Zinc proteomes, phylogenetics and evolution

Evolution has not been studied in detail with reference to the changing environment. This requires a study of the inorganic chemistry of organisms, especially metalloproteins. The evolution of organisms has been analysed many times previously using comparative studies, fossils, and molecular sequences of proteins, DNA and 16s rRNA (Zhang and Gladyshev, Chem. Rev., 2009, 109, 4828). These methods have led to the confirmation of Darwin's original proposal that evolution followed from natural selection in a changing environment often pictured as a tree. In all cases, the main tree in its upper later reaches has been well studied but its lower earlier parts are not so well defined. To approach this topic we have treated evolution as due to the intimate combination of the effect of chemical changes in the environment and in the organisms (Williams and da Silva, The Chemistry of Evolution, 2006, Elsevier). The best chemicals to examine are inorganic ions as they are common to both. As a more detailed example of the chemical study of organisms we report in this paper a bioinformatic approach to the characterization of the zinc proteomes. We deduce them from the 821 totally sequenced DNA of organisms available on NCBI, exploiting a published method developed by one of us (Andreini, Bertini and Rosato, Acc. Chem. Res., 2009, 42, 1471). Comparing the derived zinc-finger-containing proteins and zinc hydrolytic enzymes in organisms of different complexity there is a correlation in their changes during evolution related to environmental change.     

The Fox1 ferroxidase of Chlamydomonas reinhardtii: a new multicopper oxidase structural paradigm

Multicopper oxidases (MCO) contain at least four copper atoms arrayed in three distinct ligand fields supported by two canonical structural features: (1) multiples of the cupredoxin fold and (2) four unique sequence elements that include the ten histidine and one cysteine ligands to the four copper atoms. Ferroxidases are a subfamily of MCO proteins that contain residues supporting a specific reactivity towards ferrous iron; these MCOs play a vital role in iron metabolism in bacteria, algae, fungi, and mammals. In contrast to the fungal ferroxidases, e.g., Fet3p from Saccharomyces cerevisiae, the mammalian ceruloplasmin (Cp) is twice as large (six vs. three cupredoxin domains) and contains three type 1, or “blue,” copper sites. Chlamydomonas reinhardtii expresses a putative ferroxidase, Fox1, which has sequence similarity to human Cp (hCp). Eschewing the standard sequence-based modeling paradigm, we have constructed a function-based model of the Fox1 protein which replicates hCp’s six copper-site ligand arrays with an overall root mean square deviation of 1.4 Å. Analysis of this model has led also to assignment of motifs in Fox1 that are unique to ferroxidases, the strongest evidence to date that the well-characterized fungal high-affinity iron uptake system is essential to iron homeostasis in green algae. The model of Fox1 also establishes a subfamily of MCO proteins with a noncanonical copper-ligand organization. These diverse structures suggest alternative mechanisms for intramolecular electron transfer and require a new trajectory for the evolution of the MCO superfamily.     

Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.     

A proteomic approach to iron and copper homeostasis in cyanobacteria.

Cyanobacteria, which are considered to be the chloroplast precursors, are significant contributors to global photosynthetic productivity. The ample variety of membrane and soluble proteins containing different metals (mainly, iron and copper) has made these organisms develop a complex homeostasis with different mechanisms and tight regulation processes to fulfil their metal requirements in a changing environment. Cell metabolism is so adapted as to synthesize alternative proteins depending on the relative metal availabilities. In particular, plastocyanin, a copper protein, and cytochrome c(6), a haem protein, can replace each other to play the same physiological role as electron carriers in photosynthesis and respiration, with the synthesis of one protein or another being regulated by copper concentration in the medium. The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a model system because of completion of its genome sequence and the ease of its genetic manipulation, with a lot of proteomic work being done. In this review article, we focus on the functional characterization of knockout Synechocystis mutants for plastocyanin and cytochrome c(6), and discuss the ongoing proteomic analyses performed at varying copper concentrations to investigate the cyanobacterial metal homeostasis and cell response to changing environmental conditions.     

Directed Evolution of a Fluorogen-Activating Single Chain Antibody for Function and Enhanced Brightness in the Cytoplasm

Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP–fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.     

