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Dennis R Winge - One of the best experts on this subject based on the ideXlab platform.
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Cox1 mutation abrogates need for Cox23 in cytochrome c oxidase biogenesis.
Microbial cell (Graz Austria), 2016Co-Authors: Richard Glenn C Dela Cruz, Mi Young Jeong, Dennis R WingeAbstract:Cox23 is a known conserved assembly factor for cytochrome c oxidase, although its role in cytochrome c oxidase (CcO) biogenesis remains unresolved. To gain additional insights into its role, we isolated spontaneous suppressors of the respiratory growth defect in cox23∆ yeast cells. We recovered independent colonies that propagated on glycerol/lactate medium for cox23∆ cells at 37°C. We mapped these mutations to the mitochondrial genome and specifically to COX1 yielding an I101F substitution. The I101F Cox1 allele is a gain-of-function mutation enabling yeast to respire in the absence of Cox23. CcO subunit steady-state levels were restored with the I101F Cox1 suppressor mutation and oxygen consumption and CcO activity were likewise restored. Cells harboring the mitochondrial genome encoding I101F Cox1 were used to delete genes for other CcO assembly factors to test the specificity of the Cox1 mutation as a suppressor of cox23∆ cells. The Cox1 mutant allele fails to support respiratory growth in yeast lacking COX17, Cox19, Coa1, Coa2, Cox14 or Shy1, demonstrating its specific suppressor activity for cox23∆ cells.
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Analysis of Leigh Syndrome Mutations in the Yeast SURF1 Homolog Reveals a New Member of the Cytochrome Oxidase Assembly Factor Family
Molecular and Cellular Biology, 2010Co-Authors: Megan Bestwick, Mi Young Jeong, Oleh Khalimonchuk, Hyung J. Kim, Dennis R WingeAbstract:Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F249T and Y344D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G137E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G137E Shy1 mutant phenocopied shy1Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G137E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX9C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Δ cells was conducted. Respiratory function of coa4Δ cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.
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mapping the functional interaction of sco1 and cox2 in cytochrome oxidase biogenesis
Journal of Biological Chemistry, 2008Co-Authors: Kevin Rigby, Oleh Khalimonchuk, Paul A Cobine, 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 Cytochrome Oxidase Biogenesis
The Journal of biological chemistry, 2008Co-Authors: Kevin Rigby, Oleh Khalimonchuk, Paul A Cobine, Dennis R WingeAbstract:Sco1 is implicated in the copper metallation of the Cu(A) 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 (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 sco1Delta 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 sco1Delta 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, Oleh Khalimonchuk, Paul A Cobine, 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.
Peep Palumaa - One of the best experts on this subject based on the ideXlab platform.
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Modulation of redox switches of copper chaperone COX17 by Zn(II) ions determined by new ESI MS-based approach.
Antioxidants & redox signaling, 2009Co-Authors: Kairit Zovo, Peep PalumaaAbstract:Abstract COX17, a copper chaperone for cytochrome-c oxidase, contains six conserved Cys residues and exists in three oxidative states, linked with two thiol-based redox switches. The first switch leads to formation of two disulfides and occurs upon transport of COX17 into mitochondrial intermembrane space (IMS). COX172S-S is retained in the IMS and is also a functional form of the protein, which can be further oxidized to COX173S-S. According to the midpoint redox potential values, COX17 can be partially oxidized in the cytosol, which might hinder its transport into IMS. We hypothesize that Zn(II) ions might protect cytosolic COX17 from oxidation. In order to get quantitative information about the modulatory effect of Zn(II) ions on redox switches in COX17, we have used ESI MS for determination of the midpoint potentials for redox couples of COX17: COX173S-S ↔ COX172S-S (Em1) and COX172S-S ↔ COX170S-S (Em2) in the presence of Zn(II). 10 μM Zn(II) ions shift the Em2 by 21 mV and Em1 by 15 mV to more positi...
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mitochondrial copper i transfer from COX17 to sco1 is coupled to electron transfer
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Lucia Banci, Simone Ciofibaffoni, Theodoros Hadjiloi, Manuele Martinelli, Ivano Bertini, Peep PalumaaAbstract:The human protein COX17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S–S bonds and two reduced cysteines (HCOX172S-S). HCOX172S-S is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCOX172S-S, i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCOX173S-S, which can be then reduced by glutathione to apoHCOX172S-S. The HSco1/HCOX172S-S redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper–electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein–protein complex, which is instead observed in the Cu(I)HCOX172S-S/HSco1 interaction.
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A structural-dynamical characterization of human COX17.
