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Stephen G Kaler - One of the best experts on this subject based on the ideXlab platform.
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estimated birth prevalence of menkes disease and ATP7A related disorders based on the genome aggregation database gnomad
Molecular genetics and metabolism reports, 2020Co-Authors: Stephen G Kaler, Carlos Ferreira, Lung S YamAbstract:Abstract Background Previous estimates of the prevalence of Menkes disease, a lethal X-linked recessive disorder of copper metabolism, were based on confirmed clinical cases ascertained from specific populations and varied from 1 in 40,000 to 1 in 354,507. With newly available population-based allelic frequencies of DNA sequence variants, the expected birth prevalence of Menkes disease and other ATP7A-related phenotypes can be reconsidered using Hardy-Weinberg theoretical principles. Methods We reviewed the canonical ATP7A transcript in the current version of gnomAD (v2.1.1) to evaluate frequency of complete loss-of-function alleles in a diverse normal control population. As a comparator, we used the DMD locus, associated with Duchenne and Becker Muscular Dystrophy, another X-linked recessive trait. We applied Hardy-Weinberg theory and PolyPhen-2 in silico plus REVEL and CADD ensemble analyses to calculate estimated frequencies of normal and predicted deleterious ATP7A alleles. Results We identified 1106 total ATP7A variants out of 205,523 alleles in gnomAD, with missense variants most common (43.4%). Complete loss-of-function variants were found in four ATP7A alleles (frequency = 0.0000194), including three frameshift/nonsense mutations and one canonical splice donor site defect. Assuming Hardy-Weinberg equilibrium, this frequency of pathogenic alleles predicts 1 in 34,810 live male births with Menkes disease or other ATP7A-related disorders each year in the US. The same analysis for DMD loss-of-function variants predicted 1 in 7246 newborn males with Duchenne (or Becker) muscular dystrophy. We also identified nine ATP7A missense variants in gnomAD predicted as deleterious by PolyPhen-2 and stringent REVEL/CADD criteria, comprising 12 more disease-causing alleles and raising the estimated birth prevalence to 1 in 8664 and predicting 225 newborns with Menkes disease or other ATP7A-related disorders per year in the US alone. Conclusions Assuming Hardy-Weinberg equilibrium, the allelic frequency of deleterious ATP7A variants in a genomic database from a large diverse population predicts a birth prevalence of Menkes disease or ATP7A-related disorders as high as 1 in 8664 live male births. This genome-driven ascertainment of deleterious ATP7A alleles in the population implies a higher birth prevalence of Menkes disease and ATP7A-related conditions than previously appreciated. A population-based newborn screening pilot study for Menkes disease will be instrumental in confirming the prediction.
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menkes disease and other disorders related to ATP7A
2019Co-Authors: Cynthia Abou Zeid, Stephen G KalerAbstract:Abstract The ATP7A-related disorders are a heterogeneous group of diseases resulting from ATP7A (Menkes ATPase) dysfunction. They have varying phenotypes and clinical diseases, depending on the underlying genetic mutations. Some ensue from direct changes in the gene that encodes the ATP7A protein. The severity of the mutations can affect the phenotype expressed. If the mutations are severe loss-of-function mutations, the result is a lethal neurodevelopmental and systemic disease with low copper and reduced cuproenzymes activity (Menkes disease). If the mutations are milder, such as with leaky splice junction defects, some copper transport is possible and the phenotype is less severe, as in occipital horn syndrome. Two unique missense mutations that do not impair the copper transport function of ATP7A but rather its trafficking, cause a motor neuron-specific illness that develops gradually, with no evidence of abnormal copper metabolism. ATP7A function can also be affected by mutations outside of its coding gene, such as in MEDNIK and Huppke–Brendel syndromes. The resulting defects in intracellular localization of the ATP7A (and ATP7B) pump(s) can impair normal copper transport function.
