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J D Gitlin

Publications and source records attributed to J D Gitlin.

At least 37 records · Page 2Linked to original sources
PubMed1998

The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase.

Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization. Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein. Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS. Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease.

Amino Acid Sequence↗
PubMed1998

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation.

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse-chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28 degreesC resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Adenosine Triphosphatases↗
PubMed1998

Functional expression of the menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases.

Menkes disease is a fatal neurodegenerative disorder of childhood caused by the absence or dysfunction of a putative P-type ATPase encoded on the X chromosome. To elucidate the function of the Menkes disease protein, a plasmid containing the open reading frame of the human Menkes disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue. ccc2Delta yeast are deficient in copper transport into the secretory pathway, and expression of a wild type human Menkes cDNA complemented this defect, as evidenced by the restoration of copper incorporation into the multicopper oxidase Fet3p. Site-directed mutagenesis demonstrated the essential role of four specific amino acids in this process, including a conserved histidine, which is the site of the most common disease mutation in the homologous Wilson disease protein. The expression of Menkes cDNAs with successive mutations of the conserved cysteine residues in the six amino-terminal MXCXXC metal binding domains confirmed the essential role of these cysteine residues in copper transport but revealed that each of these domains is not functionally equivalent. These data demonstrate that the Menkes disease protein functions to deliver copper into the secretory pathway of the cell and that this process involves biochemical mechanisms common to previously characterized members of this P-type ATPase family.

Adenosine Triphosphatases↗
PubMed1998

HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense.

HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues. Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands. Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast. Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.

Amino Acid Sequence↗
PubMed1998

Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis.

Aceruloplasminemia is an autosomal recessive disorder characterized by progressive neurodegeneration of the retina and basal ganglia associated with specific inherited mutations in the ceruloplasmin gene. Clinical and pathologic studies in patients with aceruloplasminemia revealed a marked accumulation of iron in affected parenchymal tissues, a finding consistent with early work identifying ceruloplasmin as a ferroxidase and with recent findings showing an essential role for a homologous copper oxidase in iron metabolism in yeast. The presence of neurologic symptoms in aceruloplasminemia is unique among the known inherited and acquired disorders of iron metabolism; recent studies revealed an essential role for astrocyte-specific expression of ceruloplasmin in iron metabolism and neuronal survival in the central nervous system. Recognition of aceruloplasminemia provides new insights into the genetic and environmental determinants of copper metabolism and has important implications for our understanding of the role of copper in human neurodegenerative diseases.

Ceruloplasmin↗
PubMed1998

Aceruloplasminemia.

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism characterized by diabetes, retinal degeneration, and neurologic symptoms. Affected patients evidence marked parenchymal iron accumulation in conjunction with an absence of circulating serum ceruloplasmin and molecular genetic analysis reveals inherited mutations in the ceruloplasmin gene. Taken together with earlier studies that characterized ceruloplasmin as a ferroxidase and recent work indicating an essential role for a homologous multicopper oxidase in iron metabolism in Saccharomyces cerevisiae, these findings reveal an essential role for ceruloplasmin in human iron metabolism. The presence of neurologic symptoms in patients with aceruloplasminemia is unique among the characterized disorders of iron metabolism, and recent findings indicate that astrocyte-specific ceruloplasmin gene expression is critical for iron metabolism and neuronal survival in the retina and basal ganglia. The discovery of this disease provides new insights into the pathways of CNS iron metabolism of direct relevance to a variety of nutritional and genetic disorders of childhood.

Ceruloplasmin↗
PubMed1998

Increased very long-chain fatty acids in erythrocyte membranes of patients with aceruloplasminemia.

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism that causes neurodegeneration of the retina and basal ganglia as well as diabetes mellitus. Our previous studies suggested that increased susceptibility to plasma lipid peroxidation secondary to iron accumulation may contribute to the pathogenesis in this disease. We now have identified increases in the very long-chain fatty acids cis-17-hexacosenoic (C26:1) and hexacosanoic (C26:0) acid in the erythrocyte membranes of three family members affected with aceruloplasminemia. All of them had elevated C26:1/C22:0 and C26:0/C22:0 ratios. These findings suggest that free radicals generated in persons with aceruloplasminemia may interrupt the peroxisomal beta-oxidation of fatty acids.