The Journal of biological chemistry, 2007Co-Authors: Lucia Banci, Manuele Martinelli, Simone Ciofi-baffoni, Ivano Bertini, Anna Janicka, Henryk Kozlowski, Peep PalumaaAbstract:Abstract Human COX17 is a key mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy transducing respiratory chain. A structural and dynamical characterization of human COX17 in its various functional metallated and redox states is presented here. The NMR solution structure of the partially oxidized COX17 (COX172S-S) consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds involving Cys25-Cys54 and Cys35-Cys44, preceded by a flexible and completely unstructured N-terminal tail. In human Cu(I)COX172S-S the copper(I) ion is coordinated by the sulfurs of Cys22 and Cys23, and this is the first example of a Cys-Cys binding motif in copper proteins. Copper(I) binding as well as the formation of a third disulfide involving Cys22 and Cys23 cause structural and dynamical changes only restricted to the metal-binding region. Redox properties of the disulfides of human COX17, here investigated, strongly support the current hypothesis that the unstructured fully reduced COX17 protein is present in the cytoplasm and enters the intermembrane space (IMS) where is then oxidized by Mia40 to COX172S-S, thus becoming partially structured and trapped into the IMS. COX172S-S is the functional species in the IMS, it can bind only one copper(I) ion and is then ready to enter the pathway of copper delivery to cytochrome c oxidase. The copper(I) form of COX172S-S has features specific for copper chaperones.
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Oxidative switches in functioning of mammalian copper chaperone COX17
The Biochemical journal, 2007Co-Authors: Anastassia Voronova, Wolfram Meyer-klaucke, Annette Rompel, Bernt Krebs, Jekaterina Kazantseva, Rannar Sillard, Thomas J. Meyer, Peep PalumaaAbstract:COX17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian COX17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian COX17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian COX170S–S binds co-operatively to four Cu+; COX172S–S, with two disulfide bridges, binds to one of either Cu+ or Zn2+; and COX173S–S, with three disulfide bridges, does not bind to any metal ions. The Em (midpoint redox potential) values for two redox couples of COX17, COX173S–S↔COX172S–S (Em1) and COX172S–S↔COX170S–S (Em2), were determined to be −197 mV and −340 mV respectively. The data indicate that an equilibrium exists in the cytosol between COX170S-S and COX172S–S, which is slightly shifted towards COX170S-S. In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards COX172S–S, enabling retention of COX172S–S in the IMS and leading to the formation of a biologically competent form of the COX17 protein, COX172S–S, capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4COX17 contains a Cu4S6-type copper–thiolate cluster, which may provide safe storage of an excess of copper ions.
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Oxidative switches in functioning of mammalian copper chaperone COX17
Biochemical Journal, 2007Co-Authors: Anastassia Voronova, Wolfram Meyer-klaucke, Thomas Meyer, Annette Rompel, Bernt Krebs, Jekaterina Kazantseva, Rannar Sillard, Peep PalumaaAbstract:COX17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian COX17 share six conserved Cys residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian COX17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian COX17 0S-S} binds cooperatively four Cu +} ions; COX17 2S-S} with two disulphide bridges binds one metal ion, either Cu +} or Zn 2+}; and COX17 3S-S} with three disulphides does not bind metals. The midpoint redox potentials for two redox couples of COX17: COX17 3S-S} ↔ COX17 2S-S} (E m1}) and COX17 2S-S} ↔ COX17 0S-S} (E m2}) were determined to be E m1} = - 197 mV and E m2} = - 340 mV, respectively. The data indicates that in the cytosol exists equilibrium between COX17 0S-S} and COX17 2S-S}, which is slightly shifted towards COX17 0S-S}. In the IMS, with higher redox potential the equilibrium is shifted towards COX17 2S-S}, enabling retention of COX17 2S-S} in IMS and leading to formation of biologically competent form of protein - COX17 2S-S}, capable of copper transfer to the copper chaperone Sco1. X-ray absorption spectroscopy determined that Cu 4}COX17 contains a Cu 4}S 6}-type copper-thiolate cluster, which may provide safe storage of excessive copper ions.
Alexander Tzagoloff - One of the best experts on this subject based on the ideXlab platform.
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COX23, a Homologue of COX17, Is Required for Cytochrome Oxidase Assembly
The Journal of biological chemistry, 2004Co-Authors: Mario H. Barros, Alisha Johnson, Alexander TzagoloffAbstract:Abstract Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency. The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase. Cox23p is homologous to COX17p, a water-soluble copper protein previously implicated in the maturation of the CuA center of cytochrome oxidase. The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid. Overexpression of COX17p by itself is not a sufficient condition to rescue the mutant phenotype. Cox23p, like COX17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins. Because Cox23p and COX17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with COX17p acting downstream of Cox23p.
