General Trends in Trace Element Utilization Revealed by Comparative Genomic Analyses of Co, Cu, Mo, Ni, and Se

Trace elements are used by all organisms and provide proteins with unique coordination and catalytic and electron transfer properties. Although many trace element-containing proteins are well characterized, little is known about the general trends in trace element utilization. We carried out comparative genomic analyses of copper, molybdenum, nickel, cobalt (in the form of vitamin B₁₂), and selenium (in the form of selenocysteine) in 747 sequenced organisms at the following levels: (i) transporters and transport-related proteins, (ii) cofactor biosynthesis traits, and (iii) trace element-dependent proteins. Few organisms were found to utilize all five trace elements, whereas many symbionts, parasites, and yeasts used only one or none of these elements. Investigation of metalloproteomes and selenoproteomes revealed examples of increased utilization of proteins that use copper in land plants, cobalt in Dehalococcoides and Dictyostelium, and selenium in fish and algae, whereas nematodes were found to have great diversity of copper transporters. These analyses also characterized trace element metabolism in common model organisms and suggested new model organisms for experimental studies of individual trace elements. Mismatches in the occurrence of user proteins and corresponding transport systems revealed deficiencies in our understanding of trace element biology. Biological interactions among some trace elements were observed; however, such links were limited, and trace elements generally had unique utilization patterns. Finally, environmental factors, such as oxygen requirement and habitat, correlated with the utilization of certain trace elements. These data provide insights into the general features of utilization and evolution of trace elements in the three domains of life.     

Intracellular copper routing: the role of copper chaperones

Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the 'copper chaperones'. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.     

Posttranslational regulation of copper transporters

Copper is an essential but potentially harmful trace element involved in many enzymatic processes that require redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by posttranslational regulation of copper import and export through the copper import proteins hCTR1 and hCTR2 and the copper exporters ATP7A and ATP7B. Regulation of copper uptake and export is achieved by modulation of transporter expression, copper-dependent and copper-independent trafficking of the different transporters, posttranslational modifications, and interacting proteins. In this review we systematically discuss the contribution of these different mechanisms to the regulation of copper transport.     

铜离子稳态平衡分子机理研究进展

铜离子是生物体不可缺少的微量元素。作位酶的辅助因子,铜离子驱动着包括细胞呼吸、神经递质的传递、铁离子的摄取和抵抗氧化应激在内的重要生理过程。然而,过量时,铜离子是有害的,能损坏像DNA、蛋白质和脂肪这样的生物分子。正因为如此,生物体必须平衡细胞体内铜离子的水平。铜离子稳态平衡相关的遗传缺陷是造成Menke和Wilson疾病的原因。铜离子也被发现与癌症和神经退行性疾病有关。对酵母和其他生物体的研究发现,存在铜离子的摄取、分送、储存、排泄和抵抗毒性水平铜离子的专一机制。调控这些专一机制的铜离子信号分子是细胞平衡铜这个必不可少却又有害的离子的关键。     

Molybdoproteomes and evolution of molybdenum utilization

The trace element molybdenum (Mo) is utilized in many life forms, and it is a key component of several enzymes involved in nitrogen, sulfur, and carbon metabolism. With the exception of nitrogenase, Mo is bound in proteins to a pterin, thus forming the molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. Although a number of molybdoenzymes are well characterized structurally and functionally, evolutionary analyses of Mo utilization are limited. Here, we carried out comparative genomic and phylogenetic analyses to examine the occurrence and evolution of Mo utilization in bacteria, archaea and eukaryotes at the level of (i) Mo transport and Moco utilization trait, and (ii) Mo-dependent enzymes. Our results revealed that most prokaryotes and all higher eukaryotes utilize Mo whereas many unicellular eukaryotes including parasites and most yeasts lost the ability to use this metal. In addition, eukaryotes have fewer molybdoenzyme families than prokaryotes. Dimethylsulfoxide reductase (DMSOR) and sulfite oxidase (SO) families were the most widespread molybdoenzymes in prokaryotes and eukaryotes, respectively. A distant group of the ModABC transport system, was predicted in the hyperthermophilic archaeon Pyrobaculum. ModE-type regulation of Mo uptake occurred in less than 30% of Moco-utilizing organisms. A link between Mo and selenocysteine utilization in prokaryotes was also identified wherein the selenocysteine trait was largely a subset of the Mo trait, presumably due to formate dehydrogenase, a Mo- and selenium-containing protein. Finally, analysis of environmental conditions and organisms that do or do not depend on Mo revealed that host-associated organisms and organisms with low G + C content tend to reduce their Mo utilization. Overall, our data provide new insights into Mo utilization and show its wide occurrence, yet limited use of this metal in individual organisms in all three domains of life.     

