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.     

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.     

Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.     

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.     

Regulation of the Copper Chaperone CCS by XIAP-Mediated Ubiquitination

In order to balance the cellular requirements for copper with its toxic properties, an elegant set of mechanisms has evolved to regulate and buffer intracellular copper. The X-linked inhibitor of apoptosis (XIAP) protein was recently identified as a copper-binding protein and regulator of copper homeostasis, although the mechanism by which XIAP binds copper in the cytosol is unclear. Here we describe the identification of the copper chaperone for superoxide dismutase (CCS) as a mediator of copper delivery to XIAP in cells. We also find that CCS is a target of the E3 ubiquitin ligase activity of XIAP, although interestingly, ubiquitination of CCS by XIAP was found to lead to enhancement of its chaperone activity toward its physiologic target, superoxide dismutase 1, rather than proteasomal degradation. Collectively, our results reveal novel links among apoptosis, copper metabolism, and redox regulation through the XIAP-CCS complex.Copper is a required cofactor for critical steps in many biological processes, including aerobic respiration, iron metabolism, pigment formation, peptide amidation, neurotransmitter synthesis, connective tissue development, and protection from reactive oxygen species (29, 39, 60). Although copper is an essential nutrient, the ability of copper ions to easily exchange electrons makes copper highly toxic, so an elaborate system of transporters, chaperones, and chelators has evolved to control the intracellular and extracellular trafficking of copper. Thus, defects in copper uptake or export, at either the cellular or the organismal level, result in pathological copper deficiency or accumulation, respectively.The importance of copper in mammalian biology is illustrated by the diseases caused by mutations in the genes that encode the copper-transporting ATPases ATP7A and ATP7B. Menkes disease is caused by mutations in the gene that encodes ATP7A, which is essential to bring copper from the digestive tract to other organs. Loss-of-function mutations in ATP7A result in severe copper deficiency in all organs but the intestine and kidney, leading to musculoskeletal defects, vascular abnormalities, neurodegeneration, and usually death within the first decade of life (38). Conversely, the copper toxicosis syndrome Wilson disease is caused by mutations in the gene encoding ATP7B, which is highly similar to ATP7A but differs in its intracellular trafficking patterns and tissue distribution (35). Patients with Wilson disease accumulate copper first in the liver and later in other organs, which eventually leads to liver cirrhosis and damage to other organs if copper levels are not reduced therapeutically (8, 23).A number of other copper accumulation disorders have also been described, although their genetic and biochemical mechanisms are generally less well understood. One of the less-characterized disorders of copper accumulation occurs in a subset of Bedlington terriers that lack a functional Commd1 gene (63). COMMD1 is an ∼20-kDa protein that has been implicated in a wide variety of pathways, including nuclear factor κB signaling, response to hypoxia, sodium regulation, and copper homeostasis (7, 17, 40, 62). At least in canines, COMMD1 is required for proper hepatic copper excretion, and it can physically interact with ATP7B, suggesting a common mechanism for human Wilson disease and canine Commd1 deficiency through ATP7B-mediated copper export (18, 58).COMMD1 was independently identified as an interacting partner of the X-linked inhibitor of apoptosis (XIAP) (11). Mammalian inhibitors of apoptosis (IAPs) were originally identified as homologs of a baculovirus IAP and, as the name suggests, were thought to be primarily involved in the regulation of apoptosis (20, 36, 61). Since their initial characterization, it has become clear that IAPs regulate a wide variety of cellular processes, including mitosis, receptor-mediated signaling pathways, and copper metabolism (45, 55). XIAP binds to and ubiquitinates COMMD1 in cells, targeting it for degradation by the proteasome (11). Through this mechanism, XIAP is thought to raise intracellular copper through degradation of a key copper export protein.Surprisingly, while XIAP regulates copper homeostasis through its interaction with COMMD1, XIAP is in turn regulated by intracellular copper levels. Elevated intracellular copper leads to direct binding of copper to cysteine residues within XIAP, resulting in a distinct conformational change (42). This conformational change leads to an altered electrophoretic mobility of XIAP even under denaturing, reducing conditions, and more importantly, it decreases the stability of XIAP and impairs its ability to inhibit caspases. Thus, XIAP seems to participate in a regulatory loop, promoting its own degradation by raising intracellular copper levels. However, the mechanism by which XIAP binds to copper in the cell remains unclear. Because of its toxicity, intracellular copper is tightly controlled so that free copper is unavailable even when total copper is elevated (49). Given that copper-dependent proteins require specific copper chaperones to deliver copper to them, we hypothesized that a chaperone protein might be necessary to mediate copper delivery to XIAP as well.Through a yeast genetic screen designed to identify candidate proteins involved in delivering copper to XIAP, we identified the copper chaperone for superoxide dismutase (SOD), CCS, as an XIAP-interacting protein. We find that CCS is important for copper delivery to XIAP in mammalian cells and furthermore that CCS is a target for ubiquitination through the E3 ubiquitin ligase activity of XIAP. Surprisingly, ubiquitination of CCS by XIAP seems to be proteasome independent and, rather than triggering degradation of CCS, enhances its ability to deliver copper to its physiologic target, SOD1.     

Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin

To examine the mechanisms of copper incorporation during ceruloplasmin biosynthesis, we developed methods to resolve and identify apo and holoceruloplasmin. The identity of holoceruloplasmin was confirmed by oxidase activity staining, immunoblotting, 67Cu-ligand exchange, and 67Cu-ligand blotting. Following metabolic labeling of human liver and lung cell lines with 67Cu, newly synthesized holoceruloplasmin was detected in the culture media as two species with apparent molecular masses of 84 and 79 kDa. Pulse-chase studies demonstrate that exogenous copper is readily available for incorporation into newly synthesized ceruloplasmin and that the kinetics of apo and holoceruloplasmin synthesis and secretion are identical. Inhibition of N-linked glycosylation did not affect the rate or amount of copper incorporated into newly synthesized ceruloplasmin but did result in the secretion of a single 68-kDa holoceruloplasmin moiety. Despite differences in the kinetics of copper uptake between cell lines a linear rate of copper incorporation into newly synthesized ceruloplasmin was observed with no evidence of copper exchange following biosynthesis. Under the conditions studied, holoceruloplasmin accounted for less than 5% of the total ceruloplasmin synthesized and secreted by each cell line. The data indicate that copper is incorporated into newly synthesized ceruloplasmin early in the course of biosynthesis by a process independent of N-linked carbohydrate addition. This process of copper incorporation results in an apparent conformational change in the ceruloplasmin molecule which does not affect the secretory rate of the protein.     

Myelin movements in mature mammalian peripheral nerve fibers

Mature mouse and cat peripheral nerve fibers have been examined in vitro by time-lapse photography. Some Schmidt-Lanterman clefts which were open at the start closed later; other were seen to open and then to close, some of them more than once. The implications of these movements are considered, especially in regard to the question of the passage of materials from the endoneurial connective tissue spaces to the axon. Myelin movements other than those occurring at the Schmidt-Lanterman clefts consisted primarily of the development and frequent regression of indentations of the myelin sheath. A single evagination was seen to develop and then to recede. These myelin movements suggest that previously described invaginations and evaginations of the myelin sheath, including flaps of “redundant myelin”, are not static but rather that they are in a state of movement, forming and regressing at intervals. The possible functional significance of the development and regression of myelin sheath indentations in relationship to axoplasmic flow is discussed.