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Purification, Characterization, and Localization of Yeast COX17p, a Mitochondrial Copper Shuttle
The Journal of biological chemistry, 1997Co-Authors: J. Beers, Alexander TzagoloffAbstract:Abstract COX17p was previously shown to be essential for the expression of cytochrome oxidase in Saccharomyces cerevisiae. In the present study COX17 has been placed under the control of the GAL10 promoter in an autonomously replicating plasmid. A yeast transformant harboring the high copy construct was used to purify COX17p to homogeneity. Purified COX17p contains 0.2–0.3 mol of copper per mol of protein. The molar copper content is increased to 1.8 after incubation of COX17p in the presence of a 6-fold molar excess of cuprous chloride under reduced conditions. An antibody against COX17p was obtained by immunization of rabbits with a carboxyl-terminal peptide coupled to bovine serum albumin. The antiserum detects COX17p in both the mitochondrial and soluble protein fractions of wild type yeast and of the transformant overexpressing COX17p. Exposure of intact mitochondria to hypotonic conditions causes most of COX17p to be released as a soluble protein indicating that the mitochondrial fraction of COX17p is localized in the intermembrane space. These results are consistent with the previously proposed function of COX17p, namely in providing cytoplasmic copper for mitochondrial utilization.
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sco1 and sco2 act as high copy suppressors of a mitochondrial copper recruitment defect in saccharomyces cerevisiae
Journal of Biological Chemistry, 1996Co-Authors: D. Moira Glerum, Andrey Shtanko, Alexander TzagoloffAbstract:Abstract C129/U1 is a respiratory defective mutant of Saccharomyces cerevisiae arrested in cytochrome oxidase assembly due to a mutation in COX17, a nuclear gene encoding a low molecular weight cytoplasmic protein proposed to function in mitochondrial copper recruitment. In the present study we show that the respiratory defect of C129/U1 is rescuable by two multicopy suppressors, SCO1 and SCO2. SCO1 was earlier reported to code for a mitochondrial inner membrane protein with an essential function in cytochrome oxidase assembly (Buchwald, P., Krummeck, G., and Rodel, G. (1991) Mol. Gen. Genet. 229, 413-420). SCO2 is a homologue of SCO1, whose product is also localized in the mitochondrial membrane but is not required for respiration. SCO1 also suppresses a COX17 null mutant, indicating that overexpression of Sco1p can compensate for the absence of COX17p. In contrast, neither copper, COX17 on a multicopy plasmid, or a combination of the two is able to restore respiration in sco1 mutants. Rescue of COX17 mutants by Sco1p suggests that this mitochondrial protein plays a role either in mitochondrial copper transport or insertion of copper into the active site of cytochrome oxidase. Although SCO2 can also partially restore respiratory growth in the COX17 null mutant, rescue in this case requires addition of copper to the growth medium. SCO2 does not suppress a sco1 null mutant, although it is able to partially rescue a sco1 point mutant. We interpret the ability of SCO2 to restore respiration in COX17, but not in sco1 mutants, to indicate that Sco1p and Sco2p have overlapping but not identical functions.
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characterization of COX17 a yeast gene involved in copper metabolism and assembly of cytochrome oxidase
Journal of Biological Chemistry, 1996Co-Authors: Moira D Glerum, Andrey Shtanko, Alexander TzagoloffAbstract:Abstract Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because COX17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the COX17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the COX17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, COX17p is a cytoplasmic protein. The cytoplasmic location of COX17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of COX17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a COX17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that COX17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.
Fuyi Wang - One of the best experts on this subject based on the ideXlab platform.
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Identification of binding sites of cisplatin to human copper chaperone protein COX17 by high-resolution FT-ICR-MS
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Wei Guo, Yao Zhao, Qun Luo, Qingwu Zhang, Jianan Liu, Shaoxiang Xiong, Fuyi WangAbstract:RATIONALE COX17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. METHODS The recombinant protein apo-COX172s-s , the functional state of COX17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated COX17 adducts, of which the platination sites were identified by high-resolution Fourier transform ion cyclotron tandem mass spectrometry (FT-ICR-MS/MS) through electron capture dissociation (ECD). RESULTS Primary FT-ICR-MS showed that mono-platinated COX17 adducts were the main products, and top-down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo-COX172s-s . CONCLUSIONS This is the first report for identification of the main binding sites of cisplatin to COX17 by top-down high-resolution mass spectrometry, providing direct evidence for the competitive coordination with COX17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how COX17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd.
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Identification of binding sites of cisplatin to human copper chaperone protein COX17 by high-resolution FT-ICR-MS.
