KIN 406 Midterm 3 Flashcards
Redox reactions involve?
Addition (reduction) or removal (oxidation) of electrons
A pro-oxidant/oxidant is a?
oxidizing agent…accepts e_ from other molecules and it reduced itself
An antioxidant is a?
reducing agent…gives e- to another species and is oxidized itself
In a stable atom, electrons?
occupy each orbital with opposite spins to each other
What is a free radical?
an atom with an unpaired electron in an orbital
Why are free radicals so reactive?
Because of their unpaired electron
How do free raddicals become more stable?
By seeking to accept an e- from another molecule (could be a protein, a lipid, DNA, etc)
Redox reactions involving free radicals “stealing” electrons will continue until a terminal reaction is reached. What are two examples of these terminal reactions?
When radicals react with each other to form new nonradical species, or if it is quenched by an anti-oxidant to give a non-radical species.
What is a reactive oxygen species?
Compounds derived from partial chemical reductions of molecular oxygen
Are ROS always free radicals?
NO, because they don’t always contain unpaired electrons but are still highly reactive because chemical structure still wants to steal e-
What is ground state O2 like?
Has 2 unpaired electrons in different orbitals (diradical), however, it is stable because both electrons have identical spins…will not readily react with most molecules that e- in opposite spins
What can cause the reverse of the spin of one of electron to form reactive singlet oxygen (1O2)?
input of energy, such as radiation
How is the superoxid radical formed?
a one electron reduction of oxygen
What is superoxide usually converted to spontaneously because it is so reactive?
H2O2 (hydrogen peroxide)
Is H2O2 a free radical?
NO, because it has no upaired electrons, but it is a ROS
What is the functional difference between superoxide and hydrogen peroxide?
It is more stable than superoxide and is very long lived, so it can travel long distances, and it can readily pass through biological membranes.
What is H2O2 easily converted into?
A more highly reactive hydroxyl radical
What is the reaction for H2O2 –> hydroxyl radical?
Fenton reaction with a transition metal…Fe2+ + H2O2 —> Fe3+ + hydroxyl radical + stable hydroxide ion
What is nitric oxide?
a nitrogen based free radical/reactive nitrogen species
What is the reaction between nitric oxide and superoxide?
nitric oxide + superoxide —> peroxynitrire (ONOO-)
What is OONO-?
peroxynitrite…very long lived and very reactive
Why are free radicals, ROS, and RNS so bad?
can lethally damage proteins, DNA, and lipids which ultimately affect cell function
What are the 2 forms of antioxidants in the cell?
antioxidant enzymes and biological (chemical) antioxidants
What are the main antioxidants in the cell?
superoxide dismutase (SOD), catalase, gluthione peroxidase (GPx), and glutathione S-tranferase (GST)
Where are antioxidant enzymes synthesized, and what can induce them?
Synthesized in the body, and oxidant stress can induce their expression
3 forms of SOD?
Copper-Zinc (CuZnSOD), Manganese containing SOD (MnSOD), and extracellular SOD (EcSOD)
Where is CuSOD found?
In the cytosol
Where is MnSOD found?
in the mitochondrial matrix, makes up 15-20% of total SOD
What makes up 15-20% of SOD?
MnSOD
Where is extracellular SOC found?
A form of CuZnSOD found in the extracelllular fluid (plasma and interstitial fluid)
Where is MnSOD sythesized?
In the cytosol and transported to the mitochondria via Hsp70 and mitochondrial import stimulation factor, through TOMandTIM
What does SOD catalyze?
The reaction of superoxide and hydrogen to form hydrogen peroxide and oxygen (2 superoxide + 2 H+ –SOD–> H2O2 and O2
Where is catalse found?
in the mitochondria and other organelles
What does catalase do?
Dismutates hydrogen peroxide into water and oxygen (2H2O2 –catalase–> 2H2O + O2
Where is Gpx (glutathione peroxidase) found?
Mitochondria and cytosol
What does Gpx do?
Catalyzes the reduction of hydrogen peroxide to water by using glutathione (GSH) as an electron donor…2 GSH + H2O2 –GPX–> GSSG (glutathione disulfide) + 2H2O
What does GSH donate in the reaction with Gpx?
a pair of hydrogen ions (and electrons) to H2O2
What is GSH oxidized to?
Glutathione disulfide (GSSG)
What is the reaction for reducing GSSG back to GSH?
GSSG + NADPH + H+ –GR–> 2GSH + 2 NADP+
What is the electron donor for reducing GSSG to GSH, and the enzyme that catalyzes the reaction?
NADPH, and glutathione reductase
Which enzyme has a higher affinity for H2O2, catalase or Gpx?
GPx, and it is found in both the cytosol and mitocohdrial matrix
2 classes of nonenzymatic antioxidants?
1) antioxidants such as GSH that can be synthesized by the body 2) antioxidant enzymes that cannot be synthesized by the body and must be obtained in the diet
Main vitamin antioxidants?
Vitamin E and C
What is GSH, and where is it found?