Apoproteins↗
PubMed1998

CSF abnormalities in patients with aceruloplasminemia.

Aceruloplasminemia is a disorder of iron metabolism characterized by degeneration of the retina and basal ganglia. CSF from affected patients showed a threefold increased iron concentration that was associated with increased superoxide dismutase activity and lipid peroxidation products. These findings support the hypothesis that iron-mediated lipid peroxidation contributes to neurodegeneration in patients with aceruloplasminemia. Such measurements may have value in assessing disease progression as well as the results of iron chelation and other therapeutic interventions.

Adult↗
PubMed1997

The copper chaperone for superoxide dismutase.

Copper is distributed to distinct localizations in the cell through diverse pathways. We demonstrate here that the delivery of copper to copper/zinc superoxide dismutase (SOD1) is mediated through a soluble factor identified as Saccharomyces cerevisiae LYS7 and human CCS (copper chaperone for SOD). This factor is specific for SOD1 and does not deliver copper to proteins in the mitochondria, nucleus, or secretory pathway. Yeast cells containing a lys7Delta null mutation have normal levels of SOD1 protein, but fail to incorporate copper into SOD1, which is therefore devoid of superoxide scavenging activity. LYS7 and CCS specifically restore the biosynthesis of holoSOD1 in vivo. Elucidation of the CCS copper delivery pathway may permit development of novel therapeutic approaches to human diseases that involve SOD1, including amyotrophic lateral sclerosis.

Amino Acid Sequence↗
PubMed1997

Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.

Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

Adenosine Triphosphatases↗
PubMed1997

Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis.

To search for a mammalian homologue of ATX1, a human liver cDNA library was screened and a cDNA clone was isolated, which encodes a protein with 47% amino acid identity to Atx1p including conservation of the MTCXGC copper-binding domain. RNA blot analysis using this cDNA identified an abundant 0.5-kilobase mRNA in all human tissues and cell lines examined. Southern blot analysis using this same clone indicated that the corresponding gene exists as a single copy in the haploid genome, and chromosomal localization by fluorescence in situ hybridization detected this locus at the interface between bands 5q32 and 5q33. Yeast strains lacking copper/zinc superoxide dismutase (SOD1) are sensitive to redox cycling agents and dioxygen and are auxotrophic for lysine when grown in air, and expression of this human ATX1 homologue (HAH1) in these strains restored growth on lysine-deficient media. Yeast strains lacking ATX1 are deficient in high affinity iron uptake and expression of HAH1 in these strains permits growth on iron-depleted media and results in restoration of copper incorporation into newly synthesized Fet3p. These results identify HAH1 as a novel ubiquitously expressed protein, which may play an essential role in antioxidant defense and copper homeostasis in humans.

Amino Acid Sequence↗
PubMed1997

Use of desferrioxamine in the treatment of aceruloplasminemia.

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism resulting in neurodegeneration of the retina and basal ganglia. We report here on the treatment of a patient who developed progressive extrapyramidal symptoms that included blepharospasm, grimacing, and rigidity associated with increased iron deposition in the brain and visceral organs. Treatment for 10 months with the iron chelator desferrioxamine decreased brain iron stores, prevented progression of the neurological symptoms, and reduced plasma lipid peroxidation. These data suggest that early treatment with this chelator may be useful in such patients to diminish central nervous system iron accumulation and to prevent or ameliorate neurological symptoms associated with neurodegeneration.

Ceruloplasmin↗
PubMed1996

Biochemical characterization and intracellular localization of the Menkes disease protein.

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.

Adenosine Triphosphatases↗
PubMed1996

Ceruloplasmin gene expression in the murine central nervous system.

