Leary Flashcards
What are mitochondria
cellular organelle
Double membrane bound
Contain mitochondrial DNA which is maternally inherited
Inner vs outer mitochondrial membrane
Inner mitochondrial membrane is protein loaded, contains the ETC, and is impermeable which is needed for the proton gradient
Outer mitochondrial membrane is permeable with many holes, doesn’t have many proteins
These properties are relevant to how the organelle functions as a whole and contributes to cellular homeostasis
Where is the mtDNA, Krebs cycle, and metals
matrix of mitochondria
What are cristae? Which enzymes are concentrated there?
Inner folds of the mitochondrial membrane
Used to increase surface are to put more proteins and enzyme to produce more ATP
Enzymes of oxidative phosphorylation are concentrated within the cristae
T/F the function of the inner membrane is different depending on where it is
true
What are the reducing equivalents used in the ETC
NADH feeds into complex I and FADH2 feeds into complex II
These are needed for the ETC to function
Why might one tissue have more cristae than another
The inner membrane architecture is optimized for the tissue it functions in
Tissues that need more ATP will have more cristae
T/F mitochondria are static
False its a dynamic reticulum that responds to intracellular cues and extracellular stimuli
What are some cues that can cause the mitochondria to hyperfuse to become one organelle
Starvation
Translation inhibition
Viral infection
Conditions when differentiation is important
When would the mitochondria hyperfragment?
Cancer, bacteria, OXPHOS poisons
Conditions when cell division is important
What enzymes drive Fusion
MFN1 and MFN2
L-OPA1 vs S-OPA2
L-OPA1 is required for fission
S-OPA1 is required for fusion
Why are mitochondrial dynamics important
Adapting organelle function to a broad range of cues
Failure to appropriately balance fission and fusion will impair organelle function and cellular homeostasis
What are some basic challenges of mitochondrial content
They have a large proteome and require many proteins to function
They have their own genome which encodes crucial polypeptides
They have to coordinate expression of nuclear and mtDNA-encoded protein products (crucial to maintaining or expanding existing populations of organelles)
Describe the number of organelles and mitochondrial volume across tissues
Number varies between 1 and 1000
Fold difference ranges from 2-40%
how is the balance of mitochondria maintained
maintained between rates of biogenesis and degradation
What is mitophagy
Self-eating process in mitochondria, way to degrade mitochondria
Mitochondria alter their _____ and ____ to fulfill diverse functions
shape and number
why are fission and fusion important
Allows mitochondria to respond to internal and external cues and adapt to their environments ot fulfill the necessary conditions to survive
What are the 6 essential processes for maintaining homeostasis that the mitochondria plays a role in
ATP production
Epigenetics
Peroxisome biogenesis
Fe-S cluster synthesis
Apoptosis & cell cycle control
Regulation of cellular metal ion homeostasis
How does cytochrome C initiate apoptosis
Cytochrome C is tethered to the inner membrane under normal circumstances but when the inner membrane is compromised cytochrome C can be released and initiate apoptosis
Which processes do cytochrome c oxidase assembly affect?
ATP production and regulation of cellular metal ion homeostasis
How many subunits are encoded in nuclear and mtDNA of the mitochondria
4 complexes responsible for making the ETC and fifth for creating ATP
only complex 2 is completely from nuclear DNA and the other complexes are a mix of mtDNA and nuclear
What are some basic characteristics of mtDNA
16.6kb double stranded, circular genome
genome was compacted because it is more efficient
Present in multiple copies per organelle
Maternally inherited
In the matrix in order to protect it
Encodes 13 proteins
Lacks introns
Where is the mtDNA bottleneck during oogenesis
QC step
Can mitochondrial diseases be heterogenic
no, will only be a carrier
T/F mitochondrial diseases are extraordinarily homeogeneic
false heterogeneic
Is OXPHOS complex biogenesis different from the nucleus
fundamentally yes
What is cytochrome c oxidase
protein in complex 4 of ETC
used as a model protein
Cytochrome C oxidase structure
14 structural subunits
3 mtDNA-encoded
11 nuclear-encoded
Why use a cytochrome C oxidase as a model protein
Consists of nuclear and mtDNA encoded subunits
Rate-limiting to ATP production under a range of physiological conditions
Commonly observed metabolic lesion
Most thoroughly characterized from a genetic, biochemical and molecular biological perspective
Why are nuclear subunits important?
They are important for stability, affect rates of activity, and alter the structure of the enzyme
What are 2 important prosthetic group for cytochrome c oxidase
COX1 and COX2
Describe the assembly of cytochrome c oxidase
Assembly is highly ordered and modulated in nature
4 stages
How do mutations in mtDNA occur
spontaneously or inherited
What is the threshold effect
tissues accumulate mutant mtDNA and once they reach a threshold it starts to have an effect
Explains later onset of disease manifestation
What were the barriers or challenges with respect to diagnosing the genetic basis of these diseases?
Lack of adequately diverse and sensitive diagnostic tools
incomplete or absent sequence coverage of relevant regions of the human genome
lack of an adequate model system to study human disease
Heterogeneity
What is the genetic explanation for early onset disease?
