The Experts below are selected from a list of 438666 Experts worldwide ranked by ideXlab platform
Dennis R Winge - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
Oligomerization of Heme o Synthase in Cytochrome Oxidase Biogenesis Is Mediated by Cytochrome Oxidase Assembly Factor Coa2
The Journal of biological chemistry, 2012Co-Authors: Oleh Khalimonchuk, Hyung Ki Kim, Talina Watts, Xochitl Pérez-martínez, Dennis R WingeAbstract:The synthesis of the heme a cofactor used in cytochrome c oxidase (CcO) is dependent on the sequential action of heme o synthase (COX10) and heme a synthase (Cox15). The active state of COX10 appears to be a homo-oligomeric complex, and formation of this complex is dependent on the newly synthesized CcO subunit Cox1 and the presence of an early Cox1 assembly intermediate. COX10 multimerization is triggered by progression of Cox1 from the early assembly intermediate to downstream intermediates. The CcO assembly factor Coa2 appears important in coupling the presence of newly synthesized Cox1 to COX10 oligomerization. Cells lacking Coa2 are impaired in COX10 complex formation as well as the formation of a high mass Cox15 complex. Increasing Cox1 synthesis in coa2Δ cells restores respiratory function if COX10 protein levels are elevated. The C-terminal segment of Cox1 is important in triggering COX10 oligomerization. Expression of the C-terminal 54 residues of Cox1 appended to a heterologous matrix protein leads to efficient COX10 complex formation in coa2Δ cells, but it fails to induce Cox15 complex formation. The state of COX10 was evaluated in mutants, which predispose human patients to CcO deficiency and the neurological disorder Leigh syndrome. The presence of the D336V mutation in the yeast COX10 backbone results in a catalytically inactive enzyme that is fully competent to oligomerize. Thus, COX10 oligomerization and catalytic activation are separate processes and can be uncoupled.
-
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.
-
The role of Coa2 in hemylation of yeast Cox1 revealed by its genetic interaction with COX10.
Molecular and Cellular Biology, 2010Co-Authors: Megan Bestwick, Fabien Pierrel, Oleh Khalimonchuk, Dennis R WingeAbstract:Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2 Delta cells is suppressed either by the presence of a mutant allele of the COX10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. COX10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2 Delta cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1 Delta and shy1 Delta mutants. The suppressor activity of N196K mutant COX10 is dependent on its catalytic function and the presence of Cox15, the second enzyme involved in heme a biosynthesis. Varying the substitution at Asn196 reveals a correlation between the suppressor activity and the stabilization of the high-mass homo-oligomeric COX10 complex. We postulate that the mutant COX10 complex has enhanced efficiency in the addition of heme a to Cox1. Coa2 appears to impart stability to the oligomeric wild-type COX10 complex involved in Cox1 hemylation.
-
Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase
Molecular and cellular biology, 2009Co-Authors: Oleh Khalimonchuk, Brigitte Meunier, Megan Bestwick, Talina Watts, Dennis R WingeAbstract:Cytochrome c oxidase (CcO) is the terminal oxidase in the oxidative phosphorylation chain within mitochondria. Mammalian CcO is a 13-subunit complex in which three mitochondrion-encoded subunits (Cox1 to Cox3) form the catalytic core (14). The catalytic core is surrounded by nucleus-encoded subunits, which confer stability to the holoenzyme and likely provide sites for the regulation of its activity (25). The fully assembled yeast holoenzyme is further organized into supercomplexes with the bc1 cytochrome c reductase (22). The catalytic core subunits contain heme and copper redox cofactors (41). Cox2 binds two copper ions, forming the binuclear CuA center that is reduced by cytochrome c. Electrons from the CuA center are transferred to a low-spin heme a center in Cox1 and subsequently to a heterobimetallic heme a-copper site, designated heme a3:CuB, where molecular oxygen is bound and reduced to water (3, 45). The heme a cofactor found in CcO differs from protoheme in that a hydroxyethylfarnesyl group replaces a vinyl moiety and a pyrrole methyl group is oxidized to a formyl substituent. Heme a synthesis is catalyzed by two successive enzymes, COX10 and Cox15, that reside within the inner membrane (IM) (7, 21). COX10 