A water-soluble thiol-containing peptide that is found in high concentrations in virtually all cells. In most cells it is found ing MILLIMOLAR concentrations
What is the most important function as GSH?
to serve as a substrate for GPX
Why is the ratio of GSH:GSSG important?
high intracellular GSSG levels inactivate other important enzymes
What is the problem with cells exporting GSSG to maintain the GSH:GSSG ratio?
a loss of GSSG can reduce the ability to recycle GSH
What are GSH levels regulated by?
GSH utilization and synthesis
Where is the majority of GSH synthesized, and from what?
In the liver (90%) from the AAs cysteine, glutamate, and glycine
What is another role of GSH besides to serve as a substrate for GPX?
also important in keeping vitamins E and C (which are limited) in a reduced state
Most important fat-soluble antioxidant in the body?
vitamin E (tocopherol)
Where is tocopherol/vitamin E found?
cell membranes including inner mitochondrial membrane
How does vitamin E act as an antioxidant?
donates an electron to any lipid radical…produces a new lipid non-radical and vitamin E radical
How is a vitamin E radical reduced back to vitamin E?
gains an electron from Vitamin C OR GSH
What is the main water-soluble antioxidant found in the body?
Vitamin C/absorrbic acid
Where is vitamin C found?
in the cytosol of the cell as well as in the ECF
How does vitamin C act as an antioxidant?
Can directly act as one by donating an electron to superoxide and hydroxyl radical OR donates an electron to vitamin E radical
What is the main function of vitamin c?
to donate an electron to vitamin E radical, this forms a less reactive vitamin C radical
How can a vitamin c radical be recycled back to vitamin C?
by GSH and other means
At physiological concentrations, ROS and RNS regulate…
growth, differentiation, proliferation, and apoptosis
ROS and RNS can stimulate…
gene expression of various cytokines, transcription factors, and proteins
ROS and RNS can stimulate gene expression of various cytokines, transcription factors, and proteins by…
1) regulating kinase and phosphatase activity thereby triggering a signaling cascade through protein phosphorylation and dephosphorylation 2) regulating synthesis and breakdown of transcription factors
Transcription factors stimulates by ROS and RNS are controlled by what signalling pathway?
Nuclear Factor kB (NFkB)
Protein kinases stimulates by ROS and RNS are controlled by what signalling pathway?
Mitogen Activated Protein Kinase (MAPKinases)
Oxidant stress occurs when?
there is an imbalance between oxidant generation and antioxidant defense. Therefore, any factor than causes an increase in oxidant generation or decrease in antioxidant defense could lead to oxidative stress.
Under conditions of oxidative stress, biomolecules will be ________ in tissues and organs
oxidized
Oxidized biomolecules can be?
proteins, lipids, and nucleic acids
What are the important consequences of oxidizing proteins, lipids, and DNA?
proteins = make a variety of active molecules and structures, lipids = make up organelles and cell membranes, nucleic acids = make up DNA (change code = change proteins that are made = change cell function)
Why is mtDNA particularly sensitive to oxidative stress?
close proximity to ETC, low non-coding area, lack protective histones, insufficient repair mechanisms
Why is it important to know what is causing oxidative stress?
to make a treatment of antioxidants that can better target the stress
Main cellular sources of ROS/RNS?
mitochondria, NADPH oxidase, xanthine oxidase, myeloperoxidase, nitric oxide synthase
What precentage of the total O2 used by mitochondria is incompletely reduced to superoxide?
2-3%…although this only represents a small fraction of the total O2 utilized this can be a significant source of ROS in metabolically active tissues
How is superoxide generated?
during mitochondrial respiration E- passed down the ETC are accepted by O2 to form H2O in a final step at Complex IV. Normally, O2 undergoes 4e- reduction; however, some e- leak out of the ETC and incompletely reduce O2 to superoxide
Superoxide is considered a : a) free radical e b) ROS c) RNS d) both and b e) a.b.c
d) free radical and ROS
The antioxidant that is responsible for the breakdown of H2O2 to H2O : a0 EcSOD b) MnSOD c) CuZnSOD d) glutathione reductase e) catalase
e) catalase
The major sources of the superoxide production in the ETC are?
Complex I and Complex III
The greater the flux of e- through the ETC the greater the…
superoxide generation
Where do the electrons leaked from Complex I go?
Tend to leak towards the matrix
When the e- from Complex 1 lead into the matrix, what do they do?
They can either cause damage to mtDNA or proteins in the matrix OR be taken up by antioxidants
Where do the leaked e- from Complex III go?
either into the intermembrane space or the matrix
E- from complex 3 that are leaked into the IMS, do what?
The superoxide can leave the IMS via an anion channel and go into the cytosol OR become H2O2 from GPx and leave into the cytosol
Decreased mitochondrial content and function does what to mitochondrial ROS generation?
increases it
Increased mitochondrial content and function does what to mitochondrial ROS generation?
decreases it
Why is there decreased ROS and free radical generation from the mitochondria with increased mitochondrial content?
The metabolic load is distributed amongst more mitochondria, therefore, the flow of e- is slower, so there is less e- leak.
Where is nicotinamide adenine dinucleotide phosphate oxidase found?
in membranes (plasma, SR, vacoules)
How many subunits is NADPH oxidase?