The tonoplast copper transporter COPT5 acts as an exporter and is required for interorgan allocation of copper in Arabidopsis thaliana

Copper is an essential micronutrient for all organisms because it serves as a cofactor of several proteins involved in electron transfer. Elevated copper concentrations can cause toxic effects and organisms have established suitable mechanisms to regulate the uptake and internal distribution of copper to balance the content at an optimal concentration. In recent studies, a family of copper transporters (COPT) with high homology to other eukaryotic copper transporters (Ctr) has been identified in Arabidopsis thaliana. In this study we clarified the physiological function of COPT5. This carrier is located in the tonoplast and functions as a vacuolar copper exporter. Mutants lacking this transporter have altered copper contents in different organs when compared with wild-type plants. We were able to detect copper accumulation in the root and a decreased copper content in siliques and seeds when the COPT5 gene is mutated by T-DNA insertion. Vacuoles purified from copt5 T-DNA-insertion mutants show remarkably increased copper concentrations compared with wild-type organelles. We assume that on the cellular level COPT5 is important for copper export from the vacuole and on the level of the whole plant it is involved in the interorgan reallocation of copper ions from the root to reproductive organs.     

酵母和植物中铜的转运系统及其调控

铜是生物正常生命活动所必需的微量矿质元素。酵母和植物中有复杂的机制来调节铜的摄取、分布、螯合以及输出。本文集中讨论了酵母和植物中铜离子的转运体、铜的金属伴侣及其基因转录水平的调控。     

酵母和植物中铜的转运系统及其调控

铜是生物正常生命活动所必需的微量矿质元素。酵母和植物中有复杂的机制来调节铜的摄取、分布、螯合以及输出。本文集中讨论了酵母和植物中铜离子的转运体、铜的金属伴侣及其基因转录水平的调控。     

The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development

Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of (64)Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters.     

Occurrence of copper proteins through the three domains of life: a bioinformatic approach

In high-throughput genome-level protein investigation efforts, such as Structural Genomics, the systematic experimental characterization of metal-binding properties (i.e., the investigation of the metalloproteome) is not always pursued and remains far from trivial. In the present work, we have applied a bioinformatic approach to investigate the occurrence of (putative) copper-binding proteins in 57 different organisms spanning the entire tree of life. We found that the size of the copper proteome is generally less than 1% of the total proteome of an organism, in both eukaryotes and prokaryotes. The occurrence of copper-binding proteins is relatively scarce when compared to that of zinc-binding proteins and of non-heme iron proteins. This may be due to both poorer bioavailability (in particular with respect to iron in the ancient world) and the complexity of copper chemistry and the risks associated with it, which may have adversely affected natural selection of copper-binding proteins. The present analysis shows that there is a strong relationship between the metal coordination sphere and protein function. A network involving proteins having roles in both copper transport and respiration was identified, parts or all of which are detected in the majority of the organisms examined.     

Involvement of copper in female reproduction

Copper (Cu) is one of the essential trace metals which are necessary in maintaining the functioning of living organisms. The current knowledge on the role of copper in animal reproduction is presented in the article. Our studies have shown that complexes of copper (Cu(2+)) with gonadotropin-releasing hormone (GnRH) are even more effective in the release of LH than native GnRH. Moreover, Cu-GnRH is more potent in inducing in vivo release of FSH than LH. Copper complexes with GnRH interact with GnRH receptors (GnRHR) and modulate intracellular signaling in the gonadotrope cells of the anterior pituitary. Copper plays also a significant role in maintaining normal fetus development in mammals.     

Maintaining copper homeostasis: regulation of copper-trafficking proteins in response to copper deficiency or overload

Copper is an essential micronutrient that plays a vital role as a catalytic co-factor for a variety of metalloenzymes. The redox chemistry of copper also makes it a potentially toxic metal if not properly used. Therefore, elaborate mechanisms have evolved for controlling its cellular uptake, elimination, and distribution. In the last decade, our understanding of the systems involved in maintaining copper homeostasis has improved considerably with the characterization of copper transporters that mediate cellular copper uptake or efflux and with the identification of copper chaperones, a family of proteins required for delivering copper to specific targets in the cell. Despite the distinct roles of these proteins in copper trafficking, all seem able to respond to changes in copper status. Here, we describe recent advances in our knowledge of how copper-trafficking proteins respond to copper deficiency or overload in mammalian cells in order to maintain copper balance.     