is a farnesyl transferase that converts protoheme to heme o. Cox15 subsequently catalyzes the oxidation of the C-8 heme methyl group in a reaction that involves matrix Yah1 ferredoxin and Arh1 ferredoxin reductase (8, 11). Yeast cells lacking Cox15 contain no heme a, but show low levels of heme o, suggesting that the activities of the two enzymes are not linked (9). Likewise, Cox15 mutations in patients exhibiting fatal infantile hypertrophic cardiomyopathy result in reduced heme a but elevated heme o levels (2). In yeast, CcO biogenesis commences with Cox1 synthesis on mitochondrial ribosomes tethered to the IM by IM-associated Pet309 and Mss51 that bind to the 5′ untranslated region (UTR) of the Cox1 transcript (29, 40, 46). Mss51 has a second function in translational elongation of Cox1, and this function occurs within high-mass Mss51 complexes (∼450 and ∼400 kDa) consisting of Mss51, Cox14, and newly synthesized Cox1 (6, 33, 34). Cox1 appears to progress from the Mss51-containing complex to downstream transient assembly complexes involving Shy1 (31, 34). Yeast cells contain another Cox1 maturation factor, Coa1, which also forms an ∼440-kDa Cox1 assembly intermediate (34). The observed interactions of Coa1 with Mss51 and Shy1 suggest that it participates in the early Cox1 maturation pathway. Information on whether Coa1 is an integral component of the Mss51- or Shy1-containing Cox1 complexes is lacking. The heme a3 cofactor center appears to be inserted in Cox1 associated with the Shy1 complex (26, 35). The evidence for heme a3 site formation within the Shy1 complex is 2-fold. First, CcO assembly stalled at CuB site formation in Cox1 or at the downstream maturation of Cox2 results in accumulation of a transient Cox1 pro-oxidant intermediate that correlates with the presence of a reactive five-coordinate heme a3 cofactor (26). The pro-oxidant heme a3:Cox1 intermediate is absent in cells lacking Shy1, Coa1, or Cox1 (35). Second, isolation of CcO in Rhodobacter or Paracoccus cells lacking Surf1 (a Shy1 ortholog) reveals an enzyme complex deficient in heme a3 but not heme a (12, 37). Shy1 is not likely a heme a3-insertase, since yeast shy1Δ cells and mutant SURF1 human cells retain 10 to 15% residual CcO activity (17, 36, 47). Rather, Shy1 may be a Cox1 chaperone stabilizing the heme a3 site during Cox1 maturation. In the absence of Shy1, it is likely that the heme a3:Cox1 assembly intermediate is destabilized and only a fraction of the intermediate progresses to the final stages of CcO maturation. CuB site formation in Cox1 requires the assembly factor Cox11. CcO isolated from Rhodobacter sphaeroides cox11Δ cells lacked CuB but contained both hemes and the CuA site (23). However, heme a3 showed an altered environment by electron paramagnetic resonance (EPR) spectroscopy, most likely due to the absence of the CuB site. Assembly of CcO in Rhodobacter differs from that in yeast in that the three-subunit core enzyme can form without heme or Cu cofactors (24). In contrast, yeast cells lacking Cox11 fail to assemble CcO, and the stalled assembly complexes are largely removed by proteolysis, although residual heme a3:Cox1 intermediates persist in cox11Δ yeast cells, resulting in hydrogen peroxide sensitivity (26). The heme a center in Cox1 may be formed earlier than the CuB-heme a3 center. Studies with fibroblasts from patients with mutant COX10 or Cox15 reveal limited accumulation of the free Cox1 subunit (1, 2). In contrast, CcO-deficient patients with mutations in SURF1 revealed a Cox1 assembly intermediate with two nuclear CcO subunits, CoxIV and Va (equivalent to yeast subunits Cox5a and Cox6) (39, 43, 47). One interpretation of these results is that heme a insertion may be necessary for formation or stabilization of the S2 intermediate. In addition, studies of the assembly of the bo3 oxidase of Escherichia coli revealed that insertion of heme b (analogous to the heme a site in cytochrome oxidase) was necessary for subunit assembly (38). Two goals motivated the present work. First, we sought to elucidate the interrelationship of the various Cox1 maturation complexes involving Mss51, Coa1, and Shy1. Second, we wanted to discern the steps in which the heme a and CuB-heme a3 cofactor sites are formed during Cox1 maturation in yeast. We show here that separate Mss51-Cox1, Coa1-Cox1, and Shy1-Cox1 assembly intermediates exist and that the heme a and CuB centers are formed downstream of the Mss51-containing and Coa1-containing Cox1 intermediates.