6
What does NADPH oxidase do?
produces superoxide by catalyzing the transfer of one electron from reduced NADH or NADPH to oxygen
What cells is NADPH oxidase found in?
immune cells (neutrophils and macrophages), smooth muscle, and skeletal muscle
What does NADPH oxidase do in immune cells?
Functions in host defense by killing microbes by causing free radical damage
Where is myeloperoxidase found?
lysozymes of macrophages and neutrophils
What does myeloperoxidase do?
Involves in host defense. Uses H2O2 to oxidize L-tyrosine to produces.
Where is xanthine oxidase found?
cystolic enzyme
What does xanthine oxidase do?
Produces superoxide and H2O2 from the oxidation of xanthine and hypoxanthine . Participates in cell signaling and can act as an NADH oxidase to help maintain reducing equivalents such as NAD+
What does nitric oxide synthase do?
Catalyzes the oxidation of L-arginine to L-citrulline to release nitric oxide free radical
3 main isoforms of nitric oxide synthase?
neuronal NOS (nNOS), indiucble NOS (iNOS), endothelial NOS (eNOS)
Where is neuronal NOS found?
primarily found in skeletal muscle where it is localized to the sarcolemma region and associated with dystrophin
Where is indicible NOS found?
Primary found in macrophages and aid in host defense
Where is endothelial NOS founf?
Primarily found in endothelial cells and aids in vasodilation
Antioxidant levels in different muscle types from most to least?
Highest in TypeI, then mixed, then Type II
What type of mithochondria have a higher rate of respiration and a lower rate of ROS generation?
IMF mitochondria
What explains the lower rate of ROS generation in IMF mitochondria?
different levels of MnSOD but possibly other antioxidants AND higher levels of cytochrome c
What effect do ROS have on satellite cells?
Satellite cells incubated with H2O2 show reduced divisions in culture
DNA isolated from skeletal muscle of SOD deficient animals have _____________
increased oxidative damage
What does aging do to ROS and free radical generation and oxidative stress?
It increases them
What does aging do to cellular antioxidant defenses?
decreases them
Skeletal muscle from aged animals and humans usually shows an _______ in antioxidant enzyme activity.
increase in SOD, catalase, GPx, GST, and GR
Why is there an increase in antioxidant enzymes with age?
Very responsive to change in the oxidative environment so antioxidant enzymes increases with the increased ROS generation with age, but oxidative stress still occurs because more ROS than antioxidant enzymes
Are GSH, vitamin C, and vitamin E levels affected by age?
No
What happens to malondialdehyde , a marker of oxidative damage to lipids, with age?
it is increased
Basal mitochondrial H2O2 release is _________ in mitochondrial isolated from aged skeletal muscle
elevated
Older adults that are displaying greater fiber type changes (+40% vs -40% type II fibers) changes in mixed muscle (vastus lateralis) have ________ levels of lipid peroxidation and __________ levels of antioxidants.
higher levels of lipis\d peroxidation and lower levels of antioxidants
ETC complex deficiencies that occur in muscle during aging result in?
Increased ROS generation at the mitochondria and increased muscle damage
What happens to antioxidant levels and ROS generation in reponse to disuse?
Antioxidant enzyme levels decrease, protein oxidation levels increase, and ROS generation increase
Denervation does what to MnSOD levels in SS and IMF mitochondria?
decreases them
What does the decreased levels of MnSOD do RR ROS production, but not IMF ROS producction?
increase SS ROS
What happens to force generation when an antioxidant is taken with exercise?
It decreases
Why does optimal force generation decrease with an antioxidant?
Antioxidants cause a lack of ROS. ROS are important in generating optimal force production such as cross bridge formation.
To maintain optimal muscle force generation, what must be maintained?
An optimal cellular redox status may influence
What does regular exercise do to muscle redox status?
Elevates the level of several antioxidants (positive adaptation) and basal ROS goneration is reduced in muscle (positive adaptation)
What happens to exercise-indices signaling and antioxidant adaptation if ROS generation is blocked?
No activation of p38, ERK, NFkB (MAPK pathways) by ROS, thish inhibits the exercise-induced upregulation of these pathways which causes a decrease in antioxidant enzymes.
What does oral vitamin intake do to exercise-induced antioxidant adaptations for normal exercise?
prevents it
What happens to mitochondrial biogenesis with decreased ROS signaling from increased antioxidants?
Mitochondrial biogeneis is decreased (less PCG1-alpha and PPAR)
What are the 2 types of cell death?
necrosis and apoptosis
What are the characteristics of necrosis?
accidental form of death, usually occurs during disease or acute injury only, not part of normal development, results in rapid cellular swelling and rupture of the cell membrane, leads to major inflammatory response due to the release of intracellular material into the surrounding environment
What are the characteristic of apoptosis?
Highly conserved cell death, important part of normal tissue development and homeostasis, increased or decreased during numerous diseases. Active process regulated by various intracellular signals. Results in chromatin condensation, cell shrinking, membrane blebbing. Cell membrane remains intact, little to no inflammation
In regards to the cell membrane, how are apoptosis and necrosis different
In apoptosis, the membrane stays intact, whereas in necrosis, it blows apart
What is cell blebbing?
When the cell packages its intracellular contents into packages that express ligands that recruit phagocytes to the bleb to destroy it
What is the hallmark of apoptotic cell death?
fragmented DNA. There is also a breakdown of important structural, regulatory, and repair proteins.
