Arnab Gupta

Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance.

The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

Human copper transporters: mechanism, role in human diseases and therapeutic potential

Normal copper homeostasis is essential for human growth and development. Copper deficiency, caused by genetic mutations, inadequate diet or surgical interventions, may lead to cardiac hypertrophy, poor neuronal myelination, blood vessel abnormalities and impaired immune response. Copper overload is associated with morphological and metabolic changes in tissues and, if untreated, eventual death. Recent reports also indicate that changes in the expression of copper transporters alter the sensitivity of cancer cells to major chemotherapeutic drugs, such as cisplatin, although the mechanism behind this important phenomenon remains unclear. This review summarizes current information on the molecular characteristics of copper transporters CTR1, CTR2, ATP7A and ATP7B, their roles in mammalian copper homeostasis and the physiological consequences of their inactivation. The mechanisms through which copper transporters may influence cell sensitivity to cisplatin are discussed. Regulation of human copper homeostasis has significant therapeutic potential and requires the detailed understanding of copper transport mechanisms.

Cell‐Specific Trafficking Suggests a new role for Renal ATP7B in the Intracellular Copper Storage

Human Cu‐ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney‐specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase‐mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2–3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell‐specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.

Cellular copper levels determine the phenotype of the Arg875 variant of ATP7B/Wilson disease protein

In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly875→Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg875 phenotype. Addition of only 0.5–5 μM copper triggers the exit of ATP7B-Arg875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg875 is attributable to increased unfolding of the Arg875-containing A-domain. Copper is not required for the folding of ATP7B-Arg875 during biosynthesis, but it stabilizes protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the “disease-like” phenotype of ATP7B-Arg875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

Background: ATP7B trafficking from Golgi to vesicles is essential for copper homeostasis. Results: Mutating Ser-340/341 alters the inter-domain contacts, diminishes protein phosphorylation, and shifts ATP7B localization to vesicles. Conclusion: Spatial arrangement of functional domains determines ATP7B readiness to traffic, whereas phosphorylation may maintain the trafficking-compatible state. Significance: The proposed mechanism explains how unrelated signals trigger similar trafficking responses from ATP7B. The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.

Matrix metalloproteinase-1 (MMP-1) Promoter polymorphisms are well linked with lower stomach tumor formation in eastern Indian population.

Expression of matrix metalloproteinase-1 (MMP-1), an interstitial collagenase, plays a major role in cellular invasion during development of gastric cancer, a leading cause of death worldwide. A single-nucleotide polymorphism (SNP) −1607 1G/2G site of the MMP-1 gene promoter has been reported to alter transcription level. While the importance’s of other SNPs in the MMP-1 promoter have not yet been studied in gastric cancer, our aim was to investigate MMP-1 gene promoter polymorphisms and gastric cancer susceptibility in eastern Indian population. A total of 145 gastric cancer patients and 145 healthy controls were genotyped for MMP-1 −1607 1G/2G (rs1799750) by PCR-restriction fragment length polymorphism (RFLP), while MMP-1 −519 A/G (rs1144393), MMP-1 −422 T/A (rs475007), MMP-1 −340 T/C (rs514921) and MMP-1 −320 T/C (rs494379) were genotyped by DNA sequencing. A positive association was found with MMP-1 −422 T/A SNP that showed significant risk for regional lymph node metastasis (P = 0.021, Odd’s ratio (OR) = 3.044, Confidence intervals (CI) = 1.187–7.807). In addition, we found a significant association with lower stomach tumor formation among gastric cancer patients for three adjacent polymorphisms near the transcriptional start sites of [MMP-1 −422 T/A (P = 0.043, OR = 2.182, CI = 1.03–4.643), MMP-1 −340 T/C (P = 0.075, OR = 1.97, CI = 0.94–4.158) and MMP-1 −320 T/C (P = 0.034, OR = 2.224, CI = 1.064–40731)]. MMP-1 level in patients’ serum was correlated with MMP-1 promoter haplotypes conferring these three SNPs to evaluate the functional importance of these polymorphisms in lower stomach tumor formation and significant correlation was observed. Furthermore, MMP-1 −519 A/G polymorphism displayed poor cellular differentiation (P = 0.024, OR = 3.8, CI = 1.69–8.56) attributing a higher risk of cancer progression. In conclusion, MMP-1 proximal promoter SNPs are associated with the risk of lower stomach tumor formation and node metastasis in eastern Indian population.

