MOSC domains: ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including Molybdenum cofactor sulfurases

Using computational analysis, a novel superfamily of beta-strand-rich domains was identified in the Molybdenum cofactor sulfurase and several other proteins from both prokaryotes and eukaryotes. These MOSC domains contain an absolutely conserved cysteine and occur either as stand-alone forms such as the bacterial YiiM proteins, or fused to other domains such as a NifS-like catalytic domain in Molybdenum cofactor sulfurase. The MOSC domain is predicted to be a sulfur-carrier domain that receives sulfur abstracted by the pyridoxal phosphate-dependent NifS-like enzymes, on its conserved cysteine, and delivers it for the formation of diverse sulfur-metal clusters. The identification of this domain may clarify the mechanism of biogenesis of various metallo-enzymes including Molybdenum cofactor-containing enzymes that are compromised in human type II xanthinuria.     

Ablation of the mammalian methionine sulfoxide reductase A affects the expression level of cysteine deoxygenase

Methionine sulfoxide reductases (Msrs) are able to reduce methionine sulfoxide to methionine both in proteins and free amino acids. By their action it is possible to regulate the function of specific proteins and the cellular antioxidant defense against oxidative damage. Similarly, cysteine deoxygenase (CDO) may be involved in the regulation of protein function and antioxidant defense mechanisms by its ability to oxidized cysteine residues. The two enzymes' involvement in sulfur amino-acids metabolism seems to be connected. Lack of methionine sulfoxide reductase A (MsrA) in liver of MsrA-/- led to a significant drop in the cellular level of thiol groups and lowered the CDO level of expression. Moreover, following selenium deficient diet (applied to decrease the expression levels of selenoproteins like MsrB), the latter effect was maintained while the basal levels of thiol decreased in both mouse strains. We suggest that both enzymes are working in coordination to balance cellular antioxidant defense.     

The crystal structure of annexin Gh1 from Gossypium hirsutum reveals an unusual S3 cluster.

The three-dimensional crystal structure of recombinant annexin Gh1 from Gossypium hirsutum (cotton fibre) has been determined and refined to the final R-factor of 0.219 at the resolution of 2.1 A. This plant annexin consists of the typical 'annexin fold' and is similar to the previously solved bell pepper annexin Anx24(Ca32), but significant differences are seen when compared to the structure of nonplant annexins. A comparison with the structure of the mammalian annexin AnxA5 indicates that canonical calcium binding is geometrically possible within the membrane loops in domains I and II of Anx(Gh1) in their present conformation. All plant annexins possess a conserved tryptophan residue in the AB loop of the first domain; this residue was found to adopt both a loop-in and a loop-out conformation in the bell pepper annexin Anx24(Ca32). In Anx(Gh1), the conserved tryptophan residue is in a surface-exposed position, half way between both conformations observed in Anx24(Ca32). The present structure reveals an unusual sulfur cluster formed by two cysteines and a methionine in domains II and III, respectively. While both cysteines adopt the reduced thiolate forms and are separated by a distance of about 5.5 A, the sulfur atom of the methionine residue is in their close vicinity and apparently interacts with both cysteine sulfur atoms. While the cysteine residues are conserved in at least five plant annexins and in several mammalian members of the annexin family of proteins, the methionine residue is conserved only in three plant proteins. Several of these annexins carrying the conserved residues have been implicated in oxidative stress response. We therefore hypothesize that the cysteine motif found in the present structure, or possibly even the entire sulfur cluster, forms the molecular basis for annexin function in oxidative stress response.     

Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility

The universal stress protein (UspA) superfamily encompasses a conserved group of proteins that are found in bacteria, archaea, and eukaryotes. Escherichia coli harbors six usp genes--uspA, -C, -D, -E, -F, and -G--the expression of which is triggered by a large variety of environmental insults. The uspA gene is important for survival during cellular growth arrest, but the exact physiological role of the Usp proteins is not known. In this work we have performed phenotypic characterization of mutants with deletions of the six different usp genes. We report on hitherto unknown functions of these genes linked to motility, adhesion, and oxidative stress resistance, and we show that usp functions are both overlapping and distinct. Both UspA and UspD are required in the defense against superoxide-generating agents, and UspD appears also important in controlling intracellular levels of iron. In contrast, UspC is not involved in stress resistance or iron metabolism but is essential, like UspE, for cellular motility. Electron microscopy demonstrates that uspC and uspE mutants are devoid of flagella. In addition, the function of the uspC and uspE genes is linked to cell adhesion, measured as FimH-mediated agglutination of yeast cells. While the UspC and UspE proteins promote motility at the expense of adhesion, the UspF and UspG proteins exhibit the exact opposite effects. We suggest that the Usp proteins have evolved different physiological functions that reprogram the cell towards defense and escape during cellular stress.     