Stimuli for necrosis vs. apoptosis?
Necrosis - pathological, usually a consequence of irreversible cell injury or disease, “cell homicide.” Apoptosis - physiological and sometime pahtological, active process that is genetically controlled by a series of biochemical and molecular events, “cell suicide”
Histology of necrosis vs. apoptosis?
Necrosis: typically affects a larger number of cells, cell swelling, loss of membrane integrity. Apoptosis: typically affects a smaller number of cells, cell shrinkage, cell breaks down into membrane-bound fragments (apoptotic bodies) aka blebbing
DNA breakdown in necrosis versus apoptosis?
Necrosis = random, diffuse fragmentation. Apoptosis = orderly nuclear condensation and fragmentation
Tissue reaction in necrosis versus apoptosis?
Necrosis = inflammation with secondary injury to surrounding unaffected tissues. Apoptosis = little to no inflammation or secondary tissue injury, cell membrane signals allow for elimination by phagocytes
What is the most popular researched biological topic currently, and why?
apoptosis because it affects almost every cell in the body, and it is in normal and diseased cells
Proliferation > cell death =
accumulation of cells…cancer, autoimmune disorders
Proliferation = cell death =
homeostasis
Cell death > proliferation =
cell loss…Alzheimer’s, cardiac and muscle diseases
Inhibition of cell death leads to?
Cell accumulations…undesirable tissue growth and in this case, an accumulation of immune cells and a variety of immune disorders
What would happen to the size of the spleen and lymph nodes in a bax-/- and bak -/- mouse?
undesirable growth of these organs
Promotion of cell death leads to?
Cell loss…this leads to an undesirable tissue atrophy, retarded development, decreased immune function, and premature death
What happens to the size of the spleen and lymph nodes in Bcl-2-/- mice?
Smaller in size and smaller kidney and spleen
Where does the name caspase come from?
Cysteine ASPartic acid proteASES (caspases)
How many caspases are there?
14 have been identified
How are caspases found?
in an inactive (pro-form) and active form
2 main classes of caspases?
initiator and effector
What are the initiator caspases?
Caspase 2,8,9,12
What are the effector caspases?
Caspase 3,6,7
What do caspases do?
Cleave numerous cellular substrates that ultimately result in apoptosis…actin, poly(ADP)ribose polymerase (DNA repair enzyme), lamins (nuclear structure proteins)
How did BCl-2s get their names?
first identified as a gene that alters growth and cell death in B-cell Lymphoma (Bcl-2)
How many Bcl-2 proteins are there in the family?
25
What is the role of the Bcl-2 family?
function to inhibit or promote cell death in a variety of tissues
Where do Bcl-2 family proteins act?
on various membranes (mitochondrial, ER/SR, and nuclear)
2 main classes in the BCl-2 family?
Anti-apoptotic and pro-apoptotic
Pro-apoptotic proteins of the Bcl-2 family?
Bax, Bid, Bak
Anti-apoptotic proteins of the Bcl-2 family?
Bcl-2, Bcl-xl
What is used as a measure of a cell’s susceptibility to apoptosis?
The ratio and pro to anti-apoptotic proteins
What are some inducers of apoptosis?
high levels of free radicals, ROS, RNS. Chemotherapeutic drugs. High levels of stress hormones (glucocorticoids, catecholamines), high cystolic calcium levels. UV radiation, ethanol, growth factor withdrawal, nutrient deprivation.
Inhibitors of apoptosis?
Antioxidants (superoxide dismutase, gluthione, catalase, vitamin C, vitamin E). Various grwoth factors (IGF-I and GH)
What is the death receptor pathway?
Cytokines FasL or TNF-alpha bind to their receptors on the sarcolemma. This activates the transmembrane the Fas associated death domain, which cleaves procaspase-8 into active caspase 8. Caspase 8 cleaves procaspase 3 into caspase 3, which then begins to dismantle to cell by cleaving its substrates.
Which of the following is false in respect to muscle during aging? a) ETC deficiencies occur b) elevated lipid peroxidation c) increased catalase levels d) higher mito. ROS generation e) none of the above
e) none of the above…they are all true
During a single bout of acute exercise, it would be expected there would be: a) inc. ROS generation b) dec. ROS generation c) no change in ROS generation d) ROS generation in Type II fibers only e) none of the above, muscle does NOT generate ROS
a) increased ROS generation
What happens to the levels of p-IkB and NFbB during and post-exercise (about 4 hours)?
Exercise causes phosphorylation of IkB, which relieves IkB’s inhibition of NFkB. This allows the upregulation of pathways necessary to increase antioxidant enzymes through upregulation of specific transcription factors.
What are the 3 main ways exercise results in positive ROS-indiced adaptations relative to disease/aging?
1) transient increases in ROS generation (and signaling) 2) Lower absolute levels of ROS 3) ROS/RNS source, site of generation, and type of ROS/RNS generated in exercise are different than that of disease and aging, so it causes positive adaptation not damage
In terms of oxidative stress, what is increased in MDX mice skeletal muscle/
NO production as well as lipid (MDA) and protein (carbonyl) oxidation are increased in skeletal muscle of MDX mice
How does exercise offer some protection to lipid and protein oxidation and NO generation in muscle dystrohpy?