Modelling the self-assembly of elastomeric proteins provides insights into the evolution of their domain architectures

Elastomeric proteins have evolved independently multiple times through evolution. Produced as monomers, they self-assemble into polymeric structures that impart properties of stretch and recoil. They are composed of an alternating domain architecture of elastomeric domains interspersed with cross-linking elements. While the former provide the elasticity as well as help drive the assembly process, the latter serve to stabilise the polymer. Changes in the number and arrangement of the elastomeric and cross-linking regions have been shown to significantly impact their assembly and mechanical properties. However, to date, such studies are relatively limited. Here we present a theoretical study that examines the impact of domain architecture on polymer assembly and integrity. At the core of this study is a novel simulation environment that uses a model of diffusion limited aggregation to simulate the self-assembly of rod-like particles with alternating domain architectures. Applying the model to different domain architectures, we generate a variety of aggregates which are subsequently analysed by graph-theoretic metrics to predict their structural integrity. Our results show that the relative length and number of elastomeric and cross-linking domains can significantly impact the morphology and structural integrity of the resultant polymeric structure. For example, the most highly connected polymers were those constructed from asymmetric rods consisting of relatively large cross-linking elements interspersed with smaller elastomeric domains. In addition to providing insights into the evolution of elastomeric proteins, simulations such as those presented here may prove valuable for the tuneable design of new molecules that may be exploited as useful biomaterials.     

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.     

Understanding copper trafficking in bacteria: interaction between the copper transport protein CopZ and the N-terminal domain of the copper ATPase CopA from Bacillus subtilis

In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria. It appears that the two proteins interact in a fashion similar to that of the yeast homologue proteins [Arnesano, F., Banci, L., Bertini, I., Cantini, F., Ciofi-Baffoni, S., Huffman, D. L., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 41365-41376], although the surface potentials are reversed. A structural model for the interaction is proposed. (15)N mobility studies on the free proteins and on their complex are also reported. From these data, it appears that copper is largely transferred from CopZ to CopA, thus suggesting their possible involvement in a detoxification process. Comparing functional data of homologous proteins of other bacteria, it can be concluded that this class of proteins is involved in copper homeostasis but the specific roles are species dependent.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

The fundamental nature of life as a chemical system: the part played by inorganic elements

In this article we show why inorganic metal elements from the environment were an essential part of the origin of living aqueous systems of chemicals in flow. Unavoidably such systems have many closely fixed parameters, related to thermodynamic binding constants, for the interaction of the essential exchangeable inorganic metal elements with both inorganic and organic non-metal materials. The binding constants give rise to fixed free metal ion concentration profiles for different metal ions and ligands in the cytoplasm of all cells closely related to the Irving-Williams series. The amounts of bound elements depend on the organic molecules present as well as these free ion concentrations. This system must have predated coding which is probably only essential for reproductive life. Later evolution in changing chemical environments became based on the development of extra cytoplasmic compartments containing quite different energised free (and bound) element contents but in feed-back communication with the central primitive cytoplasm which changed little. Hence species multiplied late in evolution in large part due to the coupling with the altered inorganic environment.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus

Summary Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Comparative genomic analyses of copper transporters and cuproproteomes reveal evolutionary dynamics of copper utilization and its link to oxygen

Copper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. We found that copper use is widespread among prokaryotes, but also identified several phyla that lack cuproproteins. This is in contrast to the use of other trace elements, such as selenium, which shows more scattered and reduced usage, yet larger selenoproteomes. Copper transporters had different patterns of occurrence than cuproproteins, suggesting that the pathways of copper utilization and copper detoxification are independent of each other. We present evidence that organisms living in oxygen-rich environments utilize copper, whereas the majority of anaerobic organisms do not. In addition, among copper users, cuproproteomes of aerobic organisms were larger than those of anaerobic organisms. Prokaryotic cuproproteomes were small and dominated by a single protein, cytochrome c oxidase. The data are consistent with the idea that proteins evolved to utilize copper following the oxygenation of the Earth.     

