Section 1 - ROS part 1 Flashcards
(136 cards)
1
Q
ROS stands for _______
A
reactive oxygen species
2
Q
ROS are made inside _____? because?
A
-inside cells
-because oxygen is highly reactive
3
Q
ROS definition
A
oxygen-containing molecules that are short-lived and highly reactive
4
Q
3 types of ROS
A
-superoxide anion
-hydrogen peroxide
-hydroxyl radical
5
Q
superoxide anion + formed from ______
A
O2.-, oxygen + e-
6
Q
hydrogen peroxide + formed from ______
A
-H2O2
-superoxide anion (O2.-) + H+ = HO2., 2HO2. = H2O2 + O2
7
Q
hydroxyl radical + formed from _____
A
-HO.
-superoxide anion (O2.-) + H2O2 = O2 + OH- + OH.
8
Q
Harman (1950) hypothesized _____
A
Free Radical Theory of Aging
9
Q
Free Radical Theory of Aging mainly claims that _____
A
-increasing ROS = increase in oxidative stress = increase aging
10
Q
Free Radical Theory of Aging method
A
-irradiated people with radiation had an increase in ROS
11
Q
Free Radical Theory of Aging observed that ______
A
older animals from different species has more ROS than younger animals (consistent phenomenon)
12
Q
Free Radical Theory of Aging suggests that lifespan differences between different species is due to _______
A
different mechanisms against ROS, which cause aging
13
Q
Location of ROS production? why?
A
-mitochondria
-it is the location of oxygen consumption in the cell due to oxidative phosphorylation
14
Q
Mitochondria Metabolism summary
A
-matrix: citric acid/krebs cycle produces NADH which donates e- to electron transport chain in inner membrane.
-intermembrane space: electron transport chain pump H+ into space & H+ enters back into matrix through ATP synthase. flux of H+ produces ATP. e- enters matrix again to reduce O2 in matrix to make H2O
15
Q
pH of mitochondrial intermembrane space
A
low pH (high conc of H+)
16
Q
pH of mitochondrial matrix
A
high pH (low conc of H+)
17
Q
cytochrome c oxidase complex does what?
A
moves H+ into the intermembrane space (part of oxidative phosphorylation machinery)
18
Q
artificial liposomes with cytochrome c oxidase do what in the presence of oxygen?
A
-consumes oxygen and moves H+ into the medium (pH of the medium drops rapidly with a gradually recovery as oxygen is depleted)
19
Q
charge of mitochondrial intermembrane space
A
positive charge (protons)
20
Q
charge of mitochondrial matrix
A
negative charge
21
Q
TMRM is what?
A
fluorescent compound that follows negative charge (often used to assay mitochondrial membrane potential )
22
Q
MMP (def.)
A
mitochondrial membrane potential
23
Q
TMRM & mitochondria interaction
A
TMRM follows negative charge found inside the mitochondrial matrix
24
Q
other methods used to assay mitochondrial membrane potential
A
fluorimeter, microscopy or flow cytometry
25
Oligomycin is what?
a drug that binds ATP synthase and blocks the proton channel
26
Oligomycin + TMRM signal
-increase TMRM signal because matrix is more negative as H+ flow into the matrix through ATP synthase is blocked
27
FCCP does what?
-uncoupler that acts as a ionophore
-transports protons into matrix, disrupting ATP synthesis
28
FCCP + TMRM signal
-decrease TMRM signal rapidly because matrix is less negative as H+ enters the matrix
29
______ allows for precise localization of fluorophores with __________
-Scanning confocal laser microscope
-minimal light incidence over time
30
Why use scanning confocal laser microscope with fluorophores?