Exercise increases ROS generation in different locations than the ROS generation from neuronal NOS, like in MD. This causes positive adaptations such as increased antioxidants.
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
Caspase independent apoptosis?
AIF and EndoG and Omi
Caspase independent apoptosis?
AIF and EndoG and Omi
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
Caspase independent apoptosis?
AIF and EndoG and Omi
Caspase independent apoptosis?
AIF and EndoG and Omi
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
What type of mitochondria have more Bax protein?
10-fold higher in SS vs IMG mitochondria
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
What type of mitochondria are more susceptible to apoptotic stimuli?
IMF mito
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
What apoptosis pathways do ARC inhibt?
All 4…
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
Where is ARC highly expressed?
In skeletal muscle but absent in other healthy tissues.
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
What fibre type has the most ARC?
Type I > Type IIa > Type IIx > Type IIb
What do ARC KO mice have?
An altered fibre type distribution…more fast phenotype
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
In myonuclear apoptosis, does the entire cell die?
Nope
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
How do myonuclei supports a cytoplasmic portion of the cell?
Healthy muscle have a number of myonuclei, each supporting a cytoplasmic portion of the fiber. The nuclei number dictates the cells ability to produce proteins (i.e. myosin and actin) and therefore muscle size. Myonuclei apoptosis reduced myonuclei number and leads to skeletal muscle adaptation and remodeling. This remodeling helps maintain nuclear:cytoplasmic ratio.
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
What dictates fiber CSA and size?
The number of nuclei per fiber. Although the entire fiber may not be eliminated in response to apoptotic signaling, loss of nuclei is an important factor in muscle atrophy.
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
Bax pore formation can ultimately result in?
cyto c release, AIF release, and caspase activation
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
Death receptor pathway intitially results in?
activation of caspase 8
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
How are oxidative stress and skeletal muscle apoptosis related?
ROS can induce skeletal muscle (myotube) apoptosis
Caspase independent apoptosis?
AIF and EndoG and Omi
How is vitamin C related to skeletal muscle apoptosis and oxidative stress?
Apoptosis can be inhibited by vitamin C
What do vitamin E and GSH do in terms of oxidative stress and apoptosis?
Vitamin E can inhibit DNA fragmentation induced by H2O2, whereas GSH can reduce caspase 3 activity following UV exposure in skeletal muscle myotubes
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
What happens in the cell if GSH is depleted?
There is a decrease in CuZnSOD and MnSOD, but an increase in catalase, and there is an increase in ROS generation
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
In glutathione depletion, why does catalase increase but other antioxidants decrease?
GPx uses glutathione in the same reaction that catalase uses to reduced H2O2 to water and GSSG. Therefore, the muscle will increase catalase an a compensatory measure in response to decreased GSH. However, it isn’t enough, and there is still an increase in ROS generation
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
What happens at the mitochondria in response to GSH depletion?
An increase in mitochondrial dysfunction and apoptotic signalling…increased in mitochondrial swelling and decreased mitochondrial membrane potential and increased mitochondrial apoptotic protein release.
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
Skeletal muscle from aged mice show higher levels of __________ compared to their younger coutneraprts
active caspase-3 (cleaved)
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Young animals with accumulated mtDNA mutations in skeletal muscle also haave higher levels of _________ compared to normal mice.
active caspase-3
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
Why do mtDNA mutations lead to increased skeletal muscle apoptosis?
mtDNA mutations cause deficiencies in the ETC, which increases ROS generation. Increased ROS generation = increased apoptosis
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
Characteristics of satellite cells during aging in regards to apoptosis?
More satellite cells isolated from old animals express pro-apoptotic Bax protein compared to young animals. Satellite cell anti-apoptotic BCl-2 levels are lower in old vs young. Satellite cells isolated from old animals are also more susceptible to TNF-alpha induced apoptosis.
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
Skeletal muscle apoptosis is ______________ with aging and is exacerbated by inactivity.
elevated
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
Increased skeletal muscle apoptosis is is associated with _______ muscle weights and cross sectional area
decreased
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
What happens to mice with congenital muscular dystrophy (Lama2-/-) when there is an inhibition of apoptosis (Bax-/-)?
Reverses muscle loss, functional decline, and early death
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
Why is skeletal muscle apoptosis elevated in CHF?
Altered MHC expression (faster phenotype). There is a decrease in Bcl-2 as well as increased caspase-3 and myonuclear apoptosis (TUNEL)
A key feature that helps distinguish necrosis from apoptosis?
Plasma membrane destruction vs blebbing. Random DNA fragmentation vs. organized DNA fragmentation. Cell swelling vs. cell shrinking. Random vs. programmed used intracellular factors such as caspases.
CHF patients with apoptotic positive skeletal muscle have a 34% _________ VO2 max even though heart function is not different
lower
Which of the following is false? a) apooptosis leads to DNA fragmentation b) caspases can activate other caspases c) necrosis plays an important role during tissue development d) during disease, cells can die by apoptosis and/or necrosis e) none of the above
c) necrosis plays an important role during tissue development
Skeletal muscle apoptosis is elevated in COPD…why?
