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Scot C Leary - One of the best experts on this subject based on the ideXlab platform.
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the mitochondrial metallochaperone SCO1 maintains ctr1 at the plasma membrane to preserve copper homeostasis in the murine heart
Human Molecular Genetics, 2017Co-Authors: Zakery N Baker, Paul A Cobine, Aren Boulet, Amzad Hossain, Amr El M Zawily, Kimberly Jett, Glen F Tibbits, Michael J Petris, Scot C LearyAbstract:SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated SCO1hrt/hrt and SCO1stm/stm mice in which SCO1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived SCO1stm/stm mice. Interestingly, the reduction in copper content of SCO1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in SCO1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where SCO1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.
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human cox20 cooperates with SCO1 and sco2 to mature cox2 and promote the assembly of cytochrome c oxidase
Human Molecular Genetics, 2014Co-Authors: Myriam Bourens, Scot C Leary, Aren Boulet, Antoni BarrientosAbstract:Cytochrome c oxidase (CIV) deficiency is one of the most common respiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleus-encoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here, we have used small interference RNA and transcription activator-like effector nucleases (TALENs) technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into early CIV subassemblies. Immunoprecipitation assays using a stable COX20 knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction between COX20 and newly synthesized COX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps of COX2 maturation, stabilizing the newly synthesized protein and presenting COX2 to its metallochaperone module, which in turn facilitates the incorporation of mature COX2 into the CIV assembly line.
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novel mutations in SCO1 as a cause of fatal infantile encephalopathy and lactic acidosis
Human Mutation, 2013Co-Authors: Scot C Leary, Florin Sasarman, Paul A Cobine, Hana Antonicka, Woranontee Weraarpachai, Garry K Brown, Ruth M BrownAbstract:: Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.
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cox19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates atp7a mediated cellular copper efflux
Molecular Biology of the Cell, 2013Co-Authors: Scot C Leary, Tamiko Nishimura, Paul A Cobine, Robert M Verdijk, Ronald R De Krijger, Mark A Tarnopolsky, Dennis R Winge, Eric A ShoubridgeAbstract:SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.
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a targetable fluorescent sensor reveals that copper deficient SCO1 and sco2 patient cells prioritize mitochondrial copper homeostasis
Journal of the American Chemical Society, 2011Co-Authors: Sheel C Dodani, Scot C Leary, Paul A Cobine, Dennis R Winge, Christopher J ChangAbstract:We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a glob...
Dennis R Winge - One of the best experts on this subject based on the ideXlab platform.
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Evidence for a Pro-oxidant Intermediate in the Assembly of
2020Co-Authors: Oleh Khalimonchuk, Amanda J. Bird, Dennis R WingeAbstract:The hydrogen peroxide sensitivity of cells lacking two proteins, SCO1 and Cox11, important in the assembly of cytochrome c oxidase (CcO), is shown to arise from the transient accumulation of a pro-oxidant heme A-Cox1 stalled intermediate. The peroxide sensitivity of these cells is abrogated by a reduction in either Cox1 expression or heme A formation but exacerbated by either enhanced Cox1 expression or heme A production arising from overexpression of COX15. SCO1 and Cox11 are implicated in the formation of the CuA and CuB sites of CcO, respectively. The respective wild-type genes suppress the peroxide sensitivities of SCO1 and cox11 cells, but no cross-complementation is seen with noncognate genes. Copper-binding mutant alleles of SCO1 and Cox11 that are nonfunctional in promoting the assembly of CcO are functional in suppressing the peroxide sensitivity of their respective null mutants. Likewise, human SCO1 that is nonfunctional in yeast CcO assembly is able to suppress the peroxide sensitivity of yeast SCO1 cells. Thus, a disconnect exists between the respiratory capacity of cells and hydrogen peroxide sensitivity. Hydrogen peroxide sensitivity of SCO1 and cox11 cells is abrogated by overexpression of a novel mitochondrial ATPase Afg1 that promotes the degradation of CcO mitochondrially encoded subunits. Studies on the hydrogen peroxide sensitivity in CcO assembly mutants reveal new aspects of the CcO assembly process.
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cox19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates atp7a mediated cellular copper efflux
Molecular Biology of the Cell, 2013Co-Authors: Scot C Leary, Tamiko Nishimura, Paul A Cobine, Robert M Verdijk, Ronald R De Krijger, Mark A Tarnopolsky, Dennis R Winge, Eric A ShoubridgeAbstract:SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.
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a targetable fluorescent sensor reveals that copper deficient SCO1 and sco2 patient cells prioritize mitochondrial copper homeostasis
Journal of the American Chemical Society, 2011Co-Authors: Sheel C Dodani, Scot C Leary, Paul A Cobine, Dennis R Winge, Christopher J ChangAbstract:We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a glob...
