Plant science. Distributing nutrition.
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Arabidopsis↗
Biomedical subjects
Jonathan D Gitlin
Publications and source records attributed to Jonathan D Gitlin.
Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis.
As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.
Animals↗
Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity.
Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia, and failure to thrive, is due to inherited loss-of-function mutations in the gene encoding a copper-transporting ATPase (Atp7a) on the X chromosome. Although affected patients exhibit signs and symptoms of copper deficiency, the mechanisms resulting in neurologic disease remain unknown. We recently discovered that Atp7a is required for the production of an NMDA receptor-dependent releasable copper pool within hippocampal neurons, a finding that suggests a role for copper in activity-dependent modulation of synaptic activity. In support of this hypothesis, we now demonstrate that copper chelation exacerbates NMDA-mediated excitotoxic cell death in primary hippocampal neurons, whereas the addition of copper is specifically protective and results in a significant decrease in cytoplasmic Ca(2+) levels after NMDA receptor activation. Consistent with the known neuroprotective effect of NMDA receptor nitrosylation, we show here that this protective effect of copper depends on endogenous nitric oxide production in hippocampal neurons, demonstrating in vivo links among neuroprotection, copper metabolism, and nitrosylation. Atp7a is required for these copper-dependent effects: Hippocampal neurons isolated from newborn Mo(br) mice reveal a marked sensitivity to endogenous glutamate-mediated NMDA receptor-dependent excitotoxicity in vitro, and mild hypoxic/ischemic insult to these mice in vivo results in significantly increased caspase 3 activation and neuronal injury. Taken together, these data reveal a unique connection between copper homeostasis and NMDA receptor activity that is of broad relevance to the processes of synaptic plasticity and excitotoxic cell death.
Adenosine Triphosphatases↗
GLUT1 deficiency links nutrient availability and apoptosis during embryonic development.
GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.
Animals↗
Mechanisms of the copper-dependent turnover of the copper chaperone for superoxide dismutase.
The copper chaperone for superoxide dismutase (CCS) is an intracellular metallochaperone required for incorporation of copper into the essential antioxidant enzyme copper/zinc superoxide dismutase (SOD1). Nutritional studies have revealed that the abundance of CCS is inversely proportional to the dietary and tissue copper content. To determine the mechanisms of copper-dependent regulation of CCS, copper incorporation into SOD1 and SOD1 enzymatic activity as well as CCS abundance and half-life were determined after metabolic labeling of CCS-/- fibroblasts transfected with wild-type or mutant CCS. Wild-type CCS restored SOD1 activity in CCS-/- fibroblasts, and the abundance of this chaperone in these cells was inversely proportional to the copper content of the media, indicating that copper-dependent regulation of CCS is entirely post-translational. Although mutational studies demonstrated no role for CCS Domain I in this copper-dependent regulation, similar analysis of the CXC motif in Domain III revealed a critical role for these cysteine residues in mediating copper-dependent turnover of CCS. Further mutational studies revealed that this CXC-dependent copper-mediated turnover of CCS is independent of the mechanisms of delivery of copper to SOD1 including CCS-SOD1 interaction. Taken together these data demonstrate a mechanism determining the abundance of CCS that is competitive with the process of copper delivery to SOD1, revealing a unique post-translational component of intracellular copper homeostasis.
Animals↗
Atp7a determines a hierarchy of copper metabolism essential for notochord development.
The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.
Adenosine Triphosphatases↗
Copper and nitric oxide meet in the plasma.
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Ceruloplasmin↗
Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
Copper is an essential nutrient that plays a fundamental role in the biochemistry of the central nervous system, as evidenced by patients with Menkes disease, a fatal neurodegenerative disorder of childhood resulting from the loss-of-function of a copper-transporting P-type adenosine triphosphatase (ATPase). Despite clinical and experimental data indicating a role for copper in brain function, the mechanisms and timing of the critical events affected by copper remain poorly understood. A novel role for the Menkes ATPase has been identified in the availability of an N-methyl-D-aspartate (NMDA) receptor-dependent, releasable pool of copper in hippocampal neurons, suggesting a unique mechanism linking copper homeostasis and neuronal activation within the central nervous system. This article explores the evidence that copper acts as a modulator of neuronal transmission, and that the release of endogenous copper from neurons may regulate NMDA receptor activity. The relationship between impaired copper homeostasis and neuropathophysiology suggests that impairment of copper efflux could alter neuronal function and thus contribute to rapid neuronal degeneration.
