The Cu(II) affinity constant and reactivity of Hepcidin-25, the main iron regulator in human blood.

Hepcidin is an iron regulatory hormone that does not bind iron directly. Instead, its mature 25-peptide form (H25) contains a binding site for other metals, the so-called ATCUN/NTS (amino-terminal Cu/Ni binding site). The Cu(II)-hepcidin complex was previously studied, but due to poor solubility and difficult handling of the peptide the definitive account on the binding equilibrium was not obtained reliably. In this study we performed a series of fluorescence competition experiments between H25 and its model peptides containing the same ATCUN/NTS site and determined the Cu(II) conditional binding constant of the CuH25 complex at pH 7.4, CK7.4 = 4 ± 2 × 1014 M-1. This complex was found to be very inert in exchange reactions and poorly reactive in the ascorbate consumption test. The consequences of these findings for the putative role of Cu(II) interactions with H25 are discussed.

Global pentapeptide statistics are far away from expected distributions.

The relationships between polypeptide composition, sequence, structure and function have been puzzling biologists ever since first protein sequences were determined. Here, we study the statistics of occurrence of all possible pentapeptide sequences in known proteins. To compensate for the non-uniform distribution of individual amino acid residues in protein sequences, we investigate separately all possible permutations of every given amino acid composition. For the majority of permutation groups we find that pentapeptide occurrences deviate strongly from the expected binomial distributions, and that the observed distributions are also characterized by high numbers of outlier sequences. An analysis of identified outliers shows they often contain known motifs and rare amino acids, suggesting that they represent important functional elements. We further compare the pentapeptide composition of regions known to correspond to protein domains with that of non-domain regions. We find that a substantial number of pentapeptides is clearly strongly favored in protein domains. Finally, we show that over-represented pentapeptides are significantly related to known functional motifs and to predicted ancient structural peptides.

An Engineered Distant Homolog of Pseudomonas syringae TTSS Effector From Physcomitrella patens Can Act as a Bacterial Virulence Factor.

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in common bean (Phaseolus vulgaris). Similar to other pathogenic gram-negative bacteria, it secrets a set of type III effectors into host cells to subvert defense mechanisms. HopQ1 (for Hrp outer protein Q) is one of these type III effectors contributing to virulence of bacteria. Upon delivery into a plant cell, HopQ1 undergoes phosphorylation, binds host 14-3-3 proteins and suppresses defense-related signaling. Some plants however, evolved systems to recognize HopQ1 and respond to its presence and thus to prevent infection. HopQ1 shows homology to Nucleoside Hydrolases (NHs), but it contains a modified calcium binding motif not found in the canonical enzymes. CLuster ANalysis of Sequences (CLANS) revealed that HopQ1 and alike proteins make a distinct group of putative NHs located distantly from the classical enzymes. The HopQ1 - like protein (HLP) group comprises sequences from plant pathogenic bacteria, fungi, and lower plants. Our data suggest that the evolution of HopQ1 homologs in bacteria, fungi, and algae was independent. The location of moss HopQ1 homologs inside the fungal clade indicates a possibility of horizontal gene transfer (HGT) between those taxa. We identified a HLP in the moss Physcomitrella patens. Our experiments show that this protein (referred to as PpHLP) extended by a TTSS signal of HopQ1 promoted P. syringae growth in bean and was recognized by Nicotiana benthamiana immune system. Thus, despite the low sequence similarity to HopQ1 the engineered PpHLP acted as a bacterial virulence factor and displayed similar to HopQ1 virulence properties.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress.

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.

A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells.

Background. Members of the Runx gene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether the Runx2 gene is also expressed in testes. Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕W (v) strain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5'-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met(1) to Gly(15)) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met(1). With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein. Results. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met(1) in the deduced open reading frame of Runx2 is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein. Discussion. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a single Runx2 gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

The proline-rich region of glyceraldehyde-3-phosphate dehydrogenase from human sperm may bind SH3 domains, as revealed by a bioinformatic study of low-complexity protein segments.