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mapping the functional interaction of SCO1 and cox2 in cytochrome oxidase biogenesis
Journal of Biological Chemistry, 2008Co-Authors: Kevin Rigby, Paul A Cobine, Oleh Khalimonchuk, Dennis R WingeAbstract:Abstract SCO1 is implicated in the copper metallation of the CuA site in Cox2 of cytochrome oxidase. The structure of SCO1 in the metallated and apo-conformers revealed structural dynamics primarily in an exposed region designated loop 8. The structural dynamics of loop 8 in SCO1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif 217KKYRVYF223 on the leading edge of this loop are shown presently to be important for yeast SCO1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant SCO1 proteins failed to restore respiratory growth or cytochrome oxidase activity in SCO1Δ cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-SCO1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient SCO1-Cox2 interaction, the mutant SCO1 molecules appear compromised in a function with Cox2. The mutants failed to suppress the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of SCO1Δ cells. These studies implicate different surfaces on SCO1 for interaction or function with Cox17 and Cox2.
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mapping the functional interaction of SCO1 and cox2 in
2008Co-Authors: Kevin Rigby, Paul A Cobine, Oleh Khalimonchuk, Dennis R WingeAbstract:SCO1 is implicated in the copper metallation of the CuA site in Cox2 of cytochrome oxidase. The structure of SCO1 in the met- allated and apo-conformers revealed structural dynamics pri- marily in an exposed region designated loop 8. The structural dynamics of loop 8 in SCO1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif 217 KKYRVYF 223 on the leading edge of this loop are shown presently to be important for yeast SCO1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant SCO1 proteins failed to restore respiratory growth or cytochrome oxidase activity in SCO1 cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-SCO1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient SCO1-Cox2 interaction, the mutant SCO1 molecules appear com- promised in a function with Cox2. The mutants failed to sup- press the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of SCO1 cells. These studies implicate dif- ferent surfaces on SCO1 for interaction or function with Cox17 and Cox2.
Eric A Shoubridge - One of the best experts on this subject based on the ideXlab platform.
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cox19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates atp7a mediated cellular copper efflux
Molecular Biology of the Cell, 2013Co-Authors: Scot C Leary, Tamiko Nishimura, Paul A Cobine, Robert M Verdijk, Ronald R De Krijger, Mark A Tarnopolsky, Dennis R Winge, Eric A ShoubridgeAbstract:SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.
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human sco2 is required for the synthesis of co ii and as a thiol disulphide oxidoreductase for SCO1
Human Molecular Genetics, 2009Co-Authors: Scot C Leary, Florin Sasarman, Tamiko Nishimura, Eric A ShoubridgeAbstract:Human SCO1 and SCO2 code for essential metallochaperones with ill-defined functions in the biogenesis of the Cu A site of cytochrome c oxidase subunit II (CO II). Here, we have used patient cell lines to investigate the specific roles of each SCO protein in this pathway. By pulse-labeling mitochondrial translation products, we demonstrate that the synthesis of CO II is reduced in SCO2, but not in SCO1, cells. Despite this biosynthetic defect, newly synthesized CO II is more stable in SCO2 cells than in control cells. RNA i -mediated knockdown of mutant SC02 abolishes CO II labeling in the translation assay, whereas knockdown of mutant SCO1 does not affect CO II synthesis. These results indicate that SCO2 acts upstream of SCO1, and that it is indispensable for CO II synthesis. The subsequent maturation of CO II is contingent upon the formation of a complex that includes both SCO proteins, each with a functional CxxxC copper-coordinating motif. In control cells, the cysteines in this motif in SC01 exist as a mixed population comprised of oxidized disulphides and reduced thiols; however, the relative ratio of oxidized to reduced cysteines in SC01 is perturbed in cells from both SCO backgrounds. Overexpression of wild-type SC02, or knockdown of mutant SCO2, in SCO2 cells alters the ratio of oxidized to reduced cysteines in SCO1, suggesting that SC02 acts as a thiol-disulphide oxidoreductase to oxidize the copper-coordinating cysteines in SCO1 during CO II maturation. Based on these data we present a model in which each SCO protein fulfills distinct, stage-specific functions during CO II synthesis and Cu A site maturation.
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the p174l mutation in human SCO1 severely compromises cox17 dependent metallation but does not impair copper binding
Journal of Biological Chemistry, 2006Co-Authors: Paul A Cobine, Scot C Leary, Florin Sasarman, Eric A Shoubridge, Yihchern Horng, Fabien Pierrel, Dennis R WingeAbstract:Abstract SCO1 is a metallochaperone that is required for copper delivery to the CuA site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human SCO1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L SCO1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast SCO1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in SCO1Δ yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of SCO1, as well as the subsequent failure of CuA site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.