Adenosine Triphosphatases↗
NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
Copper is an essential transition metal with a critical role in the CNS. This requirement is underscored by Menkes disease, a fatal neurodegenerative disorder of childhood resulting from the absence or dysfunction of a copper-transporting P-type ATPase. To elucidate the cell biological mechanisms of copper homeostasis in the CNS, a polyclonal antisera against Menkes ATPase was used in immunoblot and immunohistochemical studies, demonstrating abundant expression of this copper transporter in hippocampal neurons. Consistent with this observation, immunofluorescent analysis revealed Menkes ATPase in the late Golgi of hippocampal neurons in primary culture. Glutamate receptor activation was found to result in the rapid and reversible trafficking of Menkes ATPase to neuronal processes, independent of the intracellular copper concentration and specific for activation of the NMDA- but not AMPA/kainate-type glutamate receptors. Metabolic studies revealed that trafficking of Menkes ATPase after NMDA receptor activation is associated with rapid release of copper from hippocampal neurons. Menkes ATPase is directly required for this copper efflux, because similar studies in hippocampal neurons derived from mice lacking a functional Menkes ATPase demonstrated no copper release. Together, these data reveal a critical role for Menkes ATPase in the availability of an NMDA receptor-dependent, releasable pool of copper in hippocampal neurons and demonstrate a unique mechanism linking copper homeostasis and neuronal activation within the CNS.
Adenosine Triphosphatases↗
Role of copper in the proteosome-mediated degradation of the multicopper oxidase hephaestin.
To elucidate the mechanisms of cuproprotein biosynthesis in the secretory pathway, a polyclonal antiserum was generated against hephaestin, a multicopper oxidase essential for enteric iron absorption. Immunoblot analysis and pulse-chase metabolic labeling revealed that hephaestin is synthesized as a single-chain polypeptide modified by N-linked glycosylation to a mature 161-kDa species. Cell surface biotinylation and immunofluorescent studies of polarized, differentiated colon carcinoma cells detected hephaestin on the basolateral surface under steady-state conditions. However, a decrease in the intracellular copper concentration resulted in a marked diminution in the abundance of this protein. Metabolic studies revealed no effect of decreased intracellular copper on the rate of hephaestin synthesis but a dramatic, specific, and reproducible increase in the turnover of the mature 161-kDa protein. Surprisingly, inhibitor studies revealed that this turnover occurs exclusively in the proteasome, and consistent with this finding, in vitro studies identified polyubiquitinated hephaestin under conditions abrogating copper incorporation into this protein. Taken together, these studies demonstrate the presence of a quality control system for posttranslational protein modification occurring beyond the endoplasmic reticulum that, in the case of hephaestin, directly links the rate of enteric iron uptake to nutritional copper status.
Cells, Cultured↗
A fungal multicopper oxidase restores iron homeostasis in aceruloplasminemia.
Mutations that lead to a loss of the copper-containing plasma enzyme ceruloplasmin disrupt mammalian iron homeostasis. The mechanism by which ceruloplasmin mobilizes iron from cell stores has been controversial. We demonstrate that injection of a soluble copper-containing yeast protein Fet3p can restore iron homeostasis in phlebotomized mice with a deletion of the ceruloplasmin gene. These results show the conservation of function of copper-containing proteins in eukaryotic iron metabolism.
Animals↗
The copper toxicosis gene product Murr1 directly interacts with the Wilson disease protein.
Copper toxicosis in Bedlington terriers is an autosomal recessive disorder characterized by excessive hepatic copper accumulation in association with a marked decrease in biliary copper excretion. Recent genetic data have revealed that MURR1, a single copy gene on dog chromosome 10q26, is mutated in this disorder. This gene encodes a 190-amino acid open reading frame of unknown function that is highly conserved in vertebrate species. The Wilson disease protein is a copper transporting ATPase shown to play a critical role in biliary copper excretion. Here we demonstrate that the Wilson disease protein directly interacts with the human homologue of Murr1 in vitro and in vivo and that this interaction is mediated via the copper binding, amino terminus of this ATPase. Importantly, this interaction is specific for this copper transporter, a finding consistent with the observation that impaired copper homeostasis in affected terriers is confined to the liver. Our findings reveal involvement of Murr1 in the defined pathway of hepatic biliary copper excretion, suggest a potential mechanism for Murr1 function in this process, and provide biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis.
