Purification and characterization of recombinant human mitochondrial proton-pumping nicotinamide nucleotide transhydrogenase.
Type: article
Abstract: The human mitochondrial nicotinamide nucleotide transhydrogenase (NNT) uses the proton motive force to drive hydride transfer from NADH to NADP+ and is a major contributor to the generation of mitochondrial NADPH. NNT plays a critical role in maintaining cellular redox balance. NNT-deficiency results in oxidative damage and its absence results in familial glucocorticoid deficiency. Recently it has also become clear that NNT is a tumor promoter whose presence in mouse models of non-small cell lung cancer results in enhanced tumor growth and aggressiveness. The presence of NNT mitigates the effects of oxidative stress and facilitates cancer cell proliferation, suggesting NNT-inhibition as a promising therapeutic strategy. The human NNT is a homodimer in which each subunit has a molecular weight of 114 kDa and 14 transmembrane spans. Here we report on the development of a system for isolating full-length recombinant human NNT using Escherichia coli. The purified enzyme is catalytically active, and the enzyme reconstituted into proteoliposomes pumps protons and generates a proton motive force capable of driving ATP synthesis by E. coli ATP synthase. The recombinant human NNT will facilitate structural and biochemical studies as well as provide a useful tool to develop and characterize potential anti-cancer therapeutics.
DOI: 10.48620/85121
Authors: Hong, Sangjin, Graf, Simone, von Ballmoos, Christoph, Gennis, Robert B
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6929326509
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