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normal human copper metabolism
2019Co-Authors: Cynthia Abou Zeid, Stephen G KalerAbstract:Abstract Copper is a versatile divalent metal needed as a cofactor for numerous metalloenzymes but can also induce toxic oxidative stress, for which reason its levels are tightly regulated. Multiple gene products maintain copper homeostasis in various cell types. Copper importers, such as copper transporter 1 (CTR1), are responsible for copper uptake into the cytoplasm of cells. In the intracellular milieu, copper chaperones escort the metal ion to designated cellular compartments and other copper transporters. Cytosolic proteins such as metallothioneins bind Cu+ and act as buffers to prevent the production of toxic reactive oxygen species. Finally, the copper transporters ATP7A and ATP7B have dual major functions dictated by intracellular copper levels. In normal copper states, they pump copper into the secretory pathway for metalation of cuproenzymes. When faced with excessive intracellular copper levels, they traffic to the plasma membrane and remove copper from the cell.
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the activity of menkes disease protein ATP7A is essential for redox balance in mitochondria
Journal of Biological Chemistry, 2016Co-Authors: Ashima Bhattacharjee, Haojun Yang, Megan Duffy, Emily Robinson, Arianrhod Conradantoville, Tony R Capps, Lelita T Braiterman, Michael J Wolfgang, Michael P Murphy, Stephen G KalerAbstract:Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).
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direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking
Human Molecular Genetics, 2015Co-Authors: Stephen G KalerAbstract:ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7A(P1386S) causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7A(P1386S) partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy.
Svetlana Lutsenko - One of the best experts on this subject based on the ideXlab platform.
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ATP7A-mediated transport of Cu into the secretory pathway is essential for SSAO activity.
2018Co-Authors: Haojun Yang, Jason L Burkhead, Michael J Wolfgang, Martina Ralle, Neha Dhawan, Susana Rodriguez, Jack H. Kaplan, William G. Wong, Norman Haughey, Svetlana LutsenkoAbstract:(A) Cu limitation with 10 μM TTM for 48 h decreases the SSAO activity (n = 3). (B) SSAO activity in the epididymal adipose tissue from 13-wk-old male rats fed with Cu-adequate or low-Cu diet for 8 wk (n = 7). (C) Down-regulation of ATP7A in ATP7A+/− adipocytes causes a decrease in SSAO activity (n = 4). (D) Immunocytochemistry shows that SSAO transits the ATP7A-containing compartment on its way to the plasma membrane. Top: ATP7A (green) is localized to the TGN, as evidenced by its colocalization with the TGN marker Syn6 (red); Bottom: SSAO (red) is present at the plasma membrane and inside the cells, where it colocalizes with ATP7A (green). Underlying data can be found in S1 Data; Student’s t test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.05. Cu, copper; ns, not significant; SSAO, semicarbazide-sensitive amine oxidase; Syn6, syntaxin 6; TGN, trans-Golgi network; TTM, tetrathiomolybdate; WT, wild type.
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copper metabolism ATP7A and menkes disease
eLS, 2015Co-Authors: Hannah Pierson, Svetlana Lutsenko, Zeynep TumerAbstract:ATP7A is an ATP-driven copper transport protein that plays an essential role in human health. ATP7A is critically involved in dietary copper uptake in the intestine. In addition, ATP7A delivers copper to numerous copper-dependent enzymes within the secretory pathway and facilitates copper transfer to the brain. Inactivating mutations in ATP7A are associated with severe and often lethal pathologies, such as Menkes disease, occipital horn syndrome, and distal motor neuropathy. Genetic and biochemical studies have demonstrated that disease-causing mutations disrupt ATP7A in many ways, including disruption of biosynthesis, impairment of stability, inactivation of copper transport activity and disturbance of trafficking behaviour. Cellular studies also indicate that ATP7A is a subject of complex regulation. Despite significant progress in the characterisation of ATP7A's function, cell-specific regulation of this transporter remains poorly understood. Many aspects of pathologies caused by mutations in ATP7A require further in depth studies. Key Concepts Copper is the third most abundant trace element in the body, after iron and zinc, and it is required for normal function of important copper-dependent enzymes. Copper deficiency is detrimental to the development and function of many