Copper chaperones: function, structure and copper-binding properties

 Copper is an absolute requirement for living systems and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The copper chaperones perform the dual functions of trafficking and the prevention of cytoplasmic exposure to copper ions in transit. Only a small number of copper chaperones have been identified at this time but their conservation across plant, bacterial and animal species suggests that the majority of living systems utilise these proteins for copper routing. The available data suggest that each copper-dependent protein in the cell is served by a specific copper chaperone. Although copper chaperones cannot be substituted for one another in a given cell type, copper chaperones that deliver to the same protein in different cell types appear to be functionally equivalent. The majority of the copper chaperones identified thus far have an "open-faced β-sandwich" global fold with a conserved MXCXXC metal-binding motif. Specificity for a given copper-dependent protein appears to be mediated by the residues surrounding the copper-binding motif. Copper binds to such proteins as Cu(I) in a trigonal complex with three sulfur ligands. Only the copper chaperone specific for cytochrome-c-oxidase, Cox17, deviates from this design. Received: 12 October 1998 / Accepted: 7 December 1998     

Copper chaperones: personal escorts for metal ions

Copper serves as the essential cofactor for a number of enzymes involved in redox chemistry and virtually all organisms must accumulate trace levels of copper in order to survive. However, this metal can also be toxic and a number of effective methods for sequestering and detoxifying copper prevent the metal from freely circulating inside a cell. Copper metalloenzymes are therefore faced with the challenge of acquiring their precious metal cofactor in the absence of available copper. To overcome this dilemma, all eukaryotic organisms have evolved with a family of intracellular copper binding proteins that help reserve a bioavailable pool of copper for the metalloenzymes, escort the metal to appropriate targets, and directly transfer the copper ion. These proteins have been collectively called copper chaperones. The identification of such molecules has been made possible through molecular genetic studies in the bakers' yeast Saccharomyces cerevisiae. In this review, we highlight the findings that led to a new paradigm of intracellular trafficking of copper involving the action of copper chaperones. In particular, emphasis will be placed on the ATX1 and CCS copper chaperones that act to deliver copper to the secretory pathway and to Cu/Zn superoxide dismutase in the cytosol, respectively.     

Calcium transporters and signalling in smooth muscles

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), play a crucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle cells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similar ATP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several isoforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contribution of PMCA and the Na-Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has begun to elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth muscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminal Ca, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the surface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae and cholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the dynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, close to the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can be better understood.     

Copper chaperones for cytochrome c oxidase and human disease

Biological processes in living cells are compartmentalized between lipid membranes. Integral membrane proteins often confer specific functions to these compartments and as such have a critical role in cellular metabolism and function. Cytochrome c oxidase is a macromolecular metalloprotein complex essential for the respiratory function of the cell. Elucidating the mechanisms of assembly of cytochrome c oxidase within the inner mitochondrial membrane represents a unique challenge for understanding metalloprotein biosynthesis. Elegant genetic experiments in yeast have defined several proteins required for copper delivery to cytochrome c oxidase. While the precise role of each of these proteins in copper incorporation remains unclear, recent studies have revealed that inherited mutations in two of these proteins can result in severe pathology in human infants in association with cytochrome c oxidase deficiency. Characterization of the molecular pathogenesis of these disorders offers new insights into the mechanisms of cellular copper metabolism and the role of these cytochrome c oxidase copper chaperones in human disease.     

Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.     

Sucrose transporters in plants: update on function and structure

In plants, sucrose is the major transport form for photoassimilated carbon and is both a source of carbon skeletons and energy for plant organs unable to perform photosynthesis (sink organs). As a molecule translocated over distance, sucrose has to pass through a number of membranes. Membrane transport of sucrose has therefore been considered for a long time as a major determinant of plant productivity. After several decades of physiological and biochemical experiments measuring the activity of sucrose carriers, unequivocal evidence came from the first identification of a cDNA coding a sucrose carrier (SoSUT1, Riesmeier et al. (1992) EMBO J. 11, 4705-4713). At present 20 different cDNAs encoding sucrose carriers have been identified in different plant species, in both dicots and monocots (one case). The total number is increasing rapidly and most importantly, it can be guessed from the results obtained for Arabidopsis, that in each species, sucrose transporters represent a gene family. The sequences are highly conserved and those carriers display the typical 12 transmembrane alpha-helices of members of the Major Facilitator superfamily. Yeast expression of those carriers indicate that they are all influx carriers, all cotransport sucrose and proton and that their affinity for sucrose is surprisingly similar (0.2-2 mM). All their characteristics are in agreement with those demonstrated at the physiological level in plants. These characteristics are discussed in relation to the function in plants and the few data available on the structure of those transporters in relation to their function are presented.     

