Lecture II Flashcards
Tonon
How is mitochondrial DNA transcribed?
as a polycystronic unit and the regulation occurs post-transcriptionally (possibly through mitomiRNAs
What are miRNAs?
endogenous small ncRNA that act as post-transcriptional regulators
their expression is tissue-specific
physiologically involved in cell growth, cell proliferation, and apoptosis
pathologically involved in the regulation of different genes and proteins
*they can also be biomarkers for disease
Review the biogenesis of miRNA:
https://www.youtube.com/watch?v=VCgtrtS6pzc
https://www.youtube.com/watch?v=j-zTy6vOP3M
What do miRNAs act as?
translational repressors
*recently also translational enhancers
What are mitomiRNAs?
mitochondrial microRNAs that have different functions in biological and pathological situations
expression is tissue specific
Review the biogenesis and maturation of mitomiRNAs:
https://www.youtube.com/watch?time_continue=1&v=t5jroSCBBwk&embeds_euri=https%3A%2F%2Fwww.tamirna.com%2F&feature=emb_logo
What can mitomiRNAs be involved in?
mitochondrial metabolism
fussion and fission
pathologies: Alzheimer and cardiovascular
What are some different hypothesis on how the mitomiRNAs enter the mitochondria?
specific motif at the 3’ terminal
post-translational modification
PNPase (a ribonuclease)
AGO2 via pores of the outer membrane
P-bodies
PIMILIO1 (RNA binding protein)
How is mitomiRNA isolated?
cerebellar tissue was isolated and homogenized using anti-TOM22 Ab-coated magnetic beads
solution obtains pure mitochondria
How do we remove the outer membrane from isolated mitochondria?
using a digitonin, which is a detergent that creates pores in the outer membrane
What is the last step in the isolation of mitomiRNAs?
using ribonucleases to remove the RNA remaining outside
Is it possible to detect mitomiRNA and AGO2 inside the mitochondria derived from cerebellum?
yes and it is relevant to detect specific targets of mitochondrial DNA encoded proteins that will be regulated so this can be a potential therapy for primary mitochondrial diseases
What uses a lot of ATP and requires a lot of mitochondria?
neurons
What do energy and ion balance link to?
transport
What does ion balance of neurons control?
movement
what can mitochondrial dysfunction lead to?
loss of calcium uptake
increased ROS production
triggering of apoptosis
loss of ATP synthesis
What do mitochondrial dysfunctions mostly affect?
neurons and muscles
Is the mitochondrial network localized or diffused?
it cannot be a network as the axon and dendrites are very thin
a single mitochondria travels along the dendrites and axons and undergo fusion and fission
What are the 3 levels of mitochondrial quality control in mitochondria?
molecular
organellar
cellular
How are the 3 levels of mitochondrial quality control activated?
depends on the entity of the damage
What is different about the mitochondria in terms of the control of the proteome?
they are able to control it themselves
What happens if the quality control system of the mitochondria fails to recover damage?
apoptosis is triggered via the release of cytochrome c from the inter membrane space to the cytosol
What are the 3 situations in which the control of the mitochondrial proteome is not autonomous?
proteins on the OMM: 𝛼-helix or β-barrel containing proteins gets ubiquitinated and retrotranslocated to the cytosol via VCP or p97 to be degraded (called mitochondrial associated degradation and is reminiscent of the ER associated degradation (ERAD)
proteins with disulphide bonds: if not formed properly, they are retrotranslocated to the cytosol to be degraded
translocons are engulfed and cannot import proteins from the outside and precursors are degraded before being imported into organelle
How do matrix proteins travel?
via TOM40-IM23 motor pathway
What happens with matrix proteins?
proteins of the matrix carry a presequence and travel via the TOM40-TIM23 motor pathway
after internalization, the presequence is cleaved by mitochondrial peptidase
the protein is then folded by mitochondrial chaperones (mtHsp60 and the co-chaperone myHsp10)
What are 2 main proteases involved in the degradation of misfolded matrix proteins?
LONP: highly-conserved serine-protease that degrades misfolded, damaged, or oxidized proteins
CLPXP: serine-protease CLPP + CLPX form CLPXP, which is an ATP-dependent chaperone subunit
What happens after matrix proteins are degraded by proteases?
peptidase PTRM1 peptidase digests the dipeptides into AA that are then transported outside to be recycled
What are the proteases that are involved in the IMM?
m-AAA (m-triple A): catalytic chamber faces the matrix space
i-AAA (i-triple A): catalytic chamber faces the inter membrane space
How can inner membrane proteases decide whether to degrade their target proteins when both the proteins and proteases are integral to the membrane?
the presence of the hydrophobic domain outside of the membrane
the solvent exposed domains (not integral to the membrane) are sensed by the proteases
What is the control of the mitochondrial proteome important for?
brain
What does AAA stand for?
ATPase Associated with different cellular Activities
What is usually mutated in neurodegenerative diseases?
subunits of AAA proteases
What has the mutation in i-AAA recently been linked to?
form of encephalopathy
What are the 2 proteases of the inter membrane space?
HTRA2: highly conserved and form a trimeric complex (important for the degradation of oxidized proteins)
ATP23: not well characterized (form of metalloprotease)
How does molecular quality control take place?
control of the proteome
How does organellar quality control take place?
mitochondrial dynamics
What do mitochondria look like in a tubular network?
elongated and highly connected
What do mitochondria look like in an intermediate state?
shorter tubes
What do mitochondria look like in a fragmented/fission state?
there is no network, so there are round shape structures that are individual structures
How can we visualize the mitochondrial network?
we can transfect cells with matrix-localized mito-DsRed
How can we we visualize mitochondrial fusion and fission events?
transfect cells with mito-DsRed
cotransfect a photoactivatable GFP
The mitochondrial network is very dynamic: what do fused and fragmented states depend on?
cell type
What are primary defects of fusion and fission events related to?
neurological diseases in early childhood
What is the life cycle of the mitochondria?
2 neighboring mitochondria fuse together and very soon after, a fission occurs
What is mitophagy needed for?
turnover of mitochondria
What is the triggering event for mitophagy?
depolarization
Do mitochondria prefer mitophagy or salvage?
they prefer salvage because mitophagy is energy-demanding
What are 2 prerequisites for mitophagy?
depolarization and fragmentation
In a healthy cell…
electron flats coupled with proton pumping guarantees a proton gradient that is used by ATP synthase for ATP production
What happens if there is a mitochondrial disease in which the respiratory chain is not working?
the cell uses the reversal activity of ATP synthase
ATP synthase consumes ATP to pump the proton back to the inter membrane space and re-establish the mitochondrial membrane potential (a lot of ATP is consume: it is a salvage pathway)
How can we assess mitochondrial functionality?
using TMRM (Tetramethylrhodamine, methyl ester) to visualize mitochondrial membrane potential and view the health of the mitochondria
If cells have the ability to use a salvage pathway via ATP synthase, how can we ensure they are really healthy?
we have to block ATP synthase pharmacologically (oligomycin)
Are fusion and fission spontaneous?
no, they are finely regulated
What 3 players play a role in mitochondrial fusion?
mitofusin 1 & 2 (MFN1 and MFN2) on the OM and they regulate the fusion of the OMM
optic atrophy 1 (OPA1) is on the IM and mediates the fusion of the IM
What have mutations in OPA1 been associated to?
severe form of optic atrophy in children
What have mutations in MFN2 been associated to?
genetic form of Charcot-Marie-Tooth disease, which is peripheral neuropathy