organs especially the central nervous system. Precise regulation of intracellular copper levels is vitally important because, although essential, excess copper has detrimental effects on metabolism. Several transporter molecules and carrier proteins are involved in regulating copper homeostasis. ATP7A is a member of a large family of P-type ATPases. These ATP-utilising membrane proteins pump ions across cellular membranes against a concentration gradient. ATP7A is involved in the delivery of copper to cuproenzymes in the secretory pathway and in the export of surplus copper from cells. Genetic defects in ATP7A leads to the X-linked recessive Menkes disease. Menkes disease is a multi-systemic lethal disorder, marked by neurodegenerative symptoms and connective tissue manifestations. Most of the clinical features of Menkes disease can be explained by deficiency of various copper-dependent enzymes. ATP7A mutations vary from single nucleotide changes to microscopically detectable chromosome abnormalities. Ultimate diagnostic proof of Menkes disease is the demonstration of the molecular defect in ATP7A. Menkes disease patients show progressive deterioration of brain function. Administration of copper-histidine before brain damage may slow the disease progression and result in less severe neurological symptoms. Keywords: ATP7A; ATP7A; copper; copper metabolism; Menkes disease; occipital horn syndrome
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Golgi in copper homeostasis: a view from the membrane trafficking field
Histochemistry and Cell Biology, 2013Co-Authors: Roman Polishchuk, Svetlana LutsenkoAbstract:Copper is essential for a variety of important biological processes as a cofactor and regulator of many enzymes. Incorporation of copper into the secreted and plasma membrane-targeted cuproenzymes takes place in Golgi, a compartment central for normal copper homeostasis. The Golgi complex harbors copper-transporting ATPases, ATP7A and ATP7B that transfer copper from the cytosol into Golgi lumen for incorporation into copper-dependent enzymes. The Golgi complex also sends these ATPases to appropriate post-Golgi destinations to ensure correct Cu fluxes in the body and to avoid potentially toxic copper accumulation. Mutations in ATP7A or ATP7B or in the proteins that regulate their trafficking affect their exit from Golgi or subsequent retrieval to this organelle. This, in turn, disrupts the homeostatic Cu balance, resulting in copper deficiency (Menkes disease) or copper overload (Wilson disease). Research over the last decade has yielded significant insights into the enzymatic properties and cell biology of the copper ATPases. However, the mechanisms through which the Golgi regulates trafficking of ATP7A/7B and, therefore, maintains Cu homeostasis remain unclear. This review summarizes current data on the role of the Golgi in Cu metabolism and outlines questions and challenges that should be addressed to understand ATP7A and ATP7B trafficking mechanisms in health and disease.
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Structural organization of human Cu-transporting ATPases: learning from building blocks
JBIC Journal of Biological Inorganic Chemistry, 2009Co-Authors: Amanda N. Barry, Ujwal Shinde, Svetlana LutsenkoAbstract:Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B play an essential role in human physiological function. Their primary function is to deliver copper to the secretory pathway and export excess copper from the cell for removal or further utilization. Cells employ Cu-ATPases in numerous physiological processes that include the biosynthesis of copper-dependent enzymes, lactation, and response to hypoxia. Biochemical studies of human Cu-ATPases and their orthologs have demonstrated that Cu-ATPases share many common structural and mechanistic characteristics with other members of the P-type ATPase family. Nevertheless, the Cu-ATPases have a unique coordinate environment for their ligands, copper and ATP, and additional domains that are required for sophisticated regulation of their intracellular localization and activity. Here, we review recent progress that has been made in understanding the structure of Cu-ATPases from the analysis of their individual domains and orthologs from microorganisms, and speculate about the implications of these findings for the function and regulation of human copper pumps.
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cellular multitasking the dual role of human cu atpases in cofactor delivery and intracellular copper balance
Archives of Biochemistry and Biophysics, 2008Co-Authors: Svetlana Lutsenko, Arnab Gupta, Jason L Burkhead, Vesna ZuzelAbstract: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.