Adaptor Proteins, Signal Transducing↗
Mechanisms of biosynthesis of mammalian copper/zinc superoxide dismutase.
Copper/zinc superoxide dismutase (SOD1) is an abundant intracellular enzyme with an essential role in antioxidant defense. The activity of SOD1 is dependent upon the presence of a bound copper ion incorporated by the copper chaperone for superoxide dismutase, CCS. To elucidate the cell biological mechanisms of this process, SOD1 synthesis and turnover were examined following 64Cu metabolic labeling of fibroblasts derived from CCS+/+ and CCS-/- embryos. The data indicate that copper is rapidly incorporated into both newly synthesized SOD1 and preformed SOD1 apoprotein, that each process is dependent upon CCS and that once incorporated, copper is unavailable for cellular exchange. The abundance of apoSOD1 is inversely proportional to the intracellular copper content and immunoblot and gel filtration analysis indicate that this apoprotein exists as a homodimer that is distinguishable from SOD1. Despite these distinct differences, the abundance and half-life of SOD1 is equivalent in CCS+/+ and CCS-/- fibroblasts, indicating that neither CCS nor copper incorporation has any essential role in the stability or turnover of SOD1 in vivo. Taken together, these data provide a cell biological model of SOD1 biosynthesis that is consistent with the concept of limited intracellular copper availability and indicate that the metallochaperone CCS is a critical determinant of SOD1 activity in mammalian cells. These kinetic and biochemical findings also provide an important framework for understanding the role of mutant SOD1 in the pathogenesis of familial amyotrophic lateral sclerosis.
Animals↗
Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase.
The metallochaperone Atox1 directly interacts with the copper-transporting ATPases and plays a critical role in perinatal copper homeostasis. To determine the cell biological mechanisms of Atox1 function, intracellular copper metabolism, and Menkes ATPase abundance, localization and trafficking were examined in immortalized fibroblast cell lines derived from Atox1(+/+) and Atox1(-/-) embryos. Consistent with the proposed role for Atox1 in copper delivery to the secretory pathway, a marked increase in intracellular copper content secondary to impaired copper efflux was observed in Atox1-deficient cells. Although the localization of the Menkes ATPase was identical in Atox1(+/+) and Atox1(-/-) cells under conditions of equivalent intracellular copper content, a significant impairment in copper-mediated Menkes ATPase trafficking was observed in the absence of Atox1. When quantitative confocal immunofluorescence was used, significant differences in the time and dose-dependent trafficking of the Menkes ATPase from the Golgi compartment in response to copper were observed between Atox1(+/+) and Atox1(-/-) cells. These data reveal an essential role for Atox1 in establishing the threshold for copper-dependent movement of the copper-transporting ATPases within the secretory compartment and that, in the absence of Atox1, this movement alone is not sufficient to restore normal copper efflux. Taken together, these findings provide a cell biological model for the role of this metallochaperone under the physiological conditions of copper limitation in mammalian cells.
Adenosine Triphosphatases↗
Wilson disease.
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Adenosine Triphosphatases↗
Hepatic copper metabolism: insights from genetic disease.
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Animals↗
Mechanisms of copper incorporation into human ceruloplasmin.
Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and (64)Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.
Animals↗
Ceruloplasmin metabolism and function.
Ceruloplasmin is a serum ferroxidase that contains greater than 95% of the copper found in plasma. This protein is a member of the multicopper oxidase family, an evolutionarily conserved group of proteins that utilize copper to couple substrate oxidation with the four-electron reduction of oxygen to water. Despite the need for copper in ceruloplasmin function, this protein plays no essential role in the transport or metabolism of this metal. Aceruloplasminemia is a neurodegenerative disease resulting from inherited loss-of-function mutations in the ceruloplasmin gene. Characterization of this disorder revealed a critical physiological role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores and has provided new insights into human iron metabolism and nutrition.
Ceruloplasmin↗