Carlos T. Moraes - One of the best experts on this subject based on the ideXlab platform.
-
A defect in the mitochondrial complex III, but not complex IV, triggers early ROS-dependent damage in defined brain regions
Human molecular genetics, 2012Co-Authors: Francisca Diaz, Sofia Garcia, Kyle R. Padgett, Carlos T. MoraesAbstract:We have created two neuron-specific mouse models of mitochondrial electron transport chain deficiencies involving defects in complex III (CIII) or complex IV (CIV). These conditional knockouts (cKOs) were created by ablation of the genes coding for the Rieske iron–sulfur protein (RISP) and COX10, respectively. RISP is one of the catalytic subunits of CIII and COX10 is an assembly factor indispensable for the maturation of Cox1, one of the catalytic subunits of CIV. Although the rates of gene deletion, protein loss and complex dysfunction were similar, the RISP cKO survived 3.5 months of age, whereas the COX10 cKO survived for 10–12 months. The RISP cKO had a sudden death, with minimal behavioral changes. In contrast, the COX10 cKO showed a distinctive behavioral phenotype with onset at 4 months of age followed by a slower but progressive neurodegeneration. Curiously, the piriform and somatosensory cortices were more vulnerable to the CIII defect whereas cingulate cortex and to a less extent piriform cortex were affected preferentially by the CIV defect. In addition, the CIII model showed severe and early reactive oxygen species damage, a feature not observed until very late in the pathology of the CIV model. These findings illustrate how specific respiratory chain defects have distinct molecular mechanisms, leading to distinct pathologies, akin to the clinical heterogeneity observed in patients with mitochondrial diseases.
-
cytochrome c oxidase is required for the assembly stability of respiratory complex i in mouse fibroblasts
Molecular and Cellular Biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts.
Molecular and cellular biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency
Human Molecular Genetics, 2005Co-Authors: Francisca Diaz, Christine K. Thomas, Sofia Garcia, Dayami Hernandez, Carlos T. MoraesAbstract:We have created a mouse model with an isolated cytochrome c oxidase (COX) deficiency by disrupting the COX10 gene in skeletal muscle. Missense mutations in COX10 have been previously associated with mitochondrial disorders. COX10p is a protoheme:heme-O-farnesyl transferase required for the synthesis of heme a, the prosthetic group of the catalytic center of COX. COX10 conditional knockout mice were generated by crossing a LoxP-tagged COX10 mouse with a transgenic mouse expressing cre recombinase under the myosin light chain 1f promoter. The COX10 knockout mice were healthy until approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though COX activity in COX10 KO muscles was <5% of control muscle at 2.5 months, these muscles were still able to contract at 80-100% of control maximal forces and showed only a 10% increase in fatigability, and no signs of oxidative damage or apoptosis were detected. However, the myopathy worsened with time, particularly in female animals. This COX10 KO mouse allowed us to correlate the muscle function with residual COX activity, an estimate that can help predict the progression pattern of human mitochondrial myopathies.
-
Mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency
Human molecular genetics, 2005Co-Authors: Francisca Diaz, Christine K. Thomas, Sofia Garcia, Dayami Hernandez, Carlos T. MoraesAbstract:We have created a mouse model with an isolated cytochrome c oxidase (COX) deficiency by disrupting the COX10 gene in skeletal muscle. Missense mutations in COX10 have been previously associated with mitochondrial disorders. COX10p is a protoheme:heme-O-farnesyl transferase required for the synthesis of heme a, the prosthetic group of the catalytic center of COX. COX10 conditional knockout mice were generated by crossing a LoxP-tagged COX10 mouse with a transgenic mouse expressing cre recombinase under the myosin light chain 1f promoter. The COX10 knockout mice were healthy until approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though COX activity in COX10 KO muscles was
Francisca Diaz - One of the best experts on this subject based on the ideXlab platform.