-high amount light destroys the fluorophores leading to bleaching; needs a low amount of incident light to prevent destroying fluorophores
31
byproducts of destruction of fluorophores
ROS
32
activity that increases cell ATP demand
-transport mechanism
-protein folding
33
increased ATP demand + TMRM signal
-decrease TMRM signal as H+ enters matrix to make ATP
34
Calcium controls _____ in mitochondria
pore opening (opens pore)
35
recoverable Ca2+ load + TMRM signal
-decrease TMRM signal as H+ enters matrix to make ATP
-calcium removed over time, common in muscle cells
36
excessive Ca2+ load + TMRM signal
- apoptosis
- decrease TMRM signal slightly as H+ enters matrix to make ATP until it reach permeability transition (loss of mitochondria membrane potential) and then decrease rapidly
37
PTPC stands for
permeability transition pore complex
38
PTPC does what?
serves as a gatekeeper for the mitochondrial membrane potential
39
problems with PTPC leads to ______
diseases like muscle dystrophy
40
PTPC is formed by ______
ATP synthase under the influence of Ca+
41
permeability transition is when the mitochondria does what?
mitochondria loses ability to hold H+ in intermembrane space and subsequently can't creates ATP
42
ROS and effect of PTPC
-hydrogen peroxide opens PTPC
-loss of membrane potential
43
level of ____ affects membrane permeability of mitochondria
oxidative stress and Ca+
44
increase oxidative stress/Ca+ = ______ + effect on TMRN
increase membrane permeability of mitochondria/ open pore -> TMRN staining is lost
45
To understand how a mechanism works, you need to _____
identify and characterize the proteins or molecules executing it
46
Permeability Transition Pore Complex is a ______ (descr.)
600kDa multiprotein complex on the inner membrane
47
PTPC regulates _____
exchange of ions and ATP between matrix and cytosol
48
ROS and Ca+ can lead to _____
abnormal PTPC opening, leading to mitochondrial swelling
49
PTPC opening reduces production of _____
ATP
50
PTPC opening is promoted by ______
sustained calcium, pro-oxidants (ROS), nucleotide depletion
51
PTPC opening is inhibited by ______
-Bcl2, BclXL, high ADP/ATP, cyclosporin A
52
Bcl2 proteins are _____ & function in mitochondria
-anti-apoptotic
-closes pore = ATP production continues (no apoptosis)
53
PTPC open pore & effect on ATP production
-decrease ATP production (apoptosis started)
54
ROS are _____ that modify _______ similar to _____
-cellular signalling molecules
-post-translationally many substrates
-phosphorylation
55
ROS modification of proteins crosstalks with ______
lipid modification (ex. palmitoylation)
56
ROS modification: oxidation typically _____
affect cysteine residues (ex. forms disulfide bonds)
57
result of ROS modification is ______ and it is very ______
-oxidation
-difficult to determine experimentally
58
ROS opens PTPC by ______
post-translationally modifying the regulatory protein cyclophilin D
59
cyclophilin D has this type of activity?
peptidyl-prolyl isomerase activity (changes structure of proline residues)
60
ROS modifies cyclophilin D on _____
Cys 202 (humans)/ 203 (other species)
61
Normal cell: cyclophilin D
Cys202 is S-palmitoylated
62
Cell under ischaemia (low oxygen): cyclophilin D
Cys202 is oxidized on sulfur residue with ROS
63
oxidized Cys202/203 of CyD effect on PTP
opens PTP
64
Transfection allows for the easy ____
introduction of foreign DNA sequence
65
Transfection methods (2)
-lipid treatment (using liposome to allow DNA to enter cell)
-electroporation (high voltage shocks to allow DNA to enter the cell)
66
Problem with transient transfection
not all cells will express the protein; temporary
67
Transient transfection vs. CRISPR/Cas 9
-temporary vs. permanent/may cause cell to adapt
68
Mice with knockout cyclophilin D characteristics (3)
-smaller myocardial infarction
-reduction of neuroinflammation
-resistance to neurodegeneration
69
Knockout cyclophilin D mice shows that _____
abnormal mitochondria activity plays a crucial role in disease progression
70
mutation of cys202/203 of cyclophilin D effect
inhibits mPTP opening and improves cell viability
71
proteins loaded with studied protein in western blot or other experiments serve as ______
indications that proteins are expressed at the same level in knockout and wildtype cells
72
Ionomycin does what?