COPD leads to increased breakdown of the DNA repair enzyme PARP and elevated myonuclear apoptosis. Inactivity exacerbates apoptosis in healthy subjects.
What is the major apoptotic pathway in the muscle?
The mitochondrial patheays
What does denervation do to the skeletal muscle in terms of muscle apoptosis?
Increases pro-apoptotic Bax and AIF levels while reducing anti-apoptotic BCl-2 levels in skeletal muscle. These changes are associated with elevated skeletal muscle apoptosis and atrophy. This process begins to happen within 5 days of denervation.
What does cytochrome c release cause in the mitochondrial apoptotic pathway?
Cytochrome c is released from the mito. It interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase 9 to form an apoptosome, which is essentially active Caspase 9. This leads to activation of caspase 3, which cleaves its substrates, resulting in apoptosis.
What are the 3 main ways exercise decreases apoptosis in skeletal muscle?
1) Direct change in apoptotic factors (dec. in pro-apoptotic and inc. in anti-apoptotic) 2) Changes in ROS generation and oxidative stress
What is XIAP, and what does it do?
X-linked Inhibitor of Apoptosis Protein. Inhibits the apoptosome/caspase 9 and caspase 3, resulting in the inhibition of apoptosis
Regular does what in the muscle?
Increases anti-apoptotic proteins such as Bcl-2, ARC, and XIAP in healthy, aged and hypertensive rats. Also assoicated with decreased myonuclei apoptosis and leads to positive morphological adaptations in old and hypertensive rats. In humans, moderate, regular exercise training reduces circulating apoptotic factors such as TNF-alpha and FasL in CHF patients.
What are smac/DIABLO, and what do they do?
Smac = second-mitochondria derived activator of caspases. DIABLO = direct IAP binding protein with a low isoelectric point. They bind to XIAP in the cytosol and inhibit its action, thereby allowing apoptosis to continue.
How does mitochondrial biogenesis protect against cell death?
Reduces caspase activation and AIF release
What does Omi do?
It can either translocate from the mitochondria to the nucleus and cleaves DNA (caspase independent) or it inhibits XIAP (caspase dependent)
How does mitochondrial biogenesis result in reduced mitochondrial-Ca2+ sensitivity?
Cells exposed to high levels of Ca2+ show mitochondrial rupture and loss of membrane potential (two common features during apoptosis). Cells with higher mitochondrial content are protected from these events.
What do AIF and endoG do?
AIF (apoptosis inducing factor) and EndoG (endonuclease G) translocate to the nucleus from the mitochondria and directly cleave DNA.
The anti-apoptotic protein discussed in class that’s highly expressed in slow muscle is: a) XIAP b) ARC c) Bcl-2 d) a and b e) a,b and c
b) ARC
Casapse dependent apoptosis?
Cytcohrome C, smac/DIABLO, or OMI release. XIAP.
The most important feature that differs between apoptosis in mist cells compared to muscle fibres is related to:
muscle’s multi-nucleated morphology
Caspase independent apoptosis?
AIF and EndoG and Omi
Why does mitochondrial biogenesis decrease Ca2+ sensitivity?
It enlarges the calcium sink, so for a give load of Ca2+ if there are more mitochondria it’ll be spread amongst all of them, so less swelling and release.
Mitochondrial function, apoptotic action, and pathway of Cyto C?
Shuttle e- from Complex III to Complex IV. Apoptosome formation and caspase activation. Caspase dependent.
Some intracellular conditions that damage cell proteins?
temperature at 37 degreese C or higher (denaturing conditions), many reactive small molecules (these causes oxidation, deamidation, glycation or nitrosylation). Enzymes that modify proteins such as proteases or kinases. High salt concentrations (which favor dissociation of multimers). Many fatty acids, which act like detergents. Other unfolded proteins–nascent polypeptides, damaged or mutant polypeptides and insoluble inclusions are sticky)
Mitochondrial function, apoptotic action, and pathway of EndoG?
Mitochondrial biogenesis. Direct DNA fragmentation. Caspase-independent.
Abnormal proteins that are rapidly degraded in the cell?
Incomplete proteins, missense proteins, free subunuts of multimeric complexes, postsynthetic damage, genetic engineering, protein misfolding.
Mitochondrial function, apoptotic action, and pathway of Apoptosis Inducing Factor (AIF)?
Proper function of Complex I. Direct DNA fragmentation. Caspase independent.
What is the role of the ubiquitin-proteosome system (UPS)?
Mainly responsible for basal turnover of short lived, degraded and misfolded proteins.
Mitochondrial function, apoptotic action, and pathway of smac/DIABLO?
Possible fission/fusion? Binds IAPs thereby increasing caspsase activity. Caspase-dependent.
How does the UPS recognize proteins to be degraded?
They are tagged by a poly-ubiquitin chain
Mitochondrial function, apoptotic action, and pathway of Omi/HtrA2?
Mitochondrial homeostasis. Binds IAP’s thereby increasing caspase activity for the caspase-dependent patheay. Direct DNA fragmentation for the caspase independent pathway.
3 main enzymes of the UPS
E1, E2, E3
What is the role of AIF at Complex I in the ETC?