The assembly of the cytochrome C oxidases uses a toolbox of many different factors which are essential to the assembly
Mutations to these factors could explain the early onset of the disease
mtDNA vs nuclear DNA
SCO1 vs COX
SCO1 is the yeast version of COX10
COX10 could rescue yeast SCO1 null cells but the yeast SCO1 could not rescue COX10
What might be some reasons for not being able to use yeast to study a humane mitochondrial disease gene?
Can’t interact with the correct proteins and proteins usually work in tandem
What was the visualization technique that revolutionized mitochondrial disease work
blue-native PAGE
How were SCO1 and SCO2 first identified
Was identified through the yeast PET mutant collection. Mutations in functional yeasts were related to a single enzyme or biosynthetic pathways which helped characterize different defects
They were identified as high copy suppressors of a yeast COX 17 mutant
What do mutations in human SCO result in
severe early onset disease with a fatal outcome
What are the simples ways to rescue disease caused by mutations in a given gene like SCO1 or SCO2? Problems?
Genome editing: Ethical dilemmas
Re-introduce the wild-type protein into the system: Not feasible for short-lived protein, costly, systemic delivery
Treating with copper: Was able to rescue COX deficiency in SCO2 patient cells, can rescue heart but not brain function
Why can copper treatment rescue SCO2 null cells but not SCO1 null cells
Coppers need to insert into the protein and it was shown that SCO1 acted as the insertase
How can ubiquitously expressed mutations be heterogeneic
Tissue specific:
Difference in half-life
Abundance of a given COX assembly factor
abundance of metals
Unique effects on protein
What were historic challenges in mitochondrial disease diagnosis
Clinical heterogeneity
Limited diagnostic toolbox (old tools)
Lack of reference genome sequence (reliance on large pedigrees)
Reliance on a single model organism (inadequate models)
What are on-going challenges with mitochondrial disease diagnosis
clinical heterogeneity
Multiple model organisms available
We have the human genome sequence now, however if the mutation is in the intron that may affect splicing and we do not have intron sequences
What is a mitochondrial disease treatment that is currently in phase 2 trials and could be a viable strategy? how does it function? Barrier?
Mitochondrially targeted nucleases
Promotes degradation of mutant mtDNA
The nuclease recognizes the mutation and cuts the DNA
Need to be put in via a viral delivery system which may not work for isolated organs, nuclease is very large
Could supplementation be used to treat mitochondrial disease
Protein supplement is difficult due to half-life issues and SCO2 lives in a membrane
Vitamin supplementation has minimum benefit
Thin air does not work because it results in hypoxia
What is the effect of thin air supplementation
Rescues the mitochondrial DNA, but hypoxia results in cancer and death
Can copper supplementation be used to overcome genetic defects that impede copper site biogenesis
When copper is unbound it can produce many free radicals and cause damage
Can rescue heart defects but not brain
The copper needs something protein to cross the BBB
What are some tissue-specific challenges associated with COX deficiency
Differences in half-life of the holoenzyme
Differences in abundance of a given COX assembly factor
Differences in abundance of metals essential for assembly
How can unique allelic variants in a single COX assembly factor give rise to distinct, tissue-specific forms of disease?
Unique effect on the residual function of a protein
Unique effects on the residual functions of a protein
What is cardiolipin
Cardiolipin is a unique protein to the inner mitochondrial membrane
Cardiolipin curves the membrane to promote cristae formation
Allows for mitophagy among other functions
What is the rate-limiting enzyme for cardiolipin biosynthesis
Tafazzin
It is essential in the final step of cardiolipin biosynthesis
What is Barth syndrome
Results from mutations in TAZ, the gene encoding Tafazzin
When does the mitochondria fragment or fuse
Fragment to go into a resting state and remove damaged organelles
Fuse for the complementation of gene products and requires the cooperation of the entire compartment, in response to stress and starvation
What happens when the quality control machinery cannot maintain proteostasis?
Protein folding will be impaired, when there is membrane potential damage, proteases cannot help resulting in no proton motor force and autophagy
What is the quality control mechanism for mito
Contains many proteases in every subcompartment
Processing peptidases and ATP-dependent proteases sense folding stated and ensure quality control
Why is autophagy important
Important for garbage disposal
It is important for adaptive responses as it provides nutrients and adapts to stress conditions
Where is quality control machinery concentrated? What 2 things does it fuse together)
Concentrated around the phagophore
Fuses autophagosome and lysosome
Micro & macroautophagy share a common set of essential ____ proteins
ATG (autophagy) proteins which are critical for induction of the process and autophagosome formation
5 things
What are the broad stages of autophagy
Phagophore biogenesis
Autophagosome formation
Autophagosome maturation (fusion with lysosome)
Breakdown of cargo
Recycling
Specific types of autophagy requires unique elements
Functions of PINK1 and PARKIN? What do mutations in these cause?
PINK1 recruits PARKIN to the damaged mitochondria
PARKIN poly ubiquitinates VDAC1
P62 then binds poly Ub-VDAC1
This facilitates the interaction of the organelle with core autophagic machinery
Results in parkinsons disease
Inability to clear defective mitochondria from neuronal cells contributes to the clinical progression of _________ __________
Parkinsons disease
Damages neurons
