Plate-Based Measurement of Respiration by Isolated Mitochondria

Mookerjee, Shona A.; Quinlan, Casey L.; Wong, Hoi-Shan; Dighe, Pratiksha; Brand, Martin D.

doi:10.1007/978-1-4939-7831-1_17

Cited by 10 publications

(5 citation statements)

References 16 publications

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“…Rats were euthanised by CO 2 asphyxiation followed by bilateral thoracotomy. Mitochondria were isolated from rat hindlimbs in Chappell–Perry buffer (CP-1; 100 mM KCl, 50 mM Tris HCl, 2 mM EGTA, pH 7.4) at 4°C by standard procedures [ 46 ].…”

Section: Methodsmentioning

confidence: 99%

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Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions

Lerner

Watson

et al. 2023

Biochemical Journal

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Superoxide/hydrogen peroxide production by site IQ in complex I of the electron transport chain is conventionally assayed during reverse electron transport from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells and in vivo during presumed forward electron transport. Therefore, we tested whether site IQ generates S1QEL-sensitive superoxide/hydrogen peroxide during forward electron transport (site IQf), or alternatively, whether reverse electron transport and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQ can be equally great whether reverse electron transport or forward electron transport is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during forward electron transport is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQ in cells occurs during forward electron transport, and is S1QEL-sensitive.

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Section: Methodsmentioning

confidence: 99%

“…Mitochondrial respiration was assayed at 37°C using an Agilent Seahorse XFe-24 as described previously [ 46 ], using 12.5 mM G3P as substrate.…”

Section: Methodsmentioning

confidence: 99%

Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions

Lerner

Watson

et al. 2023

Biochemical Journal

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“…Heart mitochondria were isolated from hearts of NMR and C57BL/6J mice in ice-cold heart sucrose buffer (electronic supplementary material, methods). Regardless of species, the resultant supernatant was discarded, the final mitochondria pellets were suspended in ice-cold Mitochondrial Assay Solution and were kept at high concentration (~20 mg protein/mL) on ice until use according to Mookerjee et al [50].…”

Section: Mitochondria Isolationmentioning

confidence: 99%

Naked mole-rat and Damaraland mole-rat exhibit lower respiration in mitochondria, cellular and organismal levels

Yap¹,

Wong²,

Ramanathan³

et al. 2022

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…Both phenotypes were fully rescued by re-expression of PD-L1 in TRAMP-C2- Cd274 -/- cells (Figures 4D-E). Accordingly, bioenergetic calculations ( 42 ) show that PD-L1 reduced ATP production from OXPHOS while increasing ATP production from glycolysis (Figures 4F-G). Many key parameters of mitochondrial respiration were reduced in PD-L1-expressing cells (Figure S6B) which also had reduced mitochondrial content (Figures S6C-D), without impacting mitochondrial ROS (Figure S6E).…”

Section: Resultsmentioning

confidence: 91%

More than a ligand: PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I interferon pathway

Hodgins

Abou-Hamad

Hagerman

et al. 2022

Preprint

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Targeting the PD-1/PD-L1 axis has transformed the field of immune-oncology. While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced infection with oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 suppressed type I interferon by promoting a metabolic shift characterized by enhanced glucose uptake and glycolysis rate. Lactate generated from glycolysis was the key metabolite responsible for inhibiting type I interferon responses and enhancing oncolytic virus infection in PD-L1-expressing cells. In addition to adding mechanistic insight into PD-L1 intrinsic function and showing that PD-L1 has a broader impact on immunity and cancer biology besides acting as a ligand for PD-1, our results will also help guide the numerous efforts currently ongoing to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.Once sentence summaryPD-L1 promotes oncolytic virus efficacy.

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Plate-Based Measurement of Respiration by Isolated Mitochondria

Cited by 10 publications

References 16 publications

Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions

Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions

Naked mole-rat and Damaraland mole-rat exhibit lower respiration in mitochondria, cellular and organismal levels

More than a ligand: PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I interferon pathway

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