Bacterial ATP-driven transporters of transition metals: physiological roles, mechanisms of action, and roles in bacterial virulence

Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload. Representatives from all major families of transporters participate in this task, including ion-gradient driven systems and ATP-utilizing pumps. P-type ATPases and ABC transporters both utilize the free energy of ATP hydrolysis to drive transport. Each of these very different families of transport proteins has a distinct role in maintaining transition metal homeostasis: P-type ATPases prevent intracellular overloading of both essential and toxic metals through efflux while ABC transporters import solely the essential ones. In the present review we discuss how each system is adapted to perform its specific task from mechanistic and structural perspectives. Despite the mechanistic and structural differences between P-type ATPases and ABC transporters, there is one important commonality: in many clinically relevant bacterial pathogens, transporters of transition metals are essential for virulence. Here we present several such examples and discuss how these may be exploited for future antibacterial drug development.     

Function and Regulation of the Mammalian Copper-transporting ATPases: Insights from Biochemical and Cell Biological Approaches

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.     

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.     

Plant copper chaperones

Copper chaperones, soluble copper-binding proteins, are essential for ensuring proper distribution of copper to cellular compartments and to proteins requiring copper prosthetic groups. They are found in all eukaryotic organisms. Orthologues of the three copper chaperones characterized in yeast, ATX1, CCS and COX17, are present in Arabidopsis thaliana. Plants are faced with unique challenges to maintain metal homoeostasis, and thus their copper chaperones have evolved by diversifying and gaining additional functions. In this paper we present our current knowledge of copper chaperones in A. thaliana based on the information available from the complete sequence of its genome.     

Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria

Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host–pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.     

Molecular mechanisms of copper homeostasis.

Copper is an essential trace element which plays a pivotal role in cell physiology as it constitutes a core part of important cuproenzymes. Novel components of copper homeostasis in humans have been identified recently which have been characterised at the molecular level. These include copper-transporting P-type ATPases, Menkes and Wilson proteins, and copper chaperones. These findings have paved the way towards better understanding of the role of copper deficiency or copper toxicity in physiological and pathological conditions.     

Coordination chemistry of copper proteins: how nature handles a toxic cargo for essential function

Biological copper is coordinated predominantly by just three ligand types: the side chains of histidine, cysteine, and methionine, with of course some exceptions. The arrangement of these components, however, is fascinating. The diversity provided by just these three ligands provides choices of nitrogen vs. sulfur, neutral vs. charged, hydrophilic vs. hydrophobic, susceptibility to oxidation, and degree of pH-sensitivity. In this review we examine how the total number of ligands, their spatial arrangement and solvent accessibility, the various combinations of imidazole, thiolate, and thioether donors, all work together to provide binding sites that either enable copper to carry out a function, or safely transport it in a way that prevents toxic reactivity. We separate copper proteins into two broad classes, those that utilize the metal as a cofactor, or those that traffic the metal. Enzymes and proteins that utilize copper as a cofactor use high affinity sites of high coordination numbers of 4-5 that prevent loss of the metal during redox cycling. Copper trafficking proteins, on the other hand, promote metal transfer either by having low affinity binding sites with moderate coordination number ~ 4, or by having lower coordinate binding sites of 2-3 ligands that bind with high affinity. Both strategies retain the metal but allow transfer under appropriate conditions. Analysis of studies from our own lab on model peptides, combined with those from other labs, raises an interesting hypothesis that various methionine/histidine/cysteine combinations provide organisms with dynamic, multifunctional domains on copper trafficking proteins that facilitate copper transfer under different extracellular, subcellular, and tissue-specific scenarios of pH, redox environment, and presence of other copper carriers or target proteins.     

The molecular bases for copper uptake and distribution: lessons from yeast

Copper exists in two oxidation states, cuprous (Cu1+) and cupric (Cu2+), which, respectively, can donate or accept electrons. The fact that copper has two readily interconvertible redox states makes it a catalytic co-factor for many important enzymes. Over the past years, work in a number of laboratories has clearly demonstrated that studies in yeast have served as a springboard for identifying cellular components and processes involved in copper uptake and distribution. In several cases, it has been shown that mammalian proteins are capable of functionally replacing yeast proteins, thereby revealing their remarkable functional conservation. For high-affinity copper transport into cells, it has been shown that copper transporters of the Ctr family are required. Upon entering the cell, copper is partitioned to different proteins and into different compartments within the cell. Given the potential toxicity of copper, specialized proteins bind copper after it enters the cell and subsequently donate the bound copper to their corresponding recipient proteins. Three copper-binding proteins, Ccs1, Cox17, and Atx1, have been identified that serve as "copper chaperones" to deliver copper. double dagger.