a novelbioluminescentprobefornon …€¦ · nicholls, d. g. & ferguson, s. j....
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![Page 1: A NOVELBIOLUMINESCENTPROBEFORNON …€¦ · Nicholls, D. G. & Ferguson, S. J. Bioenergetics.Fourth edition / edn, (Academic Press, Elsevier, 2013). Energy of proton gradient: ∆𝐺=](https://reader033.vdocuments.net/reader033/viewer/2022060222/5f0782c47e708231d41d57cd/html5/thumbnails/1.jpg)
A NOVEL BIOLUMINESCENT PROBE FOR NON-INVASIVE QUANTIFICATIONOF MITOCHONDRIAL MEMBRANE POTENTIAL
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Main function of mitochondria is ATP synthesis
Electrochemical proton gradient is the driving force for ATP synthesis 2
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Electron transport chain (ETC)
3Nicholls, D. G. & Ferguson, S. J. Bioenergetics. Fourth edition / edn, (Academic Press, Elsevier, 2013).
Energy of proton gradient:∆𝐺 = 𝐹∆𝜓 RT ln 𝐻𝐻 = 𝐹∆𝜓 2.3RT Δ𝑝𝐻Δψ – electric potential, MMP
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4Figures are adapted from Cell Metabolism (2015) 22, 204-206Mol. Cell Bio. (2005) 6, 318-327
Intracellular signaling• Change of gene expression
profile• Adaptation to stress• Apoptosis
Biosynthetic function• Long chain fatty acids• Branched chain amino acids• Nucleotide biosynthesis
ATP synthesis is not the only role of mitochondria in a cell
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Monitoring of mitochondria function is important to understand progression of these pathologies
Mitochondrial dysfunction is involved in many processes
MMP dysregulation is implicated in :• Aging• Alzheimer disease• Parkinson’s disease• Diabetes mellitus 2 type• Cardiomyopathy• Ischemia
Figure is adapted from Cell (2013) 153, 1194-1217
Mitochondrial membrane potential (MMP) is one of the key parameters that characterizes function of mitochondria
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Quantification of MMP relies on fluorescent dyes
Cytosol
30-60 mV
Mitochondrion
180-200 mV
O
O
O
NN
TMRM
O
O
O
NN
TMRM
Accumulation
Decrease of MMP willlead to decrease offluorescent signal.
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Intensity of the signal isproportional to the amount ofTMRM that was accumulated
∆𝜓 = 𝑅𝑇𝑧𝐹 ln 𝑇𝑀𝑅𝑀𝑇𝑀𝑅𝑀
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Drawbacks of fluorescent dyes for measuring MMP
• None of them are suitable to be used in living animals (mammals)
Methods, (2008), 46, 304-3117
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Measurement of MMP in vivo is extremely important
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• Aging• Alzheimer disease• Parkinson’s disease• Diabetes mellitus 2 type• Cardiomyopathy• Ischemia
Impossible to create relevant disease model in cell culture
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Strategy for non-invasive imaging of mitochondrial membrane potential (MMP)
Bio-orthogonal reaction
+
Bioluminescent imaging
MMP
in a context of various diseases
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PPh2R'N3
Phosphine Azide PhP
Ph
NR'
N2
Aza-ylide
+
H2O
PPh2
OR'NH2+
AminePhosphine oxide
The Staudinger reaction
Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
Hermann Staudinger
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The Staudinger ligation – the first bioorthogonal reaction
Science (2000) 287, 2007
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Using the Staudinger ligation to activate luciferin
O
OCH3
PPh
Ph
+ N3 R'
N2O
OCH3
PPh
PhN R'
Aza-ylide
CH3OH
O
N
PPhPh
R'H2O
O
NH
PPh
PhO
R'
phosphine
Ligation product
S
N
S
N CO2H
OR
R = H light
R = not H no light
Nat Chem Biol (2005) 1, 13
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Using TPP cation to deliver reagents to mitochondria
M. Murphy, Biochimica et Biophysica Acta, 1777 (2008), 1028-1031Edward J. Gane, et al. Liver international (2010), 1019-1026
Cytosol
P X
30-60 mV
3-10 X
100-500 X Mitochondrion
180-200 mV
TPP
13
- MitoQ (CoQ10)
In Phase 2 was proved to protect against liver damageduring hepatitis C
O
O
O
O H
6-10
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Design of mitochondria-activated luciferin (MAL) probe
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MMP DEPENDENT ACCUMULATION MMP DEPENDENT RATE OF LUCIFERIN RELEASE
How to make luciferin uncaging MMP dependent?1. Take two reagents that release luciferin upon reaction
2. Target them to mitochondria
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15Potentiation of reaction rate – 105 fold Only mitochondrial uncaging is observed
The bioluminescent signal intensity depends on MMP
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Reaction rate enhancement
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𝑅 = 𝑇𝑃𝑃𝑇𝑃𝑃 ≈ 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 = 𝑇𝑃𝑃𝐶𝐿𝑇𝑃𝑃𝐶𝐿 = 10 ∆. ,𝜐𝜐 = 𝑘 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿𝑘 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿 = 𝑅 𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿𝐴𝑧𝑖𝑑𝑜𝑇𝑃𝑃 𝑇𝑃𝑃𝐶𝐿 = 𝑅 = 10 ∆. = 10 ∆. ,
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The bioluminescent signal intensity depends on MMP
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Intact mitochondria Depolarized mitochondria
High rate of luciferin releaseHigh signal
Low rate of luciferin releaseLow signal
FCCP, valinomycin
Valinomycin
Nigericin
FCCPNigericin
Hyperpolarized mitochondria
Very high rate of releaseExtremely high signal
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HT-1080 luc2 cells
With MAL depolarization is detectable down to 0.5 uM
Mitochondria activated luciferin (MAL) TMRM
Detection of valinomycin induced depolarization in cells
O
O
O
NN
vs
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HT-1080 luc2 cells
Mitochondria activated luciferin-3 (MAL3) TMRM
Detection of valinomycin induced depolarization in cells
O
O
O
NN
vs
Tota
l pho
ton
flux
[p/(c
m2 *s
r)]
RFU
Response of MAL3 assay is more non-linear
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HT-1080 luc2 cells
Mitochondria activated luciferin-3 (MAL3) TMRM
Detection of hyperpolarization in cells
O
O
O
NN
vs
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Monitoring mitochondrial potential in vivo