Biomedical subjects

Scot C Leary

Publications and source records attributed to Scot C Leary.

10 recordsLinked to original sources

PubMed2007

The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis.

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.

Alleles↗

PubMed2006

A hemizygous SCO2 mutation in an early onset rapidly progressive, fatal cardiomyopathy.

Mutations in SCO2, a metallochaperone involved in mitochondrial copper delivery, are associated with early onset, fatal hypertrophic cardiomyopathy. All reported patients carry at least one copy of the common 1541G>A (E140K) mutation. Whereas patients with one copy of the E140K allele, in combination with a more deleterious mutation, follow a severe clinical course, patients homozygous for the E140K mutation have a delayed onset of disease and a more prolonged survival. Here, we have investigated a patient who appeared homozygous for the common 1541G>A mutation based on DNA sequencing and restriction enzyme analysis of a PCR product, yet presented with early onset, severe cardiomyopathy. Restriction enzyme analysis of parental DNA revealed that the mother was heterozygous for 1541G>A, while the father was homozygous wild-type. The patient showed biparental inheritance for microsatellite markers spanning the length of chromosome 22, making isodisomy unlikely. Sequencing of several single nucleotide polymorphisms within the 5'-UTR, intron and single exon of the SCO2 gene was uninformative; however, a 16 bp deletion within the intron was present in the patient and the mother, but not the father. Restriction enzyme analysis confirmed that the mother was heterozygous and that the patient was hemizygous for the deletion. Southern blot, Northern blot, and FISH analyses were consistent with the de novo deletion of one allele of SCO2 in the patient. This is the first report of hemizygosity in a SCO2 patient. The patient phenotype underscores the strikingly similar clinical course in all patients with one copy of the E140K allele. Examination of both patient and parental genotypes by thorough molecular analyses can reveal information with important implications for genetic counseling.

Cardiomyopathies↗

PubMed2006

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding.

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) 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 sco1delta 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 Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.

Binding Sites↗

PubMed2005

Human Sco1 and Sco2 function as copper-binding proteins.

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.

Aspartic Acid↗

PubMed2004

Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase.

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.

Biological Transport↗

PubMed2004

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.

Adipocytes↗

PubMed2003

Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency.

Deficiencies in the activity of cytochrome c oxidase (COX) are an important cause of autosomal recessive respiratory chain disorders. Patients with isolated COX deficiency are clinically and genetically heterogeneous, and mutations in several different assembly factors have been found to cause specific clinical phenotypes. Two of the most common clinical presentations, Leigh Syndrome and hypertrophic cardiomyopathy, have so far only been associated with mutations in SURF1 or SCO2 and COX15, respectively. Here we show that expression of COX10 from a retroviral vector complements the COX deficiency in a patient with anemia and Leigh Syndrome, and in a patient with anemia, sensorineural deafness and fatal infantile hypertrophic cardiomyopathy. A partial rescue was also obtained following microcell-mediated transfer of mouse chromosomes into patient fibroblasts. COX10 functions in the first step of the mitochondrial heme A biosynthetic pathway, catalyzing the conversion of protoheme (heme B) to heme O via the farnesylation of a vinyl group at position C2. Heme A content was reduced in mitochondria from patient muscle and fibroblasts in proportion to the reduction in COX enzyme activity and the amount of fully assembled enzyme. Mutation analysis of COX10 identified four different missense alleles, predicting amino acid substitutions at evolutionarily conserved residues. A topological model places these residues in regions of the protein shown to have important catalytic functions by mutation analysis of a prokaryotic ortholog. Mutations in COX10 have previously been reported in a single family with tubulopathy and leukodystrophy. This study shows that mutations in this gene can cause nearly the full range of clinical phenotypes associated with early onset isolated COX deficiency.

Alkyl and Aryl Transferases↗

PubMed2003

Mitochondrial biogenesis: which part of "NO" do we understand?

A recent paper by Nisoli et al. [1] provides the first evidence that elevated levels of nitric oxide (NO) stimulate mitochondrial biogenesis in a number of cell lines via a soluble guanylate-cyclase-dependent signaling pathway that activates PGC1alpha (peroxisome proliferator-activated receptor gamma coactivator-1alpha), a master regulator of mitochondrial content. These results raise intriguing possibilities for a role of NO in modulating mitochondrial content in response to physiological stimuli such as exercise or cold exposure. However, whether this signaling cascade represents a widespread mechanism by which mammalian tissues regulate mitochondrial content, and how it might integrate with other pathways that control PGC1alpha expression, remain unclear.

Adenosine Triphosphate↗

PubMed2002

Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy.

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patient's heart and fibroblast mitochondria, and levels of heme O were increased in the patient's heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency.

Age of Onset↗

PubMed2002

Chronic treatment with azide in situ leads to an irreversible loss of cytochrome c oxidase activity via holoenzyme dissociation.

Chronic treatment of cultured cells with very low levels of azide (I(50)<10 microm) leads to slow (t(12) = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl(2), Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu(A) or Cu(B). In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa(3) content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.

Azides↗