Rapid communications in mass spectrometry : RCM, 2016Co-Authors: Wei Guo, Yao Zhao, Qun Luo, Qingwu Zhang, Jianan Liu, Shaoxiang Xiong, Fuyi WangAbstract:COX17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. The recombinant protein apo-COX172s-s , the functional state of COX17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated COX17 adducts, of which the platination sites were identified by high-resolution Fourier transform ion cyclotron tandem mass spectrometry (FT-ICR-MS/MS) through electron capture dissociation (ECD). Primary FT-ICR-MS showed that mono-platinated COX17 adducts were the main products, and top-down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo-COX172s-s . This is the first report for identification of the main binding sites of cisplatin to COX17 by top-down high-resolution mass spectrometry, providing direct evidence for the competitive coordination with COX17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how COX17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Wei Guo - One of the best experts on this subject based on the ideXlab platform.
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Identification of binding sites of cisplatin to human copper chaperone protein COX17 by high-resolution FT-ICR-MS
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Wei Guo, Yao Zhao, Qun Luo, Qingwu Zhang, Jianan Liu, Shaoxiang Xiong, Fuyi WangAbstract:RATIONALE COX17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. METHODS The recombinant protein apo-COX172s-s , the functional state of COX17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated COX17 adducts, of which the platination sites were identified by high-resolution Fourier transform ion cyclotron tandem mass spectrometry (FT-ICR-MS/MS) through electron capture dissociation (ECD). RESULTS Primary FT-ICR-MS showed that mono-platinated COX17 adducts were the main products, and top-down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo-COX172s-s . CONCLUSIONS This is the first report for identification of the main binding sites of cisplatin to COX17 by top-down high-resolution mass spectrometry, providing direct evidence for the competitive coordination with COX17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how COX17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd.
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A comparative study on the interactions of human copper chaperone COX17 with anticancer organoruthenium(II) complexes and cisplatin by mass spectrometry.
Journal of inorganic biochemistry, 2016Co-Authors: Wei Guo, Linhong Zhao, Yangzhong Liu, Yao Zhao, Qun Luo, Yuanyuan Wang, Qingwu ZhangAbstract:Herein we report investigation of the interactions between anticancer organoruthenium complexes, [(η(6)-arene)Ru(en)(Cl)]PF6 (en=ethylenediamine, arene=p-cymene (1) or biphenyl (2)), and the human copper chaperone protein COX17 by mass spectrometry with cisplatin as a reference. The electrospray ionization mass spectrometry (ESI-MS) results indicate much weaker binding of the ruthenium complexes than that of cisplatin to apo-COX172s-s, the functional state of COX17. Up to tetra-platinated COX17 adducts were identified while only mono-ruthenated and a little amount of di-ruthenated COX17 adducts were detected even for the reactions with 10-fold excess of the Ru complexes. However, ESI-MS analysis coupled with liquid chromatography of tryptic digests of metalated proteins identified only three platination sites as Met4, Cys27 and His47 residues, possibly due to the lower abundance or facile dissociation of Pt bindings at other sites. Complexes 1 and 2 were found to bind to the same three residues with Met4 as the major site. Inductively coupled plasma mass spectrometry results revealed that ~7mol Pt binding to 1mol apo-COX172s-s molecules, compared to only 0.17 (1) and 0.10 (2) mol Ru to 1mol apo-COX172s-s. This is in line with the circular dichroism results that much larger unfolding extent of α-helix of apo-COX172s-s was observed upon cisplatin binding than that upon organoruthenium bindings. These results collectively indicate that COX17 might not participate in the action of these anticancer organoruthenium complexes, and further verify the distinct anticancer mechanism of the organoruthenium(II) complexes from cisplatin.
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Identification of binding sites of cisplatin to human copper chaperone protein COX17 by high-resolution FT-ICR-MS.
Rapid communications in mass spectrometry : RCM, 2016Co-Authors: Wei Guo, Yao Zhao, Qun Luo, Qingwu Zhang, Jianan Liu, Shaoxiang Xiong, Fuyi WangAbstract:COX17 is a key copper chaperone protein responsible for delivery of cuprous ions to mitochondria and has been demonstrated to be involved in the anticancer action of cisplatin. However, the binding sites of the drug to the protein have not yet been directly identified. The recombinant protein apo-COX172s-s , the functional state of COX17 transferring Cu(I), was reacted with an excess of cisplatin to produce platinated COX17 adducts, of which the platination sites were identified by high-resolution Fourier transform ion cyclotron tandem mass spectrometry (FT-ICR-MS/MS) through electron capture dissociation (ECD). Primary FT-ICR-MS showed that mono-platinated COX17 adducts were the main products, and top-down MS/MS results indicated that cisplatin bound to the Cys26 or Cys27 residue which is the binding site of cuprous ions in apo-COX172s-s . This is the first report for identification of the main binding sites of cisplatin to COX17 by top-down high-resolution mass spectrometry, providing direct evidence for the competitive coordination with COX17 of cisplatin and cuprous ions. These findings will also be helpful to understand further how COX17 facilitates cisplatin accumulation in mitochondria, and how cisplatin disturbs the transportation of cuprous ions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
