XIAP: cell death regulation meets copper homeostasis

X-linked inhibitor of apoptosis (XIAP), traditionally known as an anti-apoptotic protein, has recently been shown to be involved in copper homeostasis. XIAP promotes the ubiquitination and degradation of COMMD1, a protein that promotes the efflux of copper from the cell. Through its effects on COMMD1, XIAP can regulate copper export from the cell and potentially represents an additional intracellular sensor for copper levels. XIAP binds copper directly and undergoes a substantial conformational change in the copper-bound state. This in turn destabilizes XIAP, resulting in lowered steady-state levels of the protein. Furthermore, copper-bound XIAP is unable to inhibit caspases and cells that express this form of the protein exhibit increased rates of cell death in response to apoptotic stimuli. These events take place in the setting of excess intracellular copper accumulation as seen in copper toxicosis disorders such as Wilson's disease and establish a new relationship between copper levels and the regulation of cell death via XIAP. These findings raise important questions about the role of XIAP in the development of copper toxicosis disorders and may point to XIAP as a potential therapeutic target in these disease states.     

XIAP Is a copper binding protein deregulated in Wilson's disease and other copper toxicosis disorders

X-linked inhibitor of apoptosis (XIAP), known primarily for its caspase inhibitory properties, has recently been shown to interact with and regulate the levels of COMMD1, a protein associated with a form of canine copper toxicosis. Here, we describe a role for XIAP in copper metabolism. We find that XIAP levels are greatly reduced by intracellular copper accumulation in Wilson's disease and other copper toxicosis disorders and in cells cultured under high copper conditions. Elevated copper levels result in a profound, reversible conformational change in XIAP due to the direct binding of copper to XIAP, which accelerates its degradation and significantly decreases its ability to inhibit caspase-3. This results in a lowering of the apoptotic threshold, sensitizing the cell to apoptosis. These data provide an unsuspected link between copper homeostasis and the regulation of cell death through XIAP and may contribute to the pathophysiology of copper toxicosis disorders.     

Copper-dependent interaction of dynactin subunit p62 with the N terminus of ATP7B but not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.     

Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase-linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain-of-toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper-chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild-type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up-regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age-dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co-aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.     

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.     

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.     

X-linked inhibitor of apoptosis deficiency in the TRAMP mouse prostate cancer model

Deregulation of apoptotic pathways plays a central role in cancer pathogenesis. X-linked inhibitor of apoptosis protein (XIAP), is an antiapoptotic molecule, whose elevated expression has been observed in tumor specimens from patients with prostate carcinoma. Studies in human cancer cell culture models and xenograft tumor models have demonstrated that loss of XIAP sensitizes cancer cells to apoptotic stimuli and abrogates tumor growth. In view of these findings, XIAP represents an attractive antiapoptotic therapeutic target for prostate cancer. To examine the role of XIAP in an immunocompetent mouse cancer model, we have generated transgenic adenocarcinoma of the mouse prostate (TRAMP) mice that lack XIAP. We did not observe a protective effect of Xiap deficiency in TRAMP mice as measured by tumor onset and overall survival. In fact, there was an unexpected trend toward more aggressive disease in the Xiap-deficient mice. These findings suggest that alternative mechanisms of apoptosis resistance are playing a significant oncogenic role in the setting of Xiap deficiency. Our study has implications for XIAP-targeting therapies currently in development. Greater understanding of these mechanisms will aid in combating resistance to XIAP-targeting treatment, in addition to optimizing selection of patients who are most likely to respond to such treatment.     

Copper depletion down-regulates expression of the Alzheimer's disease amyloid-beta precursor protein gene

Alzheimer's disease is characterized by the accumulation of amyloid-beta peptide, which is cleaved from the amyloid-beta precursor protein (APP). Reduction in levels of the potentially toxic amyloid-beta has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-beta in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.     

Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression

A fall in circulating levels of cardiac survival factor insulin-like growth factor 1 (IGF-1) contributes to cardiac aging. To better understand the role of IGF-1 in cardiac aging, we examined the influence of cardiac IGF-1 overexpression on lifespan, cardiomyocyte intracellular Ca²⁺ homeostasis, protein damage, apoptosis and expression of pro- and anti-apoptotic proteins in young and old mice. Mouse survival rate was constructed by the Kaplan–Meier curve. Intracellular Ca²⁺ was evaluated by fura-2 fluorescence. Protein damage was determined by protein carbonyl formation. Apoptosis was assessed by caspase-8 expression, caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay. Pro- and anti-apoptotic proteins including Bax, p53, pp53, Bcl2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) were assessed by Western blot. Aging decreased plasma in IGF-1 levels, elevated myocyte resting intracellular Ca²⁺ levels, reduced electrically stimulated rise in intracellular Ca²⁺ and delayed intracellular Ca²⁺ decay associated with enhanced protein carbonyl formation, caspase-8 expression and caspase-3 activity in FVB mice, all of which with the exception of elevated resting intracellular Ca²⁺ were attenuated by IGF-1. Aging up-regulated expression of Bax, Bcl2 and ARC, down-regulated XIAP expression and did not affect p53, pp53 and Omi/HtrA2. The IGF-1 transgene attenuated or nullified aging-induced changes in Bax, Bcl2 and XIAP. Our data suggest a beneficial role for IGF-1 in aging-induced survival, cardiac intracellular Ca²⁺ homeostasis, protein damage and apoptosis possibly related to pro- and anti-apoptotic proteins.     

COMMD1 (copper metabolism MURR1 domain-containing protein 1) regulates Cullin RING ligases by preventing CAND1 (Cullin-associated Nedd8-dissociated protein 1) binding

Cullin RING ligases (CRLs), the most prolific class of ubiquitin ligase enzymes, are multimeric complexes that regulate a wide range of cellular processes. CRL activity is regulated by CAND1 (Cullin-associated Nedd8-dissociated protein 1), an inhibitor that promotes the dissociation of substrate receptor components from the CRL. We demonstrate here that COMMD1 (copper metabolism MURR1 domain-containing 1), a factor previously found to promote ubiquitination of various substrates, regulates CRL activation by antagonizing CAND1 binding. We show that COMMD1 interacts with multiple Cullins, that the COMMD1-Cul2 complex cannot bind CAND1, and that, conversely, COMMD1 can actively displace CAND1 from CRLs. These findings highlight a novel mechanism of CRL activation and suggest that CRL regulation may underlie the pleiotropic activities of COMMD1.     

The Schizosaccharomyces pombe Pccs protein functions in both copper trafficking and metal detoxification pathways

Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain.     

Quantitative PCR method to detect a 13-kb deletion in the MURR1 gene associated with copper toxicosis and HIV-1 replication

The recently discovered locus for copper toxicosis (CT) in Bedlington terriers (BT) has a 13-kb deletion enveloping the 187-bp exon-2 of the MURR1 gene. This MURR1 gene is not only involved with biliary copper excretion but also associated with HIV-1 replication. The microsatellite C04107 lying in an intron of the MURR1 gene is highly associated with the disease but shows haplotype diversity. The only solid molecular test for the disease is by showing the deletion in exon-2 in cDNA in liver tissue; this test is not robust on RNA from peripheral leukocytes because of their low MURR1 expression level. Because of these drawbacks, we developed a new quantitative PCR (Q-PCR) protocol. Here we show that the MURR1 exon-2/exon-3 ratio measured by Q-PCR on genomic DNA correlates perfectly with the microsatellite marker and with RT-PCR data from blood samples, buccal swabs, and liver biopsies. In view of the important role of MURR1 in cells of many tissues, this new test has a wide range of applications in comparative biomedical research. Furthermore, Q-PCR on DNA may be a new tool in general to analyze mutations that cannot be approached by standard methods.Robert P. Favier and Bart Spee contributed equally to this work.     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

Over-expression of X-Linked Inhibitor of Apoptosis Protein Modulates Multiple Aspects of Neuronal Ca2+ Signaling

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca²⁺) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca²⁺-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca²⁺ concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca²⁺ concentrations. By contrast, the rise in intracellular Ca²⁺ concentrations produced by N-methyl-d-aspartate receptor stimulation and voltage gated Ca²⁺ channel activation were markedly attenuated by XIAP over-expression. The release of Ca²⁺ from intracellular stores induced by the sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca²⁺ concentrations elicited by l-glutamate suggesting that XIAP influences Ca²⁺ signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca²⁺ by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.