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author:("klop, Leo")
1.  The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner 
PLoS ONE  2014;9(4):e92408.
The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson’s disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington’s disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.
PMCID: PMC3972230  PMID: 24691167
2.  Clinical utility gene card for: Menkes disease 
PMCID: PMC3190265  PMID: 21487442
3.  D-Serine Influences Synaptogenesis in a P19 Cell Model 
JIMD Reports  2012;6:47-53.
Recently, d-serine has been identified as an important NMDA-receptor co-agonist, which might play a role in central nervous system development. We investigated this by studying rat P19 cells, an established model for neuronal and glial differentiation. Our results show that (1) the d-serine synthesizing enzyme serine racemase was expressed upon differentiation, (2) extracellular d-serine concentrations increased upon differentiation, which was inhibited by serine racemase antagonism, and (3) inhibition of d-serine synthesis or prevention of d-serine binding to the NMDA-receptor increased synaptophysin expression and intercellular connections, supporting a role for NMDA-receptor activation by d-serine, synthesized by serine racemase, in shaping synaptogenesis and neuronal circuitry during central nervous system development. In conjunction with recent evidence from literature, we therefore suggest that d-serine deficiency might be responsible for the severe neurological phenotype seen in patients with serine deficiency disorders. In addition, this may provide a pathophysiological mechanism for a role of d-serine deficiency in psychiatric disorders.
PMCID: PMC3565666  PMID: 23430939
4.  Liver-Specific Commd1 Knockout Mice Are Susceptible to Hepatic Copper Accumulation 
PLoS ONE  2011;6(12):e29183.
Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1Δhep mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1Δhep mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1Δhep mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.
PMCID: PMC3245254  PMID: 22216203
5.  Increased concentrations of both NMDA receptor co-agonists d-serine and glycine in global ischemia: a potential novel treatment target for perinatal asphyxia 
Amino Acids  2011;43(1):355-363.
Worldwide, perinatal asphyxia is an important cause of morbidity and mortality among term-born children. Overactivation of the N-methyl-d-aspartate receptor (NMDAr) plays a central role in the pathogenesis of cerebral hypoxia–ischemia, but the role of both endogenous NMDAr co-agonists d-serine and glycine remains largely elusive. We investigated d-serine and glycine concentration changes in rat glioma cells, subjected to oxygen and glucose deprivation (OGD) and CSF from piglets exposed to hypoxia–ischemia by occlusion of both carotid arteries and hypoxia. We illustrated these findings with analyses of cerebrospinal fluid (CSF) from human newborns affected by perinatal asphyxia. Extracellular concentrations of glycine and d-serine were markedly increased in rat glioma cells exposed to OGD, presumably through increased synthesis from l-serine. Upon reperfusion glycine concentrations normalized and d-serine concentrations were significantly lowered. The in vivo studies corroborated the finding of initially elevated and then normalizing concentrations of glycine and decreased d-serine concentrations upon reperfusion These significant increases of both endogenous NMDAr co-agonists in combination with elevated glutamate concentrations, as induced by global cerebral ischemia, are bound to lead to massive NMDAr activation, excitotoxicity and neuronal damage. Influencing these NMDAr co-agonist concentrations provides an interesting treatment target for this common, devastating and currently poorly treatable condition.
PMCID: PMC3374112  PMID: 21947661
d-Serine; Glutamate; NMDA receptor; Perinatal asphyxia; Ischemia
6.  Differences in presentation and progression between severe FIC1 and BSEP deficiencies 
Journal of hepatology  2010;53(1):170-178.
Background & Aims
Progressive familial intrahepatic cholestasis (PFIC) with normal serum levels of gamma-glutamyltranspeptidase can result from mutations in ATP8B1 (encoding familial intrahepatic cholestasis 1 [FIC1]) or ABCB11 (encoding bile salt export pump [BSEP]). We evaluated clinical and laboratory features of disease in patients diagnosed with PFIC, who carried mutations in ATP8B1 (FIC1 deficiency) or ABCB11 (BSEP deficiency). Our goal was to identify features that distinguish presentation and course of these 2 disorders, thus facilitating diagnosis and elucidating the differing consequences of ATP8B1 and ABCB11 mutations.
A retrospective multi-center study was conducted, using questionnaires and chart review. Available clinical and biochemical data from 145 PFIC patients with mutations in either ATP8B1 (61 “FIC1 patients”) or ABCB11 (84 “BSEP patients”) were evaluated.
At presentation, serum aminotransferase and bile salt levels were higher in BSEP patients; serum alkaline phosphatase values were higher, and serum albumin values were lower, in FIC1 patients. Elevated white blood cell counts, and giant or multinucleate cells at liver biopsy, were more common in BSEP patients. BSEP patients more often had gallstones and portal hypertension. Diarrhea, pancreatic disease, rickets, pneumonia, abnormal sweat tests, hearing impairment, and poor growth were more common in FIC1 patients. Among BSEP patients, the course of disease was less rapidly progressive in patients bearing the D482G mutation.