May aid in the assembly of the 46 NADH Dehydrogenase (Complex I) components. May act directly in Complex I by acting as an NADH oxidase.
Role of E1 in UPS?
Activation
What are the 3 consequences of cytochrome c and AID release from the mitochondria? How does this act as an amplification step in apoptosis?
1) Apoptosis activation (caspase dependent and independent) 2) ATP depletion (messes up H+ gradient and ETC flow, so less phosphorylation potential and less ATP) 3) ROS generation (more e- leak because no cytochrome c, making ROS). By stopping the production of ATP and increasing the ROS generation, it acts as almost a postitive feedback loop that the cell is indeed supposed to die.
Role of E2 in UPS?
Conjugation
Mechanism of mitochondrial protein release involving Bax pore?
Bax becomes activated in the cytosol by numerous factors such as chemotherapy and ROS. Bax tranlocates to the outer membrane of the mitochondria, where they insert themselves in the membrane, forming a Bax/Bak pore. This pore allows cytochrome c to be released into the cytosol, causing the apoptosis ccascade to begin
Role of E3 in UPS?
Ligation
Mechanism of mitochondrial protein release involving VDAC and ANT?
translocation of Bax to the outer mitochondrial membrane causes the interaction between VDAC and ANT, which results in the formation of the megapore. The megapore allows for water and ions to enter into the matrix, causing it to swell, and then the MOM ruptures, causing the release of cyto chrome C.
What are misfolded/damages/denatured proteins marked with in order to cause ubiquitin conjugation?
Hsp70 and Hsp40
Maintained, high levels of cystolic Ca2+ activates what, which leads to what?
Actiavtes the proteolytic enzyme m-calpain, which in turn actiavtes the caspase 12 pathway, which actiavtes caspase 3
What is ubiquitin activated by?
E1 (ubiquitin activating enzyme)
What do calpains do?
Activate caspase 12 (activates caspase 3) and also cleave proteins themselves.
What happens after ubiquitin is actiated by E1?
Ub is transferred to a ubiquitin carrier protein (E2; ubiquitin conjugating enzyme). Then, proteins to be degraded and E2 both bind to a specific E3 (ubiquitin-protein ligase). This activated structure transfers the ubiquitin to the protein. This ultimately acts as a signal to target the protein tothe 26s (proteosome for degradation).
What is 26s?
a proteosome
What is 26S proteosome made up of?
19S regulatory particle and 20S core particle.
What is the structure of the proteosome?
Two 19S regulatory particles that contain a base and a lid which recognizes polyubiquinated proteins, as well as allows substrate to enter 20S.
What is the role of 19S in the proteosome?
Regulatory particle that also unfolds any folded proteins that cannot fit through the narrow channel
What is the role of the 20S core particle in the proteosome?
Contains catalytic activity
What does the proteosome do?
Peptides are released, and ub is released
What are the two types of pathologies that can arise in the ubiquitin-proteasome system?
1) Those that result from gain of activity — accelerated degradation of the protein target 2) Those that result from loss of function — stabilization of certain proteins or inhibition of 26S proteasome, resulting in reduced degradation
How does the UPS relate to cancer?
UPS is overactive in cancer, resulting in the degradation of specific proteins, such as p53, that promote apoptosis. Without these proteins, the cancer cells continue to grow. When treated with a proteasome inhibitor, there is an increase in p53.
What does the inhibition of the prtoeasome activity do in cancer?
Promotes cell cycle arrest and decreases tumour size
What is Huntingdon’s Disease characterized by?
An increase in glutamine repeats on the Huntingdon protein which leads to formation of inclusion bodies
What is proteasome activity like in Huntingdon’s Disease?
Decreased proteasome activity, contributing to aggregated protein accumulation. In contrast, activation of the proteasome clears protein aggregates
What are the 2 major E3 ubiquitin ligases in skeletal muscle?
MuRF1 (muscle ring finger protein) and MAFbx (atrogin-1, muscle atrophy f-box protein)
What do MuRF1 and MAFbx do?
These factors direct the polyubiquitination of proteins in order to target them for proteolysis by the proteasome.
What regulates MuRF1 and MAFbx?
Myostatin/TGFbeta
What happens to the UPS during denervation and immobilization?
Inactivity upregulates E3 ligases (MuRF1 and MAFbx), so more protein degradation and loss of muscle mass
What happens when MAFbx and MuRF1 are knocked out acutely?
There is less muscle atrophy, therefore, UPS and muscle specific ligases are required for muscle atrophy.
What happens if there is long term inhibition of MAFbx?
Preserves muscle mass but results in more muscle damage/dysfunction
What happens to the proteasome and MAFbx if there is a KO of MuRF1?
MAFbx and proteasome are upregulates as a compensatory measure.
What type of apoptosis does skeletal muscle undergo?
myonuclear apoptosis because it is multinucleated morphology
What does autophagy mean?
self eating
What is autophagy?
Process of degradation of long lived proteins, portions of the cytosol and organelles. Used to provide energy substrates and cellular defense
What are the 3 main types of autophagy?
Micro-autophagy, chaperone mediated autophagy, and macro-autophagy
What does autophagy do?