Evolution of Copper Transporting ATPases in Eukaryotic Organisms

Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution.

Matrix metalloproteinase3 gene promoter polymorphisms and their haplotypes are associated with gastric cancer risk in eastern Indian population.

Single nucleotide polymorphisms (SNPs) of matrix metalloproteinase3 (MMP3) promoter in the development and progression of gastric cancer of whole stomach has never been investigated in any population. We conducted a hospital‐based case‐control study to explore the MMP3 SNPs and their haplotypes with the risk of gastric cancer for the first time in eastern Indian population. A total of 218 gastric cancer patients and 175 healthy controls were genotyped for MMP3‐1612 5A/6A (rs3025058) by PCR–RFLP and rechecked 10% by DNA sequencing. MMP3‐707 A/G (rs522616) and MMP3‐375 C/G (rs617819) were genotyped by DNA sequencing among 209 patients and 154 controls. MMP3‐1612 5A6A genotype (P = 0.026, odds ratio (OR) = 1.756, confidence interval (CI) = 1.070–2.883), combined 5A5A and 5A6A genotype (P = 0.015, OR = 1.791, CI = 1.122–2.858) and 5A allele (P = 0.002, OR = 1.75, CI = 1.21–2.53) and; MMP3‐707 GG genotype (P = < 0.0001; OR = 9.612; 95% CI = 3.403–27.147), combined GG and AG genotype (P = 0.001, OR = 2.201, CI = 1.385–3.498) and G allele (P = <0.0001, OR = 2.189, CI = 1.582–3.033) conferred significant risk for gastric cancer development. Also, tobacco addicted individuals with combined 5A5A and 5A6A genotype (P = 0.005, OR = 2.952, CI = 1.377–6.327) at −1612 position of MMP3 promoter displayed a higher risk to gastric cancer development. The genotypic combinations of all three MMP3 promoter polymorphisms and their haplotypes with increasing risk allele in a dose‐dependent manner showed a potential risk for developing gastric cancer. The analyses suggested that the MMP3‐707 G/G and MMP3‐1612 5A/6A polymorphisms are potential independent predictors of gastric cancer risk development.

A novel COMMD1 mutation Thr174Met associated with elevated urinary copper and signs of enhanced apoptotic cell death in a Wilson Disease patient

Wilson disease (WD) results from accumulation of copper and caused due to mutations in ATP7B, a copper transporting ATPase. Besides regular hepatic and neurological symptoms, WD patients occasionally manifest atypical symptoms due to unknown cause. To understand the molecular etiology of atypical WD manifestations, we screened COMMD1, a gene implicated in canine copper toxicosis, in 109 WD patients including those with atypical symptoms. In a patient showing apoptotic symptoms and high urinary copper surpassing normal WD levels, we identified a novel, putative mutation in COMMD1. Two other changes were also identified in the gene. We have examined genotype-phenotype correlation between the detected changes and the atypical presentation of the WD patient.

Association of MMP7 −181A→G Promoter Polymorphism with Gastric Cancer Risk INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB)

Background: Effect of tobacco carcinogen nicotine on MMP7 transcription and gastric cancer risk remains unknown. Results: Preferential binding of pCREB, induced by nicotine, to G-allele promoter enhanced the transcription and thus aggravated gastric cancer risk. Conclusion: Nicotine shows an additive effect over MMP7 GG genotype toward gastric cancer risk. Significance: These findings may help in the clinical management of gastric cancer. Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The −181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of −181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1–3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07–5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the −181G than the −181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.