Liqiang Song - One of the best experts on this subject based on the ideXlab platform.
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mir 495 enhances the sensitivity of non small cell lung cancer cells to platinum by modulation of copper transporting p type adenosine triphosphatase a ATP7A
Journal of Cellular Biochemistry, 2014Co-Authors: Liqiang SongAbstract:Copper-transporting P-type adenosine triphosphatase A (ATP7A) is associated with platinum drug resistance in non-small cell lung cancer (NSCLC). microRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at post-transcriptional level. In this study, the aim is to explore which miRNAs might participate in the platinum resistance by targeting ATP7A in NSCLC. Using real-time PCR-based miRNA expression profiling and bioinformatics, we selected miR-495 as a candidate miRNA. EGFP reporter assay, real-time PCR, and Western blot validated that ATP7A was a direct target for miR-495. The drug sensitivity assay indicated that miR-495 enhanced the cell response to cisplatin (CDDP) in NSCLC cells, while inhibition of miR-495 led to the opposite effects. Importantly, either overexpression or knockdown of ATP7A could override the effect of miR-495 on chemosensitivity. We also demonstrated that miR-495 increased the intracellular CDDP accumulation and overexpression of ATP7A can reduce the increased drug concentration induced by miR-495. Finally, we discovered that there was a converse relationship between miR-495 and ATP7A levels in NSCLC tissues sensitive or resistant to CDDP. In conclusion, our data demonstrate that miR-495 regulates the multi-drug resistance by modulation of ATP7A expression in NSCLC and suggest that miR-495 may serve as a potential biomarker for the treatment of multi-drug resistant NSCLC patients with high ATP7A levels. J. Cell. Biochem. 115: 1234–1242, 2014. © 2013 Wiley Periodicals, Inc.
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mir 495 enhances the sensitivity of non small cell lung cancer cells to platinum by modulation of copper transporting p type adenosine triphosphatase a ATP7A
Journal of Cellular Biochemistry, 2014Co-Authors: Liqiang SongAbstract:Copper-transporting P-type adenosine triphosphatase A (ATP7A) is associated with platinum drug resistance in non-small cell lung cancer (NSCLC). microRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at post-transcriptional level. In this study, the aim is to explore which miRNAs might participate in the platinum resistance by targeting ATP7A in NSCLC. Using real-time PCR-based miRNA expression profiling and bioinformatics, we selected miR-495 as a candidate miRNA. EGFP reporter assay, real-time PCR, and Western blot validated that ATP7A was a direct target for miR-495. The drug sensitivity assay indicated that miR-495 enhanced the cell response to cisplatin (CDDP) in NSCLC cells, while inhibition of miR-495 led to the opposite effects. Importantly, either overexpression or knockdown of ATP7A could override the effect of miR-495 on chemosensitivity. We also demonstrated that miR-495 increased the intracellular CDDP accumulation and overexpression of ATP7A can reduce the increased drug concentration induced by miR-495. Finally, we discovered that there was a converse relationship between miR-495 and ATP7A levels in NSCLC tissues sensitive or resistant to CDDP. In conclusion, our data demonstrate that miR-495 regulates the multi-drug resistance by modulation of ATP7A expression in NSCLC and suggest that miR-495 may serve as a potential biomarker for the treatment of multi-drug resistant NSCLC patients with high ATP7A levels.
Lucia Banci - One of the best experts on this subject based on the ideXlab platform.
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Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy
2016Co-Authors: Fabio Arnesano, Ivano Bertini, Lucia Banci, Isabella C. Felli, Maurizio Losacco, Giovanni NatileAbstract:Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH3)2}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation
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probing the interaction of cisplatin with the human copper chaperone atox1 by solution and in cell nmr spectroscopy
Journal of the American Chemical Society, 2011Co-Authors: Fabio Arnesano, Ivano Bertini, Lucia Banci, Isabella C. Felli, Maurizio Losacco, Giovanni NatileAbstract:Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active...
