Physical interaction and functional studies of human SCO1, a mitochondrial metallochaperone

Cytochrome c oxidase (COX) is a multimeric protein complex embedded in the inner mitochondrial membrane that contributes to the electrochemical potential ultimately required for adenosine triphosphate (ATP) synthesis. Synthesis of Cytochrome c Oxidase 1 (SCO1) and SCO2 are two of many accessory proteins that facilitate the assembly of individual COX structural subunits into a functional holoenzyme complex. SCO1 and SCO2 also function to regulate cellular copper homeostasis. Both of these functions require that SCO proteins collaborate with several interacting partners. ¬With few exceptions, however, their protein partners have yet to be identified and, as a consequence, we lack mechanistic insight into SCO protein function. To address this gap in our knowledge, I used physical methods to identify interacting partners of SCO1. Physical interactions between potential interacting partners of endogenous SCO1 or overexpressed SCO1-FLAG were stabilized with a chemical crosslinker, and the protein complexes were purified using the appropriate primary antibody. Mass spectrometric analysis of the eluates provided two lists of potential interacting partners that were subsequently filtered by gene ontology function and mitochondrial localization. The final list was comprised of 85 proteins, which were rank ordered based on total peptide counts per protein. Three of these candidate interacting partners were then further characterized; COX20, tricarboxylate transport protein (SLC25A1) and mitochondrial 2-oxoglutarate/malate carrier (SLC25A11). I first confirmed the authenticity of the observed interactions by conducting the reciprocal co-immunoprecipitations in the absence of a chemical crosslinker. I then investigated the effect of transient knockdown of each protein on the abundance of COX II, a direct proxy of total COX content. Knockdown of COX20 and SLC25A1 reduced COX II levels in SCO1 and SCO2 patient fibroblasts, but did not affect COX II abundance in control cells. SLC25A11 knockdown lowered COX II abundance in both control and SCO patient cells. These data suggest that the ability of SCO1 to interact with each of these proteins somehow facilitates the maturation of the CuA site of COX II

Elesclomol restores mitochondrial function in genetic models of copper deficiency

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 115 (2018): 8161-8166, doi:10.1073/pnas.1806296115.Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.This work was supported by National Institutes of Health Awards R01GM111672 (to V.M.G.), R01 DK110195 (to B.-E.K.), and DK 44464 (to J.D.G.); Welch Foundation Grant A-1810 (to V.M.G.); and Canadian Institutes of Health Research Operating Grant MOP 133562 (to S.C.L.)

COA6 is structurally tuned to function as a thiol-disulfide oxidoreductase in copper delivery to mitochondrial cytochrome c oxidase

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coilhelix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.Fil: Soma, Shivatheja. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Naik, Mandar T. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Naik, Mandar T. Brown University. Department of Molecular Pharmacology, Physiology, and Biotechnology; United States.Fil: Boulet, Aren. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Roesler, Anna A. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Dziuba, Nathaniel. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. University of Calcutta. Department of Biochemistry; India.Fil: Yu, Qinhong. University of California. Department of Chemistry; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Chemistry; United States.Fil: Ames, James B. University of California. Department of Chemistry; United States.Fil: Leary, Scot C. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Gohil, Vishal M. Texas A&M University. Department of Biochemistry and Biophysics; United States

The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

Human SCO1 fulfills essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper (Cu) homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1(-/-) mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis

Mitochondrial dysfunction reactivates α-fetoprotein expression that drives copper-dependent immunosuppression in mitochondrial disease models

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by alpha-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that alpha-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that alpha-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.Peer reviewe

Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase

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