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Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

ABSTRACT: The adenine nucleotide translocase (ANT) of the mitochondrial inner membrane exchanges ADP for ATP. Mitochondria were isolated from human vastus lateralis muscle (n?=?9). Carboxyatractyloside titration of O2 consumption rate (Jo) at clamped [ADP] of 21??M gave ANT abundance of 0.97?±?0.14?nmol ANT/mg and a flux control coefficient of 82%?±?6%. Flux control fell to 1%?±?1% at saturating (2?mM) [ADP]. The KmADP for Jo was 32.4?±?1.8??M. In terms of the free (-3) ADP anion this KmADP was 12.0?±?0.7??M. A novel luciferase-based assay for ATP production gave KmADP of 13.1?±?1.9 ?M in the absence of ATP competition. The free anion KmADP in this case was 2.0?±?0.3??M. Targeted proteomic analyses showed significant acetylation of ANT Lysine23 and that ANT1 was the most abundant isoform. Acetylation of Lysine23 correlated positively with KmADP, r?=?0.74, P?=?0.022. The findings underscore the central role played by ANT in the control of oxidative phosphorylation, particularly at the energy phosphate levels associated with low ATP demand. As predicted by molecular dynamic modeling, ANT Lysine23 acetylation decreased the apparent affinity of ADP for ANT binding.

SUBMITTER: Willis WT

PROVIDER: S-EPMC6919326 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

Willis W T WT Miranda-Grandjean D D Hudgens J J Willis E A EA Finlayson J J De Filippis E A EA Zapata Bustos R R Langlais P R PR Mielke C C Mandarino L J LJ

Archives of biochemistry and biophysics 20180410

The adenine nucleotide translocase (ANT) of the mitochondrial inner membrane exchanges ADP for ATP. Mitochondria were isolated from human vastus lateralis muscle (n = 9). Carboxyatractyloside titration of O2 consumption rate (Jo) at clamped [ADP] of 21 μM gave ANT abundance of 0.97 ± 0.14 nmol ANT/mg and a flux control coefficient of 82% ± 6%. Flux control fell to 1% ± 1% at saturating (2 mM) [ADP]. The KmADP for Jo was 32.4 ± 1.8 μM. In terms of the free (-3) AD ...[more]

PMID: 29653079

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Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Dataset Information

Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

Publications

Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

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