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Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

ABSTRACT: Succinate dehydrogenase (SDH) requires a covalent addition of FAD for catalytic function.Mutational analyses of Sdh1 implicate C-terminal region Arg residues involvement in covalent flavinylation and SDH assembly.SDH assembly is dependent on FAD binding to Sdh1 but not covalent binding.These results document the basis for the SDH deficiency and pathology seen with mutations in human Sdh1. The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ~70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg(582) in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg(638) compromises SDH function only when present in combination with a Cys(630) substitution. Mutations of either Arg(582) or Arg(638)/Cys(630) do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC3504780 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

Kim Hyung J HJ Jeong Mi-Young MY Na Un U Winge Dennis R DR

The Journal of biological chemistry 20121007 48

<h4>Background</h4>Succinate dehydrogenase (SDH) requires a covalent addition of FAD for catalytic function.<h4>Results</h4>Mutational analyses of Sdh1 implicate C-terminal region Arg residues involvement in covalent flavinylation and SDH assembly.<h4>Conclusion</h4>SDH assembly is dependent on FAD binding to Sdh1 but not covalent binding.<h4>Significance</h4>These results document the basis for the SDH deficiency and pathology seen with mutations in human Sdh1. The enzymatic function of succina ...[more]

PMID: 23043141

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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

Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

Publications

Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

Similar Datasets

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