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human SCO1 and sco2 function as copper binding proteins
Journal of Biological Chemistry, 2005Co-Authors: Yihchern Horng, Scot C Leary, Eric A Shoubridge, Paul A Cobine, Fiona B J Young, Graham N George, Dennis R WingeAbstract:Abstract The function of human SCO1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human SCO1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human SCO1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast SCO1, are also critical for in vivo function of human SCO1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast SCO1 abrogated the Cu(II) visible transitions and in yeast SCO1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function.
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human SCO1 and sco2 have independent cooperative functions in copper delivery to cytochrome c oxidase
Human Molecular Genetics, 2004Co-Authors: Scot C Leary, Michaela Jaksch, Brett A Kaufman, Guy Hellen Guercin, Andre Mattman, Giovanna Pellecchia, Eric A ShoubridgeAbstract:: Human SCO1 and SCO2 are paralogous genes that code for metallochaperone proteins with essential, but poorly understood, roles in copper delivery to cytochrome c oxidase (COX). Mutations in these genes produce tissue-specific COX deficiencies associated with distinct clinical phenotypes, although both are ubiquitously expressed. To investigate the molecular function of the SCO proteins, we characterized the mitochondrial copper delivery pathway in SCO1 and SCO2 patient backgrounds. Immunoblot analysis of patient cell lines showed reduced levels of the mutant proteins, resulting in a defect in COX assembly, and the appearance of a common assembly intermediate. Overexpression of the metallochaperone COX17 rescued the COX deficiency in SCO2 patient cells but not in SCO1 patient cells. Overexpression of either wild-type SCO protein in the reciprocal patient background resulted in a dominant-negative phenotype, suggesting a physical interaction between SCO1 and SCO2. Chimeric proteins, constructed from the C-terminal copper-binding and N-terminal matrix domains of the two SCO proteins failed to complement the COX deficiency in either patient background, but mapped the dominant-negative phenotype in the SCO2 background to the N-terminal domain of SCO1, the most divergent part of the two SCO proteins. Our results demonstrate that the human SCO proteins have non-overlapping, cooperative functions in mitochondrial copper delivery. Size exclusion chromatography suggests that both the proteins function as homodimers. We propose a model in which COX17 delivers copper to SCO2, which in turn transfers it directly to the CuA site at an early stage of COX assembly in a reaction that is facilitated by SCO1.
Gerhard Rödel - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial Sco proteins are involved in oxidative stress defense.
Redox biology, 2018Co-Authors: Aslihan Ekim Kocabey, Luise Kost, Maria Gehlhar, Gerhard RödelAbstract:Abstract Members of the evolutionary conserved Sco protein family have been intensively studied regarding their role in the assembly of the mitochondrial cytochrome c oxidase. However, experimental and structural data, specifically the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. In our study, we addressed this putative function of Sco proteins using Saccharomyces cerevisiae as a model system. Like many eukaryotes, this yeast possesses two SCO homologs (SCO1 and SCO2). Mutants bearing a deletion of either of the two genes are not affected in their growth under oxidative stress. However, the concomitant deletion of the SOD1 gene encoding the superoxide dismutase 1 resulted in a distinct phenotype: double deletion strains lacking SCO1 or SCO2 and SOD1 are highly sensitive to oxidative stress and show dramatically increased ROS levels. The respiratory competent double deletion strain Δsco2Δsod1 paved the way to investigate the putative antioxidant function of SCO homologs apart from their role in respiration by complementation analysis. Sco homologs from Drosophila, Arabidopsis, human and two other yeast species were integrated into the genome of the double deletion mutant and the transformants were analyzed for their growth under oxidative stress. Interestingly, all homologs except for Kluyveromyces lactis K07152 and Arabidopsis thaliana HCC1 were able to complement the phenotype, indicating their role in oxidative stress defense. We further applied this complementation-based system to investigate whether pathogenic point mutations affect the putative antioxidant role of hSco2. Surprisingly, all of the mutant alleles failed to restore the ROS-sensitivity of the Δsco2Δsod1 strain. In conclusion, our data not only provide clear evidence for the function of Sco proteins in oxidative stress defense but also offer a valuable tool to investigate this role for other homologous proteins.
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hcc1 the arabidopsis homologue of the yeast mitochondrial copper chaperone SCO1 is essential for embryonic development
Journal of Experimental Botany, 2011Co-Authors: Iris Steinebrunner, Marlen Landschreiber, Udo Krausebuchholz, Juliane Teichmann, Gerhard RödelAbstract:The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to SCO1p, the respiratory-deficient yeast SCO1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from SCO1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.