Ion homeostasis, channels, and transporters: an update on cellular mechanisms

The steady-state maintenance of highly asymmetric concentrations of the major inorganic cations and anions is a major function of both plasma membranes and the membranes of intracellular organelles. Homeostatic regulation of these ionic gradients is critical for most functions. Due to their charge, the movements of ions across biological membranes necessarily involves facilitation by intrinsic membrane transport proteins. The functional characterization and categorization of membrane transport proteins was a major focus of cell physiological research from the 1950s through the 1980s. On the basis of these functional analyses, ion transport proteins were broadly divided into two classes: channels and carrier-type transporters (which include exchangers, cotransporters, and ATP-driven ion pumps). Beginning in the mid-1980s, these functional analyses of ion transport and homeostasis were complemented by the cloning of genes encoding many ion channels and transporter proteins. Comparison of the predicted primary amino acid sequences and structures of functionally similar ion transport proteins facilitated their grouping within families and superfamilies of structurally related membrane proteins. Postgenomics research in ion transport biology increasingly involves two powerful approaches. One involves elucidation of the molecular structures, at the atomic level in some cases, of model ion transport proteins. The second uses the tools of cell biology to explore the cell-specific function or subcellular localization of ion transport proteins. This review will describe how these approaches have provided new, and sometimes surprising, insights regarding four major questions in current ion transporter research. 1) What are the fundamental differences between ion channels and ion transporters? 2) How does the interaction of an ion transport protein with so-called adapter proteins affect its subcellular localization or regulation by various intracellular signal transduction pathways? 3) How does the specific lipid composition of the local membrane microenvironment modulate the function of an ion transport protein? 4) How can the basic functional properties of a ubiquitously expressed ion transport protein vary depending on the cell type in which it is expressed?     

Glycine transporters and synaptic function

Glycine is an inhibitory neurotransmitter that is mainly active in the caudal areas of the CNS. However, glycine also participates in excitatory neurotransmission since it is a co-agonist of the NMDA subtype of glutamate receptors. The concentration of glycine at synapses is mainly controlled by two sodium and chloride dependent transporters, GLYT1 and GLYT2, proteins that display a complementary distribution and activity in the nervous system. Our understanding of the physiological role of these transporters has advanced recently, thanks to the development of specific inhibitors and the generation of mice defective in the corresponding genes. In addition, the three-dimensional resolution of the structure of a bacterial homologue has shed light on the mechanisms of glycine transport. It is likely that this knowledge will prove to be useful for the development of drugs with antipsychotic, procognitive or analgesic properties.     

Zinc transporters and the cellular trafficking of zinc

Zinc is an essential nutrient for all organisms because this metal serves as a catalytic or structural cofactor for many different proteins. Zinc-dependent proteins are found in the cytoplasm and within many organelles of the eukaryotic cell including the nucleus, the endoplasmic reticulum, Golgi, secretory vesicles, and mitochondria. Thus, cells require zinc transport mechanisms to allow cells to efficiently accumulate the metal ion and distribute it within the cell. Our current knowledge of these transport systems in eukaryotes is the focus of this review.     

Mechanical cues in cellular signalling and communication

Multicellular organisms comprise an organized array of individual cells surrounded by a meshwork of biomolecules and fluids. Cells have evolved various ways to communicate with each other, so that they can exchange information and thus fulfil their specified and unique functions. At the same time, cells are also physical entities that are subjected to a variety of local and global mechanical cues arising in the microenvironment. Cells are equipped with several different mechanisms to sense the physical properties of the microenvironment and the mechanical forces arising within it. These mechanical cues can elicit a variety of responses that have been shown to play a crucial role in vivo. In this review, we discuss the current views and understanding of cell mechanics and demonstrate the emerging evidence of the interplay between physiological mechanical cues and cell-cell communication pathways.     

Interplay between mitochondria and cellular calcium signalling

Mitochondria are increasingly ascribed central roles in vital cell signalling cascades. These organelles are now recognised as initiators and transducers of a range of cell signals, including those central to activation and amplification of apoptotic cell death. Moreover, as the main source of cellular ATP, mitochondria must be responsive to fluctuating energy demands of the cell. As local and global fluctuations in calcium concentration are ubiquitous in eukaryotic cells and are the common factor in a dizzying array of intra- and inter-cellular signalling cascades, the relationships between mitochondrial function and calcium transients is currently a subject of intense scrutiny. It is clear that mitochondria not only act as local calcium buffers, thus shaping spatiotemporal aspects of cytosolic calcium signals, but that they also respond to calcium uptake by upregulating the tricarboxylic acid cycle, thus reacting metabolically to local signalling. In this chapter we review current knowledge of mechanisms of mitochondrial calcium uptake and release and discuss the consequences of mitochondrial calcium handling for cell function, particularly in conjunction with mitochondrial oxidative stress.     

Caveolae,transmembrane signalling and cellular transformation

Caveolae are -50–100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs -40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a ‘third’ proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin — a transformation-dependent v-Src substrate and caveolar marker protein — and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.     

分子伴侣结构与功能的研究进展

分子伴侣是细胞内一类能够协助其他多肽进行正常折叠、组装、转运、降解的蛋白,并在 DNA的复制、转录,细胞骨架功能,细胞内的信号转导等广泛的领域,都发挥着重要的生理作用,其结构与功能异常会导致多种相关的疾病。简要综述了分子伴侣结构与功能方面的研究进展。