dissipates MMP; Ca2+ permeabilizing drug, increase Ca+ levels (calcium opens PTP)
73
ionomycin + cyclophilin D that can't be oxidized
-ionomycin does not dissipate mitochondrial membrane potential as efficiently
74
cyclosporin A does what?
a drug that inhibits PTP by interfering with CypD activity
75
cyclophilin D does what?
activates permeability transition (opens mPTP opening)
76
In the presence of the mutant CypD (cyclophilin D), CSA (cyclosporin A) ______
is no longer able to alter PTP opening
77
Ionomycin does not dissipate MMP well if ______
Cys203(sulfur) that is expressed cannot be oxidized
78
max flux of PTP requires ______
binding of cyclophilin D (oxidized) and presence of ROS , Ca++
79
ROS modulate mitochondrial energy output by _______
altering the open state of PTP
80
how ROS is measured in vivo?
-H2DCFDA (outside cell) enter lipid bilayer and is transformed by esterases into H2DCF
-ROS reacts with it to produce fluorescent DCF probe (measured by fluorescence microscopy and flow cytometry)
81
Advantages of flow cytometry to confocal microscopy
-precise quantification of fluorophores
-can observe live cell biological processes
-1D and 2D flow cytometry
82
level of ROS is measured by ____
fluorescent DCF probe
83
amount of fluorescent DCF probe is proportional to _____
amount of ROS
84
level of ROS in healthy cells/ diseased cells
-can high or low (doesn't support Free Radical Theory of Aging)
85
ROS-induced ROS release (def.) in cardiac myocytes
-damaged mitochondria from illumination produces ROS then ROS damages another mitochondria, leading ROS release (propagation of mitochondria damage)
86
ROS-induced ROS release affects ____
mitochondrial ATP production by affecting MMP (as seen in TMRM signal)
87
ROS move from mitochondria to mitochondria, doing what?
disrupting mPTP
88
Closed pore effect on ROS production
ROS production is slow
89
Speed of OXPHOS/ATP production effect on ROS production
ROS production is fast
90
ROS scavenging (the elimination of ROS) involves ____
multiple enzymes like SOD1 and SOD2
91
_____ (ALS) is one of the more prevalent _____.
-Amyotrophic lateral sclerosis
-adult-onset neurodegenerative diseases
92
ALS disease characterization
-progressive injury and death of lower motor neurons in the spinal cord and brainstem, and upper motor neurons in the motor cortex
-rapid progression
-survival only 2-3 years after symptoms appear
93
ALS disease mutations
-20% familial ALS caused by mutaions in copper/zinc superoxide dismutase (Cu/Zn SOD, SOD1) - majority of cases are not familial
94
mutations in SOD1 are not ____
the cause of most cases of ALS
95
p66Shc adaptor protein controls what? However, futher studies show that it is not a _____
-oxidative stress response and lifespan in mammals
-lifespan gene
96
p66 is a ____
mitochondrial protein
97
loss of p66 does what?
-protected from paraquat death
-knockout mice live longer & protected from aging
98
p66 does what?
sequesters cytochrome c after the induction of apoptosis, causing an increase in ROS production because electrons are no longer passed to complex IV of electron transport chain
99
after p66 sequesters cytochrome c and increases ROS production, ______ ensue
induced permeability transition and apoptosis ensue
100
Why are p66 ko mice protected from aging?