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The P(174)L mutation in the human hSCO1 gene affects the assembly of cytochrome c oxidase.
Biochemical and Biophysical Research Communications, 2000Co-Authors: Claudia Paret, Anja Lode, Udo Krause-buchholz, Gerhard RödelAbstract:Abstract Mutations of the yeast SCO1 gene result in impaired COX assembly. Recently, heterozygous mutations in the human homologue hSCO1 have been reported in infants suffering from neonatal ketoacidotic coma and isolated COX deficiency (Valnot et al., 2000). One of the hSCO1 alleles harboured a frame shift mutation resulting in a premature stop codon, the other a missense mutation leading to a substitution of proline(174) by leucine. This position is next to the essential CXXXC motif, which is conserved in all SCO1p homologues. We used chimeric proteins with the amino-terminal portion derived from yeast SCO1p and carboxy-terminal portion including the CXXXC motif from the human hSCO1p to provide experimental evidence for the pathogenic nature of the P(174)L mutation. These chimeras are able to complement yeast SCO1 null mutants. Introduction of the P(174)L mutation affects the function of these chimeric proteins severely, as shown by impaired COX assembly and loss of COX activity.
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mitochondrial copper metabolism in yeast mutational analysis of SCO1p involved in the biogenesis of cytochrome c oxidase
Current Genetics, 1999Co-Authors: Anja Rentzsch, G Krummeckweiss, A Hofer, A Bartuschka, Kai Ostermann, Gerhard RödelAbstract:Saccharomyces cerevisiae SCO1p is believed to be involved in the transfer of copper from the carrier Cox17p to the mitochondrial cytochrome c oxidase subunits 1 and 2. We here report on the results of a mutational analysis of SCO1p. The two cysteine residues of a potential metal-binding motif (CxxxC) are essential for protein function as shown by their substitution by alanines. Chimeras consisting of SCO1p and its homolog S. cerevisiae Sco2p restrict the specificity of SCO1p function to the N-terminal half of the protein. A candidate region for conferring specificity on SCO1p is a stretch of hydrophobic amino acids, which act as a membrane anchor. In line with this suggestion is the result that alterations of individual amino acids within this region impair SCO1p function.
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immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane
Molecular Genetics and Genomics, 1991Co-Authors: Paul Buchwald, Gaby Krummeck, Gerhard RödelAbstract:The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a β-Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho0 strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.
Paul A Cobine - One of the best experts on this subject based on the ideXlab platform.
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the mitochondrial metallochaperone SCO1 maintains ctr1 at the plasma membrane to preserve copper homeostasis in the murine heart
Human Molecular Genetics, 2017Co-Authors: Zakery N Baker, Paul A Cobine, Aren Boulet, Amzad Hossain, Amr El M Zawily, Kimberly Jett, Glen F Tibbits, Michael J Petris, Scot C LearyAbstract:SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated SCO1hrt/hrt and SCO1stm/stm mice in which SCO1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived SCO1stm/stm mice. Interestingly, the reduction in copper content of SCO1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in SCO1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where SCO1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.
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the mitochondrial metallochaperone SCO1 is required to sustain expression of the high affinity copper transporter ctr1 and preserve copper homeostasis
Cell Reports, 2015Co-Authors: Chris Hlynialuk, Paul A Cobine, Aren Boulet, Binbing Ling, Zakery N Baker, Lisa D Yu, Amzad Hossain, Amr El M Zawily, Pamela J McfieAbstract:Summary 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.
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novel mutations in SCO1 as a cause of fatal infantile encephalopathy and lactic acidosis
Human Mutation, 2013Co-Authors: Scot C Leary, Florin Sasarman, Paul A Cobine, Hana Antonicka, Woranontee Weraarpachai, Garry K Brown, Ruth M BrownAbstract:: Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.
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cox19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates atp7a mediated cellular copper efflux
Molecular Biology of the Cell, 2013Co-Authors: Scot C Leary, Tamiko Nishimura, Paul A Cobine, Robert M Verdijk, Ronald R De Krijger, Mark A Tarnopolsky, Dennis R Winge, Eric A ShoubridgeAbstract:SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.
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a targetable fluorescent sensor reveals that copper deficient SCO1 and sco2 patient cells prioritize mitochondrial copper homeostasis
Journal of the American Chemical Society, 2011Co-Authors: Sheel C Dodani, Scot C Leary, Paul A Cobine, Dennis R Winge, Christopher J ChangAbstract:We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a glob...