The identification of novel genes involved in copper and oxygen metabolism

Copper (Cu) is an essential trace dietary metal which is also potentially toxic. Cells have evolved an intricate system of transporters and chaperones to carefully regulate Cu homeostasis. Cu uptake is mediated by the Cu1+-specific permease, CTR1. Cytoplasmic Cu is delivered to specific compartments via a series of metallochaperones. In the trans-Golgi network (TGN), the ATP7A or ATP7B proteins are required to pump Cu into the secretory pathway to various cuproenzymes. ATP7A also plays a protective role against Cu toxicity by trafficking to the plasma membrane to export Cu from the cell. Metallothioneins (MTs) are another type of protein that plays a protective role against Cu toxicity by sequestering excess Cu in the cytoplasm. There are four isoforms of MT in mice of which MT I and MT II are ubiquitously expressed. Our preliminary data demonstrates the essentiality of ATP7A, MTI and MTII in copper tolerance. We generated a fibroblast cell line in which Atp7a, Mt-I, and Mt-II genes were deleted (Atp7a-/Mt-), which resulted in sensitivity of these cells to sub-micromolar levels of copper. Using a genome-wide CRISPR-Cas9 deletion screen, we identified a novel gene, VHL (von Hippel-Lindau), that when deleted confers Cu tolerance to Atp7a-/Mt- cells. The VHL protein is a ubiquitin-ligase responsible for the degradation of HIF transcription factors, which are the major regulators of gene expression in response to hypoxia. We show that VHL deletion confers Cu tolerance by stimulating the expression of the ATP7B copper transporter. Pharmacological inhibition of VHL-HIF1[alpha] by Roxadustat (a prolyl hydroxylase inhibitor), which is clinically used to treat anemia, conferred resistance to copper by upregulating ATP7B. Importantly, Roxadustat was found to increase hepatic ATP7B expression in vivo. These novel data identify a previously unknown link between copper homeostasis and oxygen sensing and have potential translational implications for the treatment of Wilson disease, a copper overload condition cause by insufficient expression of ATP7B.Includes bibliographical references (pages 102-118)

Identification of copper metabolism as a KRAS-specific vulnerability in colorectal cancer