Glyceraldehyde-3-phosphate dehydrogenase from human sperm (GAPDHS) provides energy to the sperm flagellum, and is therefore essential for sperm motility and male fertility. This isoform is distinct from somatic GAPDH, not only in being specific for the testis but also because it contains an additional amino-terminal region that encodes a proline-rich motif that is known to bind to the fibrous sheath of the sperm tail. By conducting a large-scale sequence comparison on low-complexity sequences available in databases, we identified a strong similarity between the proline-rich motif from GAPDHS and the proline-rich sequence from Ena/vasodilator-stimulated phosphoprotein-like (EVL), which is known to bind an SH3 domain of dynamin-binding protein (DNMBP). The putative binding partners of the proline-rich GAPDHS motif include SH3 domain-binding protein 4 (SH3BP4) and the IL2-inducible T-cell kinase/tyrosine-protein kinase ITK/TSK (ITK). This result implies that GAPDHS participates in specific signal-transduction pathways. Gene Ontology category-enrichment analysis showed several functional classes shared by both proteins, of which the most interesting ones are related to signal transduction and regulation of hydrolysis. Furthermore, a mutation of one EVL proline to leucine is known to cause colorectal cancer, suggesting that mutation of homologous amino acid residue in the GAPDHS motif may be functionally deleterious.

Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919.

Here, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic Lactobacillus rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally localized pilus gene clusters from L. casei and Lactobacillus paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free mice, already after 2 days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days.

Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level.

Novel AlkB dioxygenases--alternative models for in silico and in vivo studies.

BACKGROUND: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1-8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. METHODOLOGY AND FINDINGS: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB(-) mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. CONCLUSIONS: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

The C2H2 zinc finger transcription factors are likely targets for Ni(II) toxicity.

Ni(II) ions are able to hydrolyze Naa-(Ser/Thr) peptide bonds in Naa-(Ser/Thr)-Xaa-His-Zaa sequences. We found that various human transcription factors contain such nickel hydrolytic patterns within C2H2 zinc finger (ZF) domains. We demonstrated the hydrolysis on two peptide models, the 3rd ZF of the Sp1 transcription factor and the 1st ZF of the ZNF302 transcription factor. The experimentally studied reaction rates indicate that the hydrolysis reaction is likely to be an element of intracellular nickel toxicity.

A widespread peroxiredoxin-like domain present in tumor suppression- and progression-implicated proteins.

BACKGROUND: Peroxide turnover and signalling are involved in many biological phenomena relevant to human diseases. Yet, all the players and mechanisms involved in peroxide perception are not known. Elucidating very remote evolutionary relationships between proteins is an approach that allows the discovery of novel protein functions. Here, we start with three human proteins, SRPX, SRPX2 and CCDC80, involved in tumor suppression and progression, which possess a conserved region of similarity. Structure and function prediction allowed the definition of P-DUDES, a phylogenetically widespread, possibly ancient protein structural domain, common to vertebrates and many bacterial species. RESULTS: We show, using bioinformatics approaches, that the P-DUDES domain, surprisingly, adopts the thioredoxin-like (Thx-like) fold. A tentative, more detailed prediction of function is made, namely, that of a 2-Cys peroxiredoxin. Incidentally, consistent overexpression of all three human P-DUDES genes in two public glioblastoma microarray gene expression datasets was discovered. This finding is discussed in the context of the tumor suppressor role that has been ascribed to P-DUDES proteins in several studies. Majority of non-redundant P-DUDES proteins are found in marine metagenome, and among the bacterial species possessing this domain a trend for a higher proportion of aquatic species is observed. CONCLUSIONS: The new protein structural domain, now with a broad enzymatic function predicted, may become a drug target once its detailed molecular mechanism of action is understood in detail.

Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization.

BACKGROUND: The correct folding and dimerization of tubulins, before their addition to the microtubular structure, needs a group of conserved proteins called cofactors A to E. The biochemical analysis of cofactors gave an insight to their general functions, however not much is known about the domain structure and detailed, molecular function of these proteins. RESULTS: Combining modelling and fold prediction tools, we present 3D models of all cofactors, including several previously unannotated domains of cofactors B-E. Apart from the new HEAT and Armadillo domains in cofactor D and an unusual spectrin-like domain in cofactor C, we have identified a new subfamily of ubiquitin-like domains in cofactors B and E. Together, these observations provide a reliable, molecular level model of cofactor complex. CONCLUSION: Distant homology searches allowed the identification of unknown regions of cofactors as self-reliant domains and allow us to present a detailed hypothesis of how a cofactor complex performs its function.