
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
What happens after DUBs remove the polyubiquitinated chain?
protein is inserted into the proteosomal structure that is composed of 4 rings
What are the 4 ring structures in the proteosome?
2 ๐ผ-subunits (regulatory) and the bottom and periphery
2 ฮฒ subunits (active catalytic) in the middle
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
What are the 3 types of autophagy in mammalian cells?
macroautophagy
chaperone-mediated autophagy
microautophagy
What is macroautophagy?
degradation os large dimension material (organelles, bacteria, or large protein aggregates
What is chaperone-mediated autophagy?
used when the cells need to degrade in a fast manner small proteins or small aggregates
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
What are tau and ๐ผ-sinuclein proteins?
proteins that accumulate in Alzheimerโs disease and when they are unfolded or misfolded, they are targeted for chaperone-mediated autophagy
What is microphagy?
occurs in stress condition in order to get rid of damaged material quickly
*cargo directly enters the lysosome
What is needed to trigger autophagy?
membranes to engulf cargoes
machineries (kinases)
What are the membranes involved in autophagy derived from?
ER
Golgi
ERGIC
endosomes
What is the kinase complex involved in autophagy?
ULK1/2 complex
What does the ULK1/2 complex do?
triggers the formation of the autophagosome when it is dephosphorylated
What does ULK1/2 recruit?
components of the ATG system
What does the ULK1/2 complex engage?
LC3
What do the ATG system and LC3 help ULK1/2 do?
form the autophagosome and engulf the cargo
Where and what does LC3 bind?
cargo inside the autophagosome
*LC3-I becomes LC3-II (de-lipated form)
Describe the autophagy mechanism:
LC3-I binds phospholipids (phosphoethanolamine) and becomes the de-lipidated form of LC3-II (the one interacting with the cargo)
What can the autophagosome directly fuse with?
the lysosome for degredation
or endosome to form an โamphisomeโ, which will later fuse with the lysosome
What is the receptor, p62, essential for?
bind the de-lipated form of LC3-II
What is p62?
prototype of a family of receptors that are called Sequestrome Like Receptors (SLRs)
they bridge between ubiquitinated cargo and LC3-II
Describe p62:
contains:
polymerization domain (PB1)
ubiquitin residues binding domain (UBA)
LC3 interaction domain (LIR)
Where are LC3 and p62 located? What happens to them/
they are inside autophagosomes, and they are degraded when they fuse with the lysosome
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
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
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
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)
Using choroquine treatment, what does the accumulation of p62 and LC3-II infer?
autophagy is working fine
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
What happened when Prof. Maltecca and her team gave Chloroquine to patients?
LC3-II band was big (implying autophagy was increased)
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)
How can the basal state results be interpreted correctly?
using blockers or inducers
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 proteinsโ degradation
What can compensate for the impairment of the proteasome?
autophagy
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)
What is ubiquitination?
mark that targets the nascent chains to the proteasome and marks it for degredation
What does it mean if ubiquitination happens co-translationally?
nascent polypeptide chains get ubiquitinated as they are being translated
What are 2 main pathways in co-translational ubiquitination?
ribosomal quality control (RQC)
cotranslational quality control
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
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
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
WHat kind of translational complexes can co-translational ubiquitination be associated with?
stalled translational complex (RQC)
active translational complex
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
What is active translational complex?
misfolding causes ubiquitination
translation is active and the ribosome is translated
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
What does ARSACS stand for?
Autosomal Recessive Spastic Ataxia of Chatlevoix-Saguenay
What is ARSACS characterized by?
cerebellar atrophy by a progressive degeneration of cerebellar Purkinje cells
autosomal recessive disorder
What does the atrophy of the cerebllum lead to?
death and degeneration of Purkinje cells
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
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
What is special about sacsin?
it is the 2nd largest human protein and highly expressed in nervous tissue and the CNS
What is the determined function of sacsin?
regulate cytoskeleton, neurofilaments, and intermediate filaments of the cytoskeleton of neurons in Purkinje cells
What happens when the ARSACS patients have a missense mutation?
the protein is never fully synthesized
What happens when the ARSACS patients have a frameshift mutation?
mRNA is degraded
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
What does active translation mean?
polysome
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
How are nascent chains detected?
using immunoprecipitation and N-terminal antibody
What happens when there is a mutation in the Sacs gene?
cells go through nonsense mediated decay (NMD)