KRAS est parmi les gènes les plus fréquemment mutés dans les cancers humains, tel que ~ 45% des cancers colorectaux (CCR). Malgré les efforts déployés pour réduire son potentiel oncogénique, KRAS muté est fréquemment associé à la résistance aux médicaments et est extrêmement difficile à cibler sur le plan thérapeutique. Les protéines à la surface cellulaire sont souvent dérégulées dans les cancers et sont des cibles thérapeutiques attrayantes en raison de leur accessibilité aux anticorps. Nous avons séquençé les ARNm de cellules épithéliales intestinales exprimant KRAS muté et observé que ces dernières présentaient des changements importants dans les gènes codant pour des protéines de surface cellulaire. Par conséquent, notre objectif était d'identifier de nouvelles cibles thérapeutiques exprimées à la surface de cellules transformées par l’oncogène KRAS. En utilisant une approche de pointe en protéomique de surface cellulaire, nous avons identifié plusieurs protéines différentiellement exprimées dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. Nous avons ensuite effectué un crible CRISPR/Cas9 basé sur les protéines de surface cellulaire, qui a révélé que la perte de la protéine Atp7a affectait de manière différentielle les cellules épithéliales intestinales, en fonction de leur statut KRAS. De façon intéressante, nous avons constaté que ATP7A était régulé à la hausse dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. ATP7A a un double rôle dans les cellules; alors qu'il est essentiel pour la maturation des enzymes dépendantes du cuivre (Cu), ATP7A protège les cellules d'une toxicité excessive induite par le Cu (cuproptose). Chez l'homme, les mutations dans ATP7A entraînent des troubles caractérisés par des déficiences systémiques dans le transport et les niveaux de Cu. Chez les animaux et dans les modèles de culture cellulaire, tel que les cellules épithéliales intestinales, les niveaux intracellulaires de Cu sont directement corrélés avec l'abondance post-transcriptionnelle d'ATP7A. Dans le même ordre d'idées, nous avons observé que les cellules de CCR avec KRAS muté avaient relativement plus de Cu intracellulaire, et la surexpression d'ATP7A protégeait les cellules KRAS muté de la cuproptose, par rapport à leurs homologues de type sauvage. Nous avons également observé que la croissance in vivo des xénogreffes KRAS mutées était réduite lorsque les souris étaient nourries avec un régime pauvre en Cu. Le Cu est utilisé par plusieurs enzymes qui régulent des fonctions cellulaires critiques, notamment la respiration mitochondriale, la motilité cellulaire et la prolifération. Nous montrons que les cellules mutantes KRAS étaient plus sensibles au chélateur de Cu, ammonium tetrathiomolybdate (TTM), par rapport aux cellules de type sauvage. De plus, les cellules avec KRAS muté traitées avec le TTM ont présenté des activités réduites de MEK1/2 dépendant du Cu et de l'enzyme de la chaîne de transport d'électrons mitochondriale, cytochrome c oxidase (CCO). Nous avons été surpris de constater que le transporteur de Cu de haute affinité, CTR1, est régulé à la baisse dans les cellules avec KRAS muté, et avons donc émis l'hypothèse que les cellules KRAS mutées doivent absorber le Cu par d'autres moyens. Ainsi, nous avons constaté que la macropinocytose agit comme une voie non canonique d'approvisionnement en Cu dans les cellules avec KRAS muté. Le traitement de cellules in vivo avec l'inhibiteur de la macropinocytose, EIPA, a inhibé l'expression d'ATP7A et diminué le Cu biodisponible dans les xénogreffes KRAS mutées. En conclusion, nos résultats montrent que les cellules avec KRAS muté augmentent les niveaux de Cu et d'ATP7A pour soutenir la tumorigenèse en augmentant l'activité cuproenzymatique et diminuant la cuproptose. Cette étude est pertinente pour le cancer, car les tissus tumoraux contiennent fréquemment des niveaux de Cu plus élevés que les tissus normaux. Des études récentes ont mis en évidence un potentiel de repositionnement du chélateur de Cu TTM, qui est disponible en clinique et utilisé pour traiter les troubles du Cu. Nos résultats démontrent que la biodisponibilité du Cu pourrait être exploitée pour traiter le CCR avec KRAS muté avec de tels inhibiteurs. Les travaux futurs comprennent l'identification de stratégies combinatoires qui peuvent être améliorer les effets anti-cancéreux de la chélation du Cu.KRAS is amongst the most frequently mutated genes driving human cancers, including ~ 45% of colorectal cancers (CRC). Despite intense efforts to curb its oncogenic potential, mutant KRAS is frequently associated with drug resistance and is extremely challenging to target therapeutically. Cell-surface proteins are often spatially dysregulated in cancers and are attractive therapeutic targets due to their easy accessibility. We performed RNA sequencing of mutant KRAS-expressing intestinal epithelial cells and observed that cells undergoing transformation exhibited dramatic changes in cell surface-coding genes. Therefore, our goal was to identify novel druggable targets expressed at the cell surface of mutant KRAS-transformed cells. Using a cutting-edge cell surface proteomics approach, we identified several differentially expressed proteins at the surface of KRAS-mutant cells compared to wild-type counterparts. We then performed a cell surface based CRISPR/Cas9 screen, which revealed that loss of the copper exporter Atp7a differentially affected the fitness of intestinal epithelial cells, depending on their KRAS status. Interestingly, we found that ATP7A was upregulated in KRAS-mutant cells compared to wild-type counterparts. ATP7A has a dual role in cells; while it is essential for maturation of copper (Cu)-dependent enzymes, ATP7A protects cells from excess Cu-induced toxicity (cuproptosis). In humans, ATP7A mutations result in disorders characterized by systemic deficiencies in Cu transport and levels. In animals and in tissue culture models, including intestinal epithelial cells, intracellular Cu levels are directly correlated with the post-transcriptional abundance of ATP7A. In line with this, we observed that KRAS-mutant CRC cells and tissues had relatively more intracellular Cu, and ATP7A-overexpression protected KRAS-mutant cells from cuproptosis, compared to wild-type counterparts. We also observed that in vivo growth of KRAS-mutant xenografts was reduced when mice were fed a Cu-deficient diet. Cu is utilized by several enzymes that regulate critical cellular functions including mitochondrial respiration, cell motility and proliferation. We show that KRAS-mutant cells were more sensitive to the Cu chelating drug ammonium tetrathiomolybdate (TTM), compared to wild-type cells. Moreover, TTM-treated KRAS-mutant cells displayed reduced activities of Cu-dependent MEK1/2 and mitochondrial electron transport chain enzyme, cytochrome c oxidase (CCO). We were surprised to find that the high-affinity CTR1 importer is downregulated in KRAS-mutant cells, and so we hypothesized that KRAS cells must uptake Cu through alternate means. In accordance with this, we found that macropinocytosis acts as a non-canonical Cu-supply route in KRAS-mutant cells. In vivo, treating cells with the macropinocytosis inhibitor EIPA, inhibited the expression of ATP7A and decreased bioavailable Cu in KRAS xenografts. In conclusion, our results show that KRAS-mutant cells increase Cu and ATP7A levels, likely to support tumorigenesis by elevating cuproenzymatic activity and parallelly dealing with cuproptosis. This study is relevant to cancer as tumor tissues and patients contain higher Cu levels than normal controls. Recent studies have highlighted a potential for repurposing the clinically available copper chelator TTM, which is used to treat Cu disorders. Our results demonstrate that copper bioavailability could be exploited to treat KRAS-mutated CRC with such inhibitors. Future work includes identification of combinatorial strategies that may be synthetic lethal to copper chelation