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cellular copper distribution a mechanistic systems biology approach
Cellular and Molecular Life Sciences, 2010Co-Authors: Lucia Banci, Ivano Bertini, Francesca Cantini, Simone CiofibaffoniAbstract:Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.
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Solution Structures of the Actuator Domain of ATP7A and ATP7B, the Menkes and Wilson Disease Proteins
Biochemistry, 2009Co-Authors: Lucia Banci, Manuele Migliardi, Francesca Cantini, Ivano Bertini, Giovanni Natile, Fiorentin Nushi, Antonio RosatoAbstract:ATP7A and ATP7B are two human P1B-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorilated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fo...
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an nmr study of the interaction of the n terminal cytoplasmic tail of the wilson disease protein with copper i hah1
Journal of Biological Chemistry, 2009Co-Authors: Lucia Banci, Chiara Massagni, Manuele Migliardi, Francesca Cantini, Ivano Bertini, Antonio RosatoAbstract:Abstract ATP7B is a human P1B-type ATPase that has a crucial role in maintaining copper(I) homeostasis. Mutations in the corresponding gene are the cause of Wilson disease. Among its various distinguishing features is a long (∼630 amino acids) N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We expressed the entire tail as a single construct in Escherichia coli and investigated its interaction with its copper chaperone (i.e. HAH1) by solution NMR spectroscopy. We observed that all six of the metal-binding domains were metallated by Cu(I)-HAH1, with the first, the second, and the fourth domains forming an adduct with it. This behavior is different from that of the highly similar human ATPase ATP7A, in which only two domains form such an adduct. The distinct behaviors of the different domains were analyzed in terms of the energetics of Cu(I) transfer, hinting at a specific role of the interaction with copper(I)-HAH1 in the overall functional process.
Ivano Bertini - One of the best experts on this subject based on the ideXlab platform.
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Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy
2016Co-Authors: Fabio Arnesano, Ivano Bertini, Lucia Banci, Isabella C. Felli, Maurizio Losacco, Giovanni NatileAbstract:Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH3)2}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation
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probing the interaction of cisplatin with the human copper chaperone atox1 by solution and in cell nmr spectroscopy
Journal of the American Chemical Society, 2011Co-Authors: Fabio Arnesano, Ivano Bertini, Lucia Banci, Isabella C. Felli, Maurizio Losacco, Giovanni NatileAbstract:Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active...
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cellular copper distribution a mechanistic systems biology approach
Cellular and Molecular Life Sciences, 2010Co-Authors: Lucia Banci, Ivano Bertini, Francesca Cantini, Simone CiofibaffoniAbstract:Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.
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Solution Structures of the Actuator Domain of ATP7A and ATP7B, the Menkes and Wilson Disease Proteins
Biochemistry, 2009Co-Authors: Lucia Banci, Manuele Migliardi, Francesca Cantini, Ivano Bertini, Giovanni Natile, Fiorentin Nushi, Antonio RosatoAbstract:ATP7A and ATP7B are two human P1B-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorilated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fo...
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an nmr study of the interaction of the n terminal cytoplasmic tail of the wilson disease protein with copper i hah1
Journal of Biological Chemistry, 2009Co-Authors: Lucia Banci, Chiara Massagni, Manuele Migliardi, Francesca Cantini, Ivano Bertini, Antonio RosatoAbstract:Abstract ATP7B is a human P1B-type ATPase that has a crucial role in maintaining copper(I) homeostasis. Mutations in the corresponding gene are the cause of Wilson disease. Among its various distinguishing features is a long (∼630 amino acids) N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We expressed the entire tail as a single construct in Escherichia coli and investigated its interaction with its copper chaperone (i.e. HAH1) by solution NMR spectroscopy. We observed that all six of the metal-binding domains were metallated by Cu(I)-HAH1, with the first, the second, and the fourth domains forming an adduct with it. This behavior is different from that of the highly similar human ATPase ATP7A, in which only two domains form such an adduct. The distinct behaviors of the different domains were analyzed in terms of the energetics of Cu(I) transfer, hinting at a specific role of the interaction with copper(I)-HAH1 in the overall functional process.