Regulation of Redox Signaling by Selenoproteins

The unique chemistry of oxygen has been both a resource and threat for life on Earth for at least the last 2.4 billion years. Reduction of oxygen to water allows extraction of more metabolic energy from organic fuels than is possible through anaerobic glycolysis. On the other hand, partially reduced oxygen can react indiscriminately with biomolecules to cause genetic damage, disease, and even death. Organisms in all three superkingdoms of life have developed elaborate mechanisms to protect against such oxidative damage and to exploit reactive oxygen species as sensors and signals in myriad processes. The sulfur amino acids, cysteine and methionine, are the main targets of reactive oxygen species in proteins. Oxidative modifications to cysteine and methionine can have profound effects on a protein’s activity, structure, stability, and subcellular localization. Non-reversible oxidative modifications (oxidative damage) may contribute to molecular, cellular, and organismal aging and serve as signals for repair, removal, or programmed cell death. Reversible oxidation events can function as transient signals of physiological status, extracellular environment, nutrient availability, metabolic state, cell cycle phase, immune function, or sensory stimuli. Because of its chemical similarity to sulfur and stronger nucleophilicity and acidity, selenium is an extremely efficient catalyst of reactions between sulfur and oxygen. Most of the biological activity of selenium is due to selenoproteins containing selenocysteine, the 21st genetically encoded protein amino acid. The most abundant selenoproteins in mammals are the glutathione peroxidases (five to six genes) that reduce hydrogen peroxide and lipid hydroperoxides at the expense of glutathione and serve to limit the strength and duration of reactive oxygen signals. Thioredoxin reductases (three genes) use nicotinamide adenine dinucleotide phosphate to reduce oxidized thioredoxin and its homologs, which regulate a plethora of redox signaling events. Methionine sulfoxide reductase B1 reduces methionine sulfoxide back to methionine using thioredoxin as a reductant. Several selenoproteins in the endoplasmic reticulum are involved in the regulation of protein disulfide formation and unfolded protein response signaling, although their precise biological activities have not been determined. The most widely distributed selenoprotein family in Nature is represented by the highly conserved thioredoxin-like selenoprotein W and its homologs that have not yet been assigned specific biological functions. Recent evidence suggests selenoprotein W and the six other small thioredoxin-like mammalian selenoproteins may serve to transduce hydrogen peroxide signals into regulatory disulfide bonds in specific target proteins.     

Origin and evolution of the protein-repairing enzymes methionine sulphoxide reductases

Abstract The majority of extant life forms thrive in an O(2)-rich environment, which unavoidably induces the production of reactive oxygen species (ROS) during cellular activities. ROS readily oxidize methionine (Met) residues in proteins/peptides to form methionine sulphoxide [Met(O)] that can lead to impaired protein function. Two methionine sulphoxide reductases, MsrA and MsrB, catalyse the reduction of the S and R epimers, respectively, of Met(O) in proteins to Met. The Msr system has two known functions in protecting cells against oxidative damage. The first is to repair proteins that have lost activity due to Met oxidation and the second is to function as part of a scavenger system to remove ROS through the reversible oxidation/reduction of Met residues in proteins. Bacterial, plant and animal cells lacking MsrA are known to be more sensitive to oxidative stress. The Msr system is considered an important cellular defence mechanism to protect against oxidative stress and may be involved in ageing/senescence. MsrA is present in all known eukaryotes and eubacteria and a majority of archaea, reflecting its essential role in cellular life. MsrB is found in all eukaryotes and the majority of eubacteria and archaea but is absent in some eubacteria and archaea, which may imply a less important role of MsrB compared to MsrA. MsrA and MsrB share no sequence or structure homology, and therefore probably emerged as a result of independent evolutionary events. The fact that some archaea lack msr genes raises the question of how these archaea cope with oxidative damage to proteins and consequently of the significance of msr evolution in oxic eukaryotes dealing with oxidative stress. Our best hypothesis is that the presence of ROS-destroying enzymes such as peroxiredoxins and a lower dissolved O(2) concentration in those msr-lacking organisms grown at high temperatures might account for the successful survival of these organisms under oxidative stress.     

Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes.

Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.     

The FHA domain is a modular phosphopeptide recognition motif.

FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.     

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

Solution structure and dynamics of the reduced and oxidized forms of the N-terminal domain of PilB from Neisseria meningitidis

The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.     

Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases

Oxidative damage to proteins is considered to be one of the major causes of aging and age-related diseases, and thus mechanisms have evolved to prevent or reverse these modifications. Methionine is one of the major targets of reactive oxygen species (ROS), where it is oxidized to methionine sulfoxide (MetO). Recently, evidence has accumulated suggesting that methionine (Met) oxidation may play an important role in the development and progression of neurodegenerative diseases like Alzheimer's and Parkinson's diseases. Oxidative alteration of Met to Met(O) is reversed by the methionine sulfoxide reductases (consisting of MsrA enzymes that reduce S-MetO and MsrB enzymes that reduce R-MetO, respectively). A major biological role of the Msr system is suggested by the fact that the MsrA null mouse (MT) exhibits a neurological disorder in the form of ataxia ("tip toe walking"), is more sensitive to oxidative stress, and has a shorter life span (by approximately 40%) than wild-type (WT) mice. By their action, the Msr enzymes can regulate protein function, be involved in signal-transduction pathways, and prevent cellular accumulation of faulty proteins. Malfunction of the Msr system can lead to cellular changes resulting in compromised antioxidant defense, enhanced age-associated diseases involving neurodegeneration, and shorter life span. In this review, the function and possible roles of the Msr system in prokaryotes and eukaryotes, in general, and in neurodegenerative diseases, in particular, will be discussed.     

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotie RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved time-tional amino acids between archaeal and eukaryotic Piwi domains suggested their functional similarity. Our results provide new clues to the evolution of RNA silencing pathways.