Metalation and Maturation of Zinc Ectoenzymes: A Perspective

Numerous zinc ectoenzymes are folded and activated in the compartments of the early secretory pathway, such as the ER and the Golgi apparatus, before reaching their final destination. During this process, zinc must be incorporated into the active site; therefore, metalation of the nascent protein is indispensable for the expression of the active enzyme. However, to date, the molecular mechanism underlying this process has been poorly investigated. This is in sharp contrast to the physiological and pathophysiological roles of zinc ectoenzymes, which have been extensively investigated over the past decades. This manuscript concisely outlines the present understanding of zinc ectoenzyme activation through metalation by zinc and compares this with copper ectoenzyme activation, in which elaborate copper metalation mechanisms are known. Moreover, based on the comparison, several hypotheses are discussed. Approximately 80 years have passed since the first zinc enzyme was identified; therefore, it is necessary to improve our understanding of zinc ectoenzymes from a biochemical perspective, which will further our understanding of their biological roles

THE REGULATION OF THE INTESTINAL COPPER EXPORTER IS COORDINATED WITH SYSTEMIC COPPER HOMEOSTASIS

Copper (Cu) plays key catalytic and regulatory roles in biochemical reactions essential for normal growth, development, and health. Defects in Cu metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of Cu. While it is well established that the enterocyte is a key regulatory point for Cu absorption into the body, how the intestine responds to systemic Cu requirements is poorly understood. Here, we demonstrate that fine-tuned Cu homeostasis is required for normal growth and development in C. elegans. Moreover, we show that CUA-1, the ATP7A/B homolog in worms, localizes to lysosome-like organelles (gut granules) in the intestine under Cu-overload conditions for Cu detoxification, while Cu-deficiency results in a redistribution of CUA-1 to basolateral membranes for Cu efflux to peripheral tissues. Defects in gut granule biogenesis exhibit result in abnormal Cu sequestration and increased susceptibility to toxic Cu levels. Our studies establish that CUA-1 is a key intestinal Cu exporter, and that its trafficking is regulated in response to systemic Cu status in worms. In addition, while the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation in mammals. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Importantly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Altogether, our results implicate CUA-1/ATP7A Cu exporter in the intestine as a key modulator for organismal Cu homeostasis in metazoans

Cu homeostasis in bacteria: The ins and outs

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells

Inflammation and hypoxia : novel regulators of mammalian copper homeostasis in macrophages

Title from PDF of title page (University of Missouri--Columbia, viewed on March 8, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Michael J. Petris.Vita.Ph. D. University of Missouri--Columbia 2008.Copper is an essential cofactor of enzymes involved in a variety of important metabolic processes including ATP production, iron transport, and antioxidant defense. The maintenance of copper homeostasis requires a balance of copper uptake and export, as well as the appropriate partitioning of copper between the cytoplasm, mitochondria and secretory compartments. Although many of the proteins involved in copper homeostasis have been identified, it is unknown whether specific pathophysiological conditions lead to compensatory changes in the intracellular copper distribution. In this study, we identify changes in copper homeostasis in response to pro-inflammatory mediators. We also show a novel role for the copper-transporting ATPase, ATP7A, in the bactericidal activity of RAW264.7 macrophage cell. We also identify striking alterations in copper homeostasis in response to hypoxia in RAW264.7 macrophage cells. In response to hypoxia, we observe a change in the hierarchy of intracellular distribution favoring delivery of copper to ATP7A and to the secretory pathway, as evidenced by enhanced activity of the ferroxidase, ceruloplasmin, and by copper-dependent trafficking of ATP7A in hypoxic macrophages in vitro and in vivo. Our study underscores the potential for pathophysiological conditions to regulate adaptive responses involving altered copper distribution to cuproenzymes.Includes bibliographical reference

Copper Homeostasis in the Filamentous Ascomycete Aspergillus nidulans.