Severe forms of FIC1 and BSEP deficiency differed. BSEP patients manifested more severe hepatobiliary disease, while FIC1 patients showed greater evidence of extrahepatic disease.
PMCID: PMC3042805  PMID: 20447715
cholestasis; genetics; transport protein; pediatrics; P-type ATPase; ATP binding cassette protein; ATP8B1; FIC1; ABCB11; BSEP
7.  The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression 
Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-011-0743-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3249196  PMID: 21667063
ATP7A; Copper; COMMD1; Menkes disease; Pharmacological chaperones; Protein folding
8.  Clusterin facilitates COMMD1 and I-κB degradation to enhance NF-κB activity in prostate cancer cells 
Molecular cancer research : MCR  2010;8(1):119-130.
Secretory clusterin (sCLU) is a stress-activated, cytoprotective chaperone that confers broad-spectrum cancer treatment resistance and its targeted inhibitor (OGX-011) is currently in Phase II trials for prostate, lung, and breast cancer. However, molecular mechanisms by which sCLU inhibits treatment-induced apoptosis in prostate cancer remain incompletely defined. We report that sCLU increases NF-κB nuclear translocation and transcriptional activity by serving as a ubiquitin binding protein that enhances COMMD1 and I-κB proteasomal degradation by interacting with members of the SCF-βTrCP E3 ligase family. Knockdown of sCLU in prostate cancer cells stabilizes COMMD1 and I-κB, thereby sequestrating NF-κB in the cytoplasm and decreasing NF-κB transcriptional activity. Comparative microarray profiling of sCLU over-expressing and knockdown prostate cancer cells confirmed that the expression of many NF-κB regulated genes positively correlate with sCLU levels. We propose that elevated levels of sCLU promote prostate cancer cell survival by facilitating degradation of COMMD1 and I-κB, thereby activating the canonical NF-κB pathway.
PMCID: PMC2808437  PMID: 20068069
prostate cancer; clusterin; COMMD1; I-κB; NF-κB
9.  COMMD1 disrupts HIF-1α/β dimerization and inhibits human tumor cell invasion 
The Journal of Clinical Investigation  2010;120(6):2119-2130.
The gene encoding COMM domain–containing 1 (COMMD1) is a prototypical member of the COMMD gene family that has been shown to inhibit both NF-κB– and HIF-mediated gene expression. NF-κB and HIF are transcription factors that have been shown to play a role in promoting tumor growth, survival, and invasion. In this study, we demonstrate that COMMD1 expression is frequently suppressed in human cancer and that decreased COMMD1 expression correlates with a more invasive tumor phenotype. We found that direct repression of COMMD1 in human cell lines led to increased tumor invasion in a chick xenograft model, while increased COMMD1 expression in mouse melanoma cells led to decreased lung metastasis in a mouse model. Decreased COMMD1 expression also correlated with increased expression of genes known to promote cancer cell invasiveness, including direct targets of HIF. Mechanistically, our studies show that COMMD1 inhibits HIF-mediated gene expression by binding directly to the amino terminus of HIF-1α, preventing its dimerization with HIF-1β and subsequent DNA binding and transcriptional activation. Altogether, our findings demonstrate a role for COMMD1 in tumor invasion and provide a detailed mechanism of how this factor regulates the HIF pathway in cancer cells.
PMCID: PMC2877941  PMID: 20458141
10.  Distinct Wilson-disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B 
Gastroenterology  2007;133(4):1316-1326.
Wilson disease is characterized by hepatic copper overload and caused by mutations in the gene encoding the copper transporting P-type ATPase ATP7B. ATP7B interacts with COMMD1, a protein that is deleted in Bedlington terriers with hereditary copper toxicosis. Here we characterized the implications of the interaction between COMMD1 and ATP7B in relation to the pathogenesis of Wilson disease.
GST pull-down experiments, co-immunoprecipitations, immunofluoresensce microscopy, site-directed mutagenesis and biosynthetic labeling experiments were performed to characterize the interaction between COMMD1 and ATP7B and the effects of Wilson disease causing mutations.
COMMD1 specifically interacted with the amino-terminal region of ATP7B. This interaction was independent of intracellular copper levels and of the expression of the copper chaperone ATOX1. Four Wilson disease patient derived mutations in this region of ATP7B significantly increased its binding to COMMD1. Two of these mutations also resulted in mislocalization and increased degradation rate of ATP7B. Although COMMD1 did not affect copper-induced trafficking of ATP7B, it markedly decreased the stability of newly synthesized ATP7B.
Our data implicate COMMD1 in the pathogenesis of Wilson disease and indicate that COMMD1 exerts its regulatory role in copper homeostasis through the regulation of ATP7B stability.
PMCID: PMC2857755  PMID: 17919502
11.  COMMD1 Promotes pVHL and O2-Independent Proteolysis of HIF-1α via HSP90/70 
PLoS ONE  2009;4(10):e7332.