Works with the UPS to remove damaged factors in the cell, and is not limited by the size of the protein or factor like UPS. Plays a major role in metabolism, cell cycle, differentiation and cell death
What is one of the key roles of autophagy?
Promotes cell survival by clearing the cell of damaging proteind and organelles
What is the defining feature of autophagy?
Development and presence of a double membrane vesicle
Progression in the development of the autolysosome?
Phagophore –> autophagsome (double membrane) –> fusion of lysosome to autophagosome to form an autolysosome with the aid of LAMP-2A
What are atgs?
Autophagy-related genes
What is LC3?
Atg8…microtubule-associated protein 1 light chain 3B, a ubiquitin-like protein that forms the autophagosome membrane
What is p62?
an adaptor protein that binds both ubiquitin and LC3
What is LAMP2?
aids in autophagosome and lysosome fusion
What are key factors regulating autophagy?
atg, LC3/ATG8, P62. LAMP2
What two kinase complexes regulate the activation and induction of autophagosome formation?
Beclin1 complex and mTOR substrate complex.
What is the Beclin1 complex?
a macromolecular complex that once activated generates phosphatidylinositol-3-phosphate, which promotes autophagosomal membrane generation
What is the mTOR substrate complex?
Highly regulated by ULK1, which is regulated by mTOR and AMPK activity. Activated of mTOR substrate complex promotes autophagy.
Through what pathways does energy deprivation cause autophagy?
Energy deprivation activates AMPK, which activates the mTOR substrate complex, leading to autopgasomal nucleation and and autophagosome formation.
Through what pathways for insulin and other growth factor signalling inhibit autophagy?
Activates class I PI3K-AKT, which activates mTOR signalling complex I, which inhibits the mTOR substrate complex.
Through what pathways does starvation and rapamycin increase autophagy?
They inhibit the mTOR signalling complex I, which usually inhibits the mTOR signalling complex, so an inhibitor of an inhibitor = autophagy activation.
What are the 2 ubiquitin-like conjugation systems regulate elongation and maturation of the autophagosome?
ATG5-ATG12 conjugation system and LC3/ATG8 Conjugation System
What is the ATG5-ATG12 conjugation system?
This complex is formed by conjugation of ATG5 to ATG12 by ATG7 (E1-like enzyme) and ATG10 (E2 like enzyme). This elongates the autophagic membrane.
What is the LC3/ATG8 conjugation system?
LC3 is cleaved to generate LC3I, conjugation of PE (a phospholipid) with LC3I occurs with the aid of ATG7 (E-1 like enzyme) and ATG3 (E2-like enzyme), which results in elongation of the autophagic membrane
What ATG is key in the formation of the autophagosome?
ATG7
Animals deficient in ATG7 have what?
Severley reduced autophagosome formation and autophagy
What does ATG4 do?
Cleaves ATG5-ATG12 and outer LC3 from autophagsome membrane, leaving a phospholipid bimembrane with LC3 on the inside
What is key in selective autophagy?
LC3 on the inside of the autophagosome binds with p62, which binds with ubiquinated proteins and organelles, so they are marked for degradtion by the lysosome
What can initiate the targeting of mitochondria for degradation by autophagy?
Damage and/or loss of mitochondrial membrane potential
What is PINK1?
Molecule found in low levels on mitochondria, but accumulates on outer membrane during low membrane potential
What is PARKIN?
E3 ligase normally found in cytosol but translocates to mitochondria upon loss of membrane potential. It then promotes ubiquination of mitochondrial protein (VDAC)
What are the two ways recognition of targeted mitochondria by the autophagsome occurs?
P62: binds to UB proteins, which can then be tethered to LCS on the autophagosome membrane. OR, BNIP3/Nix: autophagy specific receptors that are expressed in damaged mitochondria. They can interact directly with LC3 (do not require ubiquination)
In mice that are knocked out for Atg7, what is seen?
An upregulation of LC3 and p62, with centralized nuclei and damages fibers…autophagy is required for muscle maintenance
What aspects of autophagy are down regulated in aging?
Upstream signals (insulin, glucagon) are altered during aging leading to decreaesd activation of autophagy. Altered function of the lyosome impairs clearance of targeted factors.
Reduction in autophagy lead to what in aging?
Accumulation of abnormal mitochondria (swollen, lacking cristae, dysmorphic). Aged animals that have reduced autophagy have impaired mitochondria oxygen consumption, without altered ETC complex expression.
Relationship of autophagy and muscular dystrophy?
MD have reduced autophagy and show an accumulation of damaged/abnormal mitochondria and SR. This is associated with increased apoptosis.
What does starvation do in MD?
Removes the damaged organelles and reduces apoptosis.
Following starvation in mice with MD, what is upregulated?
Autophagic flux (LC3II/I), Beclin-1 and BNIP3 following starvation in MD mice
Knockdown of Beclin-1 elminates what?
Protective effect of starvation (not BNIP3)
Exercise and autophagy relationship?
Autophagy is induced by exercise and regulated by BCl-2:Beclin interaction
Mice lacking 3 phosphorylation residues on the Bcl-2 protein or Beclin-1 partial KO (+/-) show defects in?
Starvation and exercise induced muscle autophagy
Mice with deficient autophagy shows deficits in…
running endurance and altered glucose regulation