-
Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-1α in Complex III and Complex IV Eficient Cells.
Cells, 2020Co-Authors: Amy Saldana-caboverde, Sofia Garcia, Nadee Nissanka, Anne Lombès, Francisca DiazAbstract:Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or COX10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1–5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1α during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1α regulate the assembly of respiratory complexes.
-
A defect in the mitochondrial complex III, but not complex IV, triggers early ROS-dependent damage in defined brain regions
Human molecular genetics, 2012Co-Authors: Francisca Diaz, Sofia Garcia, Kyle R. Padgett, Carlos T. MoraesAbstract:We have created two neuron-specific mouse models of mitochondrial electron transport chain deficiencies involving defects in complex III (CIII) or complex IV (CIV). These conditional knockouts (cKOs) were created by ablation of the genes coding for the Rieske iron–sulfur protein (RISP) and COX10, respectively. RISP is one of the catalytic subunits of CIII and COX10 is an assembly factor indispensable for the maturation of Cox1, one of the catalytic subunits of CIV. Although the rates of gene deletion, protein loss and complex dysfunction were similar, the RISP cKO survived 3.5 months of age, whereas the COX10 cKO survived for 10–12 months. The RISP cKO had a sudden death, with minimal behavioral changes. In contrast, the COX10 cKO showed a distinctive behavioral phenotype with onset at 4 months of age followed by a slower but progressive neurodegeneration. Curiously, the piriform and somatosensory cortices were more vulnerable to the CIII defect whereas cingulate cortex and to a less extent piriform cortex were affected preferentially by the CIV defect. In addition, the CIII model showed severe and early reactive oxygen species damage, a feature not observed until very late in the pathology of the CIV model. These findings illustrate how specific respiratory chain defects have distinct molecular mechanisms, leading to distinct pathologies, akin to the clinical heterogeneity observed in patients with mitochondrial diseases.
-
cytochrome c oxidase is required for the assembly stability of respiratory complex i in mouse fibroblasts
Molecular and Cellular Biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts.
Molecular and cellular biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency
Human Molecular Genetics, 2005Co-Authors: Francisca Diaz, Christine K. Thomas, Sofia Garcia, Dayami Hernandez, Carlos T. MoraesAbstract:We have created a mouse model with an isolated cytochrome c oxidase (COX) deficiency by disrupting the COX10 gene in skeletal muscle. Missense mutations in COX10 have been previously associated with mitochondrial disorders. COX10p is a protoheme:heme-O-farnesyl transferase required for the synthesis of heme a, the prosthetic group of the catalytic center of COX. COX10 conditional knockout mice were generated by crossing a LoxP-tagged COX10 mouse with a transgenic mouse expressing cre recombinase under the myosin light chain 1f promoter. The COX10 knockout mice were healthy until approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though COX activity in COX10 KO muscles was <5% of control muscle at 2.5 months, these muscles were still able to contract at 80-100% of control maximal forces and showed only a 10% increase in fatigability, and no signs of oxidative damage or apoptosis were detected. However, the myopathy worsened with time, particularly in female animals. This COX10 KO mouse allowed us to correlate the muscle function with residual COX activity, an estimate that can help predict the progression pattern of human mitochondrial myopathies.
Sofia Garcia - One of the best experts on this subject based on the ideXlab platform.
-
Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-1α in Complex III and Complex IV Eficient Cells.
Cells, 2020Co-Authors: Amy Saldana-caboverde, Sofia Garcia, Nadee Nissanka, Anne Lombès, Francisca DiazAbstract:Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or COX10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1–5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1α during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1α regulate the assembly of respiratory complexes.