Aceruloplasminemia is an autosomal recessive disorder resulting in neurodegeneration of the retina and basal ganglia in association with iron accumulation in these tissues. To begin to define the mechanisms of central nervous system iron accumulation and neuronal loss in this disease, cDNA clones encoding murine ceruloplasmin were isolated and characterized. RNA blot analysis using these clones detected a 3.7-kb ceruloplasmin-specific transcript in multiple murine tissues including the eye and several regions of the brain. In situ hybridization of systemic tissues revealed cell-specific ceruloplasmin gene expression in hepatocytes, the splenic reticuloendothelial system and the bronchiolar epithelium of the lung. In the central nervous system, abundant ceruloplasmin gene expression was detected in specific populations of astrocytes within the retina and the brain as well as the epithelium of the choroid plexus. Analysis of primary cell cultures confirmed that astrocytes expressed ceruloplasmin mRNA and biosynthetic studies revealed synthesis and secretion of ceruloplasmin by these cells. Taken together these results demonstrate abundant cell-specific ceruloplasmin expression within the central nervous system which may account for the unique clinical and pathologic findings observed in patients with aceruloplasminemia.

Amino Acid Sequence↗
PubMed1996

Increased plasma lipid peroxidation in patients with aceruloplasminemia.

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism due to mutations in the ceruloplasmin gene. Although the presence of these mutations reveals an essential role for ceruloplasmin in human biology, the mechanisms of tissue injury in this disease are unknown. We report here on the identification of increased plasma lipid peroxidation in multiple affected family members with aceruloplasminemia. Consistent with the absence of serum ceruloplasmin, plasma ferroxidase activity was markedly reduced and serum ferritin was significantly increased. Plasma lipid peroxidation was determined as thiobarbituric acid-reactive products (TBA products) in plasma samples from control, heterozygote, and affected patients. Basal levels of lipid peroxides were three times control values in patients with aceruloplasminemia and were significantly increased in these patients in the presence of copper ions and hydrogen peroxide. In each case these increases were suppressed by the addition of exogenous ceruloplasmin. These data suggest that increased susceptibility to lipid peroxidation may contribute to the unique neuropathology observed in patients with aceruloplasminemia and imply a role for free radical-mediated tissue injury in degenerative disorders of the basal ganglia.

Adult↗
PubMed1996

Genetic and molecular basis for copper toxicity.

Recent studies resulted in the cloning of the genes responsible for Menkes syndrome and Wilson disease. Despite the distinct clinical phenotypes of these disorders, each gene encodes a highly homologous member of the cation-transport P-type ATPase family. The remarkable evolutionary conservation of these proteins in bacteria, yeast, plants, and mammals reveals a fundamental protein structure essential for copper export in all life forms. Characterization of a molecular defect in the rat homologue of the Wilson ATPase in the Long-Evans Cinnamon rat identifies an animal model of Wilson disease and will permit experimental analysis of the precise role of this ATPase in copper transport, the effects of specific inherited mutations on transport function, and the cellular and molecular mechanisms of tissue injury resulting from copper accumulation. Finally, recent molecular genetic analysis of a distinct group of patients with low serum ceruloplasmin and basal ganglia symptoms identified a series of mutations in the ceruloplasmin gene. The presence of these mutations in conjunction with the clinical and pathologic findings clarifies the essential biological role of this abundant copper protein in metal metabolism and identifies aceruloplasminemia as a novel autosomal recessive disorder of iron metabolism.

Adenosine Triphosphatases↗
PubMed1996

Characterization of a nonsense mutation in the ceruloplasmin gene resulting in diabetes and neurodegenerative disease.

We report here on the characterization of a mutation in the ceruloplasmin gene in a 45 year old woman with insulin-dependent diabetes mellitus who presented with the recent onset of gait disturbance and dysarthria. Physical examination revealed an ataxic gait, scanning speech and retinal degeneration. Magnetic resonance imaging of the brain was consistent with increased basal ganglia iron content and laboratory studies revealed a low serum iron concentration and no detectable serum ceruloplasmin. Nucleotide sequence analysis of the ceruloplasmin gene from this patient revealed a G to A substitution in exon 15 resulting in a nonsense mutation at amino acid 858 (Trp858ter). The patient's younger, neurologically asymptomatic brother was also found to be homozygous for this mutation. Taken together the clinical and genetic data support the concept of an essential and unique role for ceruloplasmin in human iron metabolism. Identification of this kindred extends the spectrum of ceruloplasmin gene mutations resulting in this autosomal recessive, late-onset neurodegenerative disease and highlights the importance of recognizing aceruloplasminemia as a genetic cause of diabetes and neurologic disease.

Amino Acid Sequence↗