237 p.El objetivo de esta tesis doctoral ha sido el estudio de la homeostasis del cobre en el hongo filamentoso Aspergillus nidulans. El cobre es un oligoelemento necesario para la vida, pero en cantidades superiores a las necesarias tiene efectos nocivos. Debido a esta propiedad el cobre ha sido utilizado como antimicrobiano en la agricultura en cantidades inconmensurables generando un problema medioambiental. Los resultados de la tesis están divididos en tres capítulos: En el primero, por una parte se detallan los resultados de un experimento de RNA-seq que compara el transcriptoma del hongo en condiciones vegetativas y condiciones de toxicidad por cobre, y por la otra, se describe el sistema de internalización de cobre de alta afinidad que posee el organismo, las proteínas responsables de dicha internalización, su regulación y su sublocalización celular. El segundo capítulo se centra en el mecanismo de detoxificación que el hongo posee para evitar un exceso de cobre dentro de la célula. Se describe el método de detoxificación que posee el hongo, la proteína que lleva a cabo el proceso, su regulación, su localización subcelular y la regulación de todo el sistema mediante un factor de transcripción. En el último capítulo, se describe la metodología seguida para buscar posibles inhibidores de posibles ¿targets¿ del sistema de homeostasis del cobre para potenciar el efecto de fungicidas basados en cobre y así poder bajar las dosis de cobre

Investigating Copper Acquisition And Delivery via Transporters and a Pharmacological Chaperone in Copper-Deficient Worms and Mice

Copper (Cu) is a key micronutrient required for a variety of essential biochemical pathways. Systemic or tissue-specific Cu-deficiencies, caused by insufficient dietary Cu uptake or mutations in Cu transporting genes, result in impaired growth, neuropathy, ataxia, hypopigmentation, osteoporosis and anemia-like symptoms in mammals. How organisms regulate Cu homeostasis at the systemic levels in response to Cu deficiencies remain elusive. In this study, we use Caenorhabditis elegans (C. elegans), a genetically tractable, multi-tissue metazoan to explore Cu homeostasis and investigate these unknowns. The high-affinity Cu transporters encoded by CTR family genes are required for dietary Cu uptake and maintaining systemic Cu balance from yeast to mammals. However, little is known about Cu acquisition mechanisms in C. elegans. We identified ten CTR ortholog genes in C. elegans; of these, chca-1 was functionally characterized. Cu availability regulates transcription of chca-1 in both the intestine and hypodermis, and chca-1 is essential for normal growth, and reproduction in the worm. Additionally, altered Cu balance caused by the loss of CHCA-1 results in defects in Cu-responsive avoidance behavior. Identification of this CTR-like gene in C. elegans, which appears to be essential for normal Cu homeostasis in the worm, illustrates the importance of Cu delivery via CHCA-1 for normal metazoan development and behavioral phenotypes. In addition, we show that a Cu-binding pharmacological chaperone, elesclomol (ES), fully restores the developmental defects and Cu deficiencies in chca-1-depleted worms, as well as the lethality in worms lacking cua-1 expression (Cu exporter ATP7A ortholog), suggesting ES is able to efficiently deliver Cu from dietary sources to peripheral tissues through the intestine in C. elegans. Our study was further expanded to mammalian models such as cardiac-specific Ctr1-depleted (Ctr1hrt/hrt) mice. We found that ES administration fully restores the postnatal lethality, developmental defects and cardiac hypertrophy found in Ctr1hrt/hrt mice, as well as rescuing the secondary systemic Cu homeostasis responses, including aberrant ATP7A protein levels in the liver and intestine. Moreover, ES shows the potential ability to transport Cu across the blood-brain-barrier in in vitro studies. These results illustrate the capability of ES to rescue systemic Cu deficiency in worms and mice, independent of the presence of functional Cu transporters, and shed light on the therapeutic usage of ES in Cu-deficient human diseases

A Commemorative Issue in Honor of Professor Nick Hadjiliadis: Metal Complex Interactions with Nucleic Acids and/or DNA

This Special Issue of the International Journal of Molecular Science comprises a comprehensive study on “Metal Complex Interactions with Nucleic Acids and/or DNA”. This Special Issue has been inspired by the important contribution of Prof. Nick Hadjiliadis to the field of palladium or/and platinum/nucleic acid interactions. It covers a selection of recent research and review articles in the field of metal complex interactions with nucleic acids and/or DNA. Moreover, this Special Issue on "Metal Complexes Interactions with Nucleic Acids and/or DNA" provides an overview of this increasingly diverse field, presenting recent developments and the latest research with particular emphasis on metal-based drugs and metal ion toxicity