Genomics, metagenomics and proteomics in biomining microorganisms

The use of acidophilic, chemolithotrophic microorganisms capable of oxidizing iron and sulfur in industrial processes to recover metals from minerals containing copper, gold and uranium is a well established biotechnology with distinctive advantages over traditional mining. A consortium of different microorganisms participates in the oxidative reactions resulting in the extraction of dissolved metal values from ores. Considerable effort has been spent in the last years to understand the biochemistry of iron and sulfur compounds oxidation, bacteria-mineral interactions (chemotaxis, quorum sensing, adhesion, biofilm formation) and several adaptive responses allowing the microorganisms to survive in a bioleaching environment. All of these are considered key phenomena for understanding the process of biomining. The use of genomics, metagenomics and high throughput proteomics to study the global regulatory responses that the biomining community uses to adapt to their changing environment is just beginning to emerge in the last years. These powerful approaches are reviewed here since they offer the possibility of exciting new findings that will allow analyzing the community as a microbial system, determining the extent to which each of the individual participants contributes to the process, how they evolve in time to keep the conglomerate healthy and therefore efficient during the entire process of bioleaching.     

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.     

A periplasmic iron-binding protein contributes toward inward copper supply

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c6 when grown under copper conditions (150 nm) in which wild-type cells use plastocyanin rather than cytochrome c6. Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copper-plastocyanin is impaired, but iron-ferredoxin is unaffected in DeltafutA2 grown in 150 nm copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in DeltafutA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of DeltafutA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in DeltafutA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.     

Iron and copper metabolism

Iron and copper are essential nutrients, excesses or deficiencies of which cause impaired cellular functions and eventually cell death. The metabolic fates of copper and iron are intimately related. Systemic copper deficiency generates cellular iron deficiency, which in humans results in diminished work capacity, reduced intellectual capacity, diminished growth, alterations in bone mineralization, and diminished immune response. Copper is required for the function of over 30 proteins, including superoxide dismutase, ceruloplasmin, lysyl oxidase, cytochrome c oxidase, tyrosinase and dopamine-beta-hydroxylase. Iron is similarly required in numerous essential proteins, such as the heme-containing proteins, electron transport chain and microsomal electron transport proteins, and iron-sulfur proteins and enzymes such as ribonucleotide reductase, prolyl hydroxylase phenylalanine hydroxylase, tyrosine hydroxylase and aconitase. The essentiality of iron and copper resides in their capacity to participate in one-electron exchange reactions. However, the same property that makes them essential also generates free radicals that can be seriously deleterious to cells. Thus, these seemingly paradoxical properties of iron and copper demand a concerted regulation of cellular copper and iron levels. Here we review the most salient characteristics of their homeostasis.     

Evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits

Among different coronavirus genera, the receptor-binding S1 subunits of their spike proteins differ in primary, secondary, and tertiary structures. This study identified shared structural topologies (connectivity of secondary structural elements) in S1 domains of different coronavirus genera. The results suggest that coronavirus S1 subunits share a common evolutionary origin but have attained diverse sequences and structures following extensive divergent evolution. The results also increase understanding of the structures and functions of coronavirus S1 domains whose tertiary structures are currently unknown.     

The dynamics and evolutionary potential of domain loss and emergence

The wealth of available genomic data presents an unrivaled opportunity to study the molecular basis of evolution. Studies on gene family expansions and site-dependent analyses have already helped establish important insights into how proteins facilitate adaptation. However, efforts to conduct full-scale cross-genomic comparisons between species are challenged by both growing amounts of data and the inherent difficulty in accurately inferring homology between deeply rooted species. Proteins, in comparison, evolve by means of domain rearrangements, a process more amenable to study given the strength of profile-based homology inference and the lower rates with which rearrangements occur. However, adapting to a constantly changing environment can require molecular modulations beyond reach of rearrangement alone. Here, we explore rates and functional implications of novel domain emergence in contrast to domain gain and loss in 20 arthropod species of the pancrustacean clade. Emerging domains are more likely disordered in structure and spread more rapidly within their genomes than established domains. Furthermore, although domain turnover occurs at lower rates than gene family turnover, we find strong evidence that the emergence of novel domains is foremost associated with environmental adaptation such as abiotic stress response. The results presented here illustrate the simplicity with which domain-based analyses can unravel key players of nature's adaptational machinery, complementing the classical site-based analyses of adaptation.