The Copper Metabolism MURR1 Domain containing 1 protein COMMD1 has been associated with copper homeostasis, NF-κB signaling, and sodium transport. Recently, we identified COMMD1 as a novel protein in HIF-1 signaling. Mouse embryos deficient for Commd1 have increased expression of hypoxia/HIF-regulated genes i.e. VEGF, PGK and Bnip3. Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis, which control angiogenesis, erythropoiesis, glycolysis and cell survival/proliferation under normal and pathologic conditions. Although HIF activity is mainly controlled by ubiquitination and protein degradation by the von Hippel Lindau (pVHL) tumor suppressor gene other mechanisms have recently been identified that regulate HIF signaling independently of pVHL.
Principal Findings
Here we characterized the mechanism by which COMMD1 regulates HIF-1α protein degradation. We show that COMMD1 competes with the chaperone heat shock protein HSP90β for binding to the NH2-terminal DNA-binding and heterodimerization domain of HIF-1α to regulate HIF-1α stability together with HSP70. Inhibition of HSP90 activity with 17-Allylamino-17-demethoxygeldanamycin (17-AAG) increased COMMD1-mediated HIF-1α degradation independent of ubiquitin and pVHL.
These data reveal a novel role for COMMD1 in conjunction with HSP90β/HSP70 in the ubiquitin and O2-independent regulation of HIF-1α.
PMCID: PMC2750754  PMID: 19802386
12.  COMMD1 expression is controlled by critical residues that determine XIAP binding 
The Biochemical journal  2009;417(2):601-609.
COMM domain-containing (or COMMD) proteins participate in several cellular processes, ranging from NF-κB regulation, copper homeostasis, sodium transport and adaptation to hypoxia. The best studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP functions as its ubiquitin ligase. In this study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMM domain containing proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant unable to bind to XIAP demonstrated complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiologic protein levels due to increased ubiquitination, but this regulatory event is circumvented when expressing a mutant form that cannot bind XIAP. Altogether, our findings indicate that COMMD1 expression is primarily controlled by protein ubiquitination and its interaction with IAP proteins plays an essential role.
PMCID: PMC2606926  PMID: 18795889
COMMD1; COMMD proteins; XIAP; IAP; ubiquitin
Molecular pharmacology  2008;74(3):697-704.
Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) upregulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme, glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunits. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Since GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into considerations that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through upregulation of human copper transporter 1 (hCtr1), which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. While rates of Cu transport were also upregulated in the transfected cells, these cells exhibited biochemical signature of Cu deficiency, suggesting that GSH functions as an intracellular Cu-chelator and that overexpression of GSH can alter Cu metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes bioavailable Cu pool, leading to upregulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications that modulation of intracellular Cu pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.
PMCID: PMC2583459  PMID: 18523133
14.  Transcription Factor Sp1 Plays an Important Role in the Regulation of Copper Homeostasis in Mammalian Cells 
Molecular pharmacology  2008;74(3):705-713.
Copper is an essential metal nutrient, yet Cu overload is toxic. Here, we report that human copper transporter 1 (hCtr1) plays an important role in the maintenance of Cu homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under Cu-depleted conditions and down-regulated under Cu-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N-terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular Cu contents down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1-binding sites are involved in the basal and Cu concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that zinc finger domain of Sp1 functions as a sensor of Cu that regulates hCtr1 up-and-down in response to Cu concentration variations. Our results demonstrate that mammalian Cu homeostasis is maintained at the hCtr1 mRNA level which is regulated by the Sp1 transcription factor.
PMCID: PMC2574735  PMID: 18483225
15.  Increased Activity of Hypoxia-Inducible Factor 1 Is Associated with Early Embryonic Lethality in Commd1 Null Mice▿  
Molecular and Cellular Biology  2007;27(11):4142-4156.
COMMD1 (previously known as MURR1) belongs to a novel family of proteins termed the copper metabolism gene MURR1 domain (COMMD) family. The 10 COMMD family members are well conserved between vertebrates, but the functions of most of the COMMD proteins are unknown. We recently established that COMMD1 is associated with the hepatic copper overload disorder copper toxicosis in Bedlington terriers. Recent in vitro studies indicate that COMMD1 has multiple functions, including sodium transport and NF-κB signaling. To elucidate the function of Commd1 in vivo, we generated homozygous Commd1 null (Commd1−/−) mice. Commd1−/− embryos died in utero between 9.5 and 10.5 days postcoitum (dpc), their development was generally retarded, and placenta vascularization was absent. Microarray analysis identified transcriptional upregulation of hypoxia-inducible factor 1 (HIF-1) target genes in 9.5-dpc Commd1−/− embryos compared to normal embryos, a feature that was associated with increased Hif-1α stability. Consistent with these observations, COMMD1 physically associates with HIF-1α and inhibits HIF-1α stability and HIF-1 transactivation in vitro. Thus, this study identifies COMMD1 as a novel regulator of HIF-1 activity and shows that Commd1 deficiency in mice leads to embryonic lethality associated with dysregulated placenta vascularization.
PMCID: PMC1900009  PMID: 17371845

Results 1-15 (15)