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Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

ABSTRACT: Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.

SUBMITTER: Kmita K

PROVIDER: S-EPMC4426432 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

Kmita Katarzyna K Wirth Christophe C Warnau Judith J Guerrero-Castillo Sergio S Hunte Carola C Hummer Gerhard G Kaila Ville R I VR Zwicker Klaus K Brandt Ulrich U Zickermann Volker V

Proceedings of the National Academy of Sciences of the United States of America 20150420 18

Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or m ...[more]

PMID: 25902503

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Project description:RATIONALE:Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. OBJECTIVES:To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. METHODS AND RESULTS:Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. CONCLUSIONS:Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Project description:Background/aimDysregulation of mitochondrial pathways is implicated in several diseases, including cancer. Notably, mitochondrial respiration and mitochondrial biogenesis are favored in some invasive cancer cells, such as osteosarcoma. Hence, the aim of the current work was to investigate the effects of 2-methoxyestradiol (2-ME), a potent anticancer agent, on the mitochondrial biogenesis of osteosarcoma cells.Materials and methodsHighly metastatic osteosarcoma 143B cells were treated with 2-ME separately or in combination with L-lactate, or with the solvent (non-treated control cells). Protein levels of α-syntrophin and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) were determined by western blotting. Impact of 2-ME on mitochondrial mass, regulation of cytochrome c oxidase I (COXI) expression, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) was determined by immunofluorescence analyses. Inhibition of sirtuin 3 (SIRT3) activity by 2-ME was investigated by fluorescence assay and also, using molecular docking and molecular dynamics simulations.ResultsL-lactate induced mitochondrial biogenesis pathway via up-regulation of COXI. 2-ME inhibited mitochondrial biogenesis via regulation of PGC-1α, COXI, and SIRT3 in a concentration-dependent manner as a consequence of nuclear recruitment of neuronal nitric oxide synthase and nitric oxide generation. It was also proved that 2-ME inhibited SIRT3 activity by binding to both the canonical and allosteric inhibitor binding sites. Moreover, regardless of the mitochondrial biogenesis pathway, 2-ME affected the expression of SDHA.ConclusionHerein, mitochondrial biogenesis pathway regulation and SDHA were presented as novel targets of 2-ME, and moreover, 2-ME was demonstrated as a potent inhibitor of SIRT3. L-lactate was confirmed to exert pro-carcinogenic effects on osteosarcoma cells via the induction of the mitochondrial biogenesis pathway. Thus, L-lactate level may be considered as a prognostic biomarker for osteosarcoma.

Dataset Information

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

Publications

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

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