-
A defect in the mitochondrial complex III, but not complex IV, triggers early ROS-dependent damage in defined brain regions
Human molecular genetics, 2012Co-Authors: Francisca Diaz, Sofia Garcia, Kyle R. Padgett, Carlos T. MoraesAbstract:We have created two neuron-specific mouse models of mitochondrial electron transport chain deficiencies involving defects in complex III (CIII) or complex IV (CIV). These conditional knockouts (cKOs) were created by ablation of the genes coding for the Rieske iron–sulfur protein (RISP) and COX10, respectively. RISP is one of the catalytic subunits of CIII and COX10 is an assembly factor indispensable for the maturation of Cox1, one of the catalytic subunits of CIV. Although the rates of gene deletion, protein loss and complex dysfunction were similar, the RISP cKO survived 3.5 months of age, whereas the COX10 cKO survived for 10–12 months. The RISP cKO had a sudden death, with minimal behavioral changes. In contrast, the COX10 cKO showed a distinctive behavioral phenotype with onset at 4 months of age followed by a slower but progressive neurodegeneration. Curiously, the piriform and somatosensory cortices were more vulnerable to the CIII defect whereas cingulate cortex and to a less extent piriform cortex were affected preferentially by the CIV defect. In addition, the CIII model showed severe and early reactive oxygen species damage, a feature not observed until very late in the pathology of the CIV model. These findings illustrate how specific respiratory chain defects have distinct molecular mechanisms, leading to distinct pathologies, akin to the clinical heterogeneity observed in patients with mitochondrial diseases.
-
cytochrome c oxidase is required for the assembly stability of respiratory complex i in mouse fibroblasts
Molecular and Cellular Biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts.
Molecular and cellular biology, 2006Co-Authors: Francisca Diaz, Sofia Garcia, Hirokazu Fukui, Carlos T. MoraesAbstract:Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.
-
mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency
Human Molecular Genetics, 2005Co-Authors: Francisca Diaz, Christine K. Thomas, Sofia Garcia, Dayami Hernandez, Carlos T. MoraesAbstract:We have created a mouse model with an isolated cytochrome c oxidase (COX) deficiency by disrupting the COX10 gene in skeletal muscle. Missense mutations in COX10 have been previously associated with mitochondrial disorders. COX10p is a protoheme:heme-O-farnesyl transferase required for the synthesis of heme a, the prosthetic group of the catalytic center of COX. COX10 conditional knockout mice were generated by crossing a LoxP-tagged COX10 mouse with a transgenic mouse expressing cre recombinase under the myosin light chain 1f promoter. The COX10 knockout mice were healthy until approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though COX activity in COX10 KO muscles was <5% of control muscle at 2.5 months, these muscles were still able to contract at 80-100% of control maximal forces and showed only a 10% increase in fatigability, and no signs of oxidative damage or apoptosis were detected. However, the myopathy worsened with time, particularly in female animals. This COX10 KO mouse allowed us to correlate the muscle function with residual COX activity, an estimate that can help predict the progression pattern of human mitochondrial myopathies.
Eric L. Hegg - One of the best experts on this subject based on the ideXlab platform.
-
Regulation of the Heme A Biosynthetic Pathway DIFFERENTIAL REGULATION OF HEME A SYNTHASE AND HEME O SYNTHASE IN SACCHAROMYCES CEREVISIAE
The Journal of biological chemistry, 2008Co-Authors: Zhihong Wang, Yuxin Wang, Eric L. HeggAbstract:The assembly and activity of cytochrome c oxidase is dependent on the availability of heme A, one of its essential cofactors. In eukaryotes, two inner mitochondrial membrane proteins, heme O synthase (COX10) and heme A synthase (Cox15), are required for heme A biosynthesis. In this report, we demonstrate that in Saccharomyces cerevisiae the transcription of COX15 is regulated by Hap1, a transcription factor whose activity is positively controlled by intracellular heme concentration. Conversely, COX10, the physiological partner of COX15, does not share the same regulatory mechanism with COX15. Interestingly, protein quantification identified an 8:1 protein ratio between Cox15 and COX10. Together, these results suggest that heme A synthase and/or heme O synthase might play a new, unidentified role in addition to heme A biosynthesis.
-
Heme O synthase and heme A synthase from Bacillus subtilis and Rhodobacter sphaeroides interact in Escherichia coli.
Biochemistry, 2004Co-Authors: Brienne M. Brown, Julia A Cricco, Zhihong Wang, Kenneth R. Brown, Eric L. HeggAbstract:Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water. Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors. Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (COX10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli. Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo. Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact. Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS. Because of the strong sequence similarity and evolutionary relationship between R. sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well.