-p66 can no longer induce apoptosis through amplification of ROS production that promotes aging
101
p66 ko mice are protected from ____ (2) but have ____
- obesity and cancer
-defective cold adaption (lack uncoupling protein function for thermogenesis)
102
ROS production at complex I and II happens when ____
the steady oxygen supply is lost and the flow of electrons through electron transport chain is disrupted (traffic jams)
103
ROS produced at complex I and II do what?
chemically modify proteins close to mitochondria
104
HyPer7 GFP-based probe does what?
detect H2O2 (that leads to ROS) within mitochondria upon injury and on the leading edge of moving cells
105
antimycin does what?
blocks mitochondrial complex III
106
mitochondria + antimycin
increase ROS production
107
mitochondria, when undergoing forced respiration in the presence of galactose (instead of glucose/glycolysis) do what?>
increase ROS production
108
mitochondria produce ROS if ______ or if _____
-their electron transport chain is impaired (blocking ETS)
-cellular energy levels drop (blocking glycolysis)
109
this is not true but theoretically, deletion of SOD2/SOD1 should ____
induce mitochondrial ROS and decrease lifespan
110
KO of SOD2 does what?
-decreases oxygen consumption, decreasing mitochondrial activity
-increase lifespan in C. elegans
111
isp-1 is what?
an ortholog of human ubiquinol-cytochrome c reductase which is involved in ETC
112
KO of isp-1 does what?
increase survival more than sod-2 KO, while decreasing mitochondrial activity
113
KO of isp-1 and sod-2 together do what?
decrease mitochondrial function to the point of no survival
114
the KO of isp-1 and sod-2 shows that a bit of ____
ROS (oxidative stress) is good as it turns on the cell protective mechanism, allowing for good cellular function
115
mitochondrial ROS perform a dual function as _____
signaling molecules that can also lead to life span extension via activation of antioxidant defense mechanisms
116
in KO SOD organisms, antioxidant systems are ____
more active so higher levels of ROS are needed before reaching oxidative stress
117
over expression of ___ extends life span but deletion of ___ has often no. effect in C. elegans with the exception of _____
-catalase
-SOD
-SOD-2
118
substrates of ROS include PTPC and ___
key cellular signalling proteins
119
proteins controlled by ROS are often ____
sulfenylated (like phosphorylation)
120
sulfenylation is a ____
post-translational modification (oxidized thiol)
121
How to detect sulfenylated proteins?
-n-ethylmaleimide/iodoactemide block other thiols
-sulfenylated proteins were tagged with dimedone and dettected with an antibody
122
signal of oxidized cys increases when?
incubated at low temp
123
VCL is what?
vinculin western signal (loading control)
124
N-acetylcysteine is what?
-antioxidant that will desulfenylate non-specifically)
125
UCP1 is what?
-a mitochondrial transmembrane protein that decrease proton gradient developed by OXPHOS
126
UCP1 (uncoupling protein 1) is involved in _____
non-shivering thermogenesis in brown adipose tissue
127
in the presence of antioxidants, body temp ____
drops because UCP (which is involved in thermogenesis) is not oxidized (sulfenylated) by ROS
128
Sulfenylated UCP changes focus of mitochondria on _____ rather than _____
-heat generation at UCP (leak)
-work at mPTP/ATPase (work)
129
when oxidized/sulfenylated, UCP1 uncouples the ______ and causes _____
-mitochondrial proton gradient
-mitochondrial heat production to spike
130
temperature of mitochondria
50 C
131
Mito Thermo Yellow allows us to _____, with decrease in fluorescence meaning _____
-measure temperature within cells
-temp increase
132
peroxisomes make heat via _____ due to the lack of _____
-B-chain oxidation of fatty acids which produce ROS
-respiratory chain
133
hypoxia and hyperoxia conditions produce excess _____
ROS
134
normoxia conditions still has ___
some ROS as it is needed for cellular activity
135
under really low levels of oxygen, ROS production is ___
blunted
136
Different tissues within the body have different _____ so mitochondria in vivo doesn't ____
-oxygen supplies and demand
-have as much oxygen as in vitro
