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Inflammation and oxidative damage contribute significantly to the development of cardiovascular diseases (CVD). Postprandial oxidative stress and inflammation are characterized by an increased susceptibility of the organism toward oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. Micronutrients modulate immune system and exert a protective action by reducing oxidized low-density lipoprotein (ox-LDL) level. The aim of the present study was to evaluate the postprandial plasma ox-LDL level and the gene expression of 13 genes related to oxidative stress (HOSp) and human inflammasome pathways (HIp), after a tocopherol-enriched Mediterranean meal (TEM), and a Western high-fat meal (HFM). Moreover, Mediterranean Adequacy Index was calculated to define the quality of both meals. We set up a randomized and crossover trial in healthy human volunteers. Ox-LDL level was measured by enzyme-linked immunosorbent assay and the gene expression of 13 genes related to HOSp and HIp by qRT-PCR. Ox-LDL levels significantly decreased comparing HFM versus TEM (p

Motivation Signaling and metabolic pathways are finely regulated by a network of protein phosphorylation events. Unraveling the nature of this intricate network, composed of kinases, target proteins and their interactions, is therefore of crucial importance. Although thousands of kinase-specific phosphorylations (KsP) have been annotated in model organisms their kinase-target network is far from being complete, with less studied organisms lagging behind. Results In this work, we achieved an automated and accurate identification of kinase domains, inferring the residues that most likely contribute to peptide specificity. We integrated this information with the target peptides of known human KsP to predict kinase-specific interactions in other eukaryotes through a deep neural network, outperforming similar methods. We analyzed the differential conservation of kinase specificity among eukaryotes revealing the high conservation of the specificity of tyrosine kinases. With this approach we discovered 1590 novel KsP of potential clinical relevance in the human proteome. Availability and implementation http://akid.bio.uniroma2.it Supplementary information Supplementary data are available at Bioinformatics online.

Large-scale interaction studies contribute the largest fraction of protein interactions information in databases. However, co-purification of non-specific or indirect ligands, often results in data sets that are affected by a considerable number of false positives. For the fraction of interactions mediated by short linear peptides, we present here a combined experimental and computational strategy for ranking the reliability of the inferred partners. We apply this strategy to the family of 14-3-3 domains. We have first characterized the recognition specificity of this domain family, largely confirming the results of previous analyses, while revealing new features of the preferred sequence context of 14-3-3 phospho-peptide partners. Notably, a proline next to the carboxy side of the phospho-amino acid functions as a potent inhibitor of 14-3-3 binding. The position-specific information about residue preference was encoded in a scoring matrix and two regular expressions. The integration of these three features in a single predictive model outperforms publicly available prediction tools. Next we have combined, by a naïve Bayesian approach, these "peptide features" with "protein features", such as protein co-expression and co-localization. Our approach provides an orthogonal reliability assessment and maps with high confidence the 14-3-3 peptide target on the partner proteins.

Background and Aim.p53 activity plays a role in muscle homeostasis and skeletal muscle differentiation; all pathways that lead to sarcopenia are related to p53 activities. We investigate the allelic frequency of the TP53 codon 72 in exon 4 polymorphism in the Italian female population and the association with appendicular skeletal muscle mass index in normal weight (NW), normal weight obese (NWO), and preobese-obese (Preob-Ob) subjects.Methods.We evaluated anthropometry, body composition, and p53 polymorphism in 140 women distinguished in NW, NWO, and Preob-Ob.Results.Arg*/Arg*genotype increases sarcopenia risk up to 20% (Arg*/Arg*genotype OR = 1.20; 95% CI = 0.48–2.9;proallele*carriers OR = 0.83; 95% CI = 0.83–2.06). The risk of being sarcopenic forArg*/Arg*genotype in NWO and Preob-Ob is 31% higher than NW carriers ofproallele*(RR = 0,31, 95% CI = 0,15–0,66,P= 0,0079). We developed a model able to predict sarcopenia risk based on age, body fat, and p53 polymorphism.Conclusion. Our study evidences that genotyping TP53 polymorphism could be a useful new genetic approach, in association with body composition evaluations, to assess sarcopenia risk.

Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed ERO for ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000) J. Biol. Chem. 275, 4827-4833). Here, we report the cloning and characterization of a novel human member of this family, ERO1-Lbeta. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lbeta gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 degrees C and to alleviate the "unfolded protein response" when expressed in ero1-1 cells indicates that ERO1-Lbeta is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lbeta display different tissue distributions. Furthermore, only ERO1-Lbeta transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super- antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.

Lysophospholipids may play an important protective role during primary infection of Mycobacterium tuberculosis (MTB) by enhancing innate antimycobacterial immune response of both macrophages and alveolar epithelial cells. Here, we show that treatment with lysophosphatidic acid (LPA) of mice aerogenically infected with MTB immediately after infection results in a significant early reduction of pulmonary CFUs and of histopathological damage in comparison with control mice. In contrast, treatment of acute disease does not result in any improvement of both microbiological and histopathological parameters. Altogether, these results show that LPA treatment can exert protective effect if administrated during primary infection, only.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling pathway known as the unfolded protein response (UPR), which leads to the transcriptional activation of factors involved in ER protein folding, to a transitory inhibition of protein synthesis and to an upregulation of the ER-associated degradation pathway. In order to identify new genes regulated during the UPR we have used an RNA fingerprinting technique to analyze the gene expression profiles in cells treated with DTT or tunicamycin, two strong UPR inducers. We isolated two novel transcripts upregulated by both treatments. The selective regulation of these genes during the UPR was confirmed in different cell lines and under various UPR-inducing conditions. These studies highlighted interesting aspects of the gene expression during the UPR, including a selective downregulation of members of the hsp70 family.

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

MOTIVATION: Polymerase chain reaction (PCR)-based RNA fingerprinting is a powerful tool for the isolation of differentially expressed genes in studies of neoplasia, differentiation or development. Arbitrarily primed RNA fingerprinting is capable of targeting coding regions of genes, as opposed to differential display techniques, which target 3' non-coding cDNA. In order to be of general use and to permit a systematic survey of differential gene expression, RNA fingerprinting has to be standardized and a number of highly efficient and selective arbitrary primers must be identified. RESULTS: We have applied a rational approach to generate a representative panel of high-efficiency oligonucleotides for RNA fingerprinting studies, which display marked affinity for coding portions of known genes and, as shown by preliminary results, of novel ones. The choice of oligonucleotides was driven by computer simulations of RNA fingerprinting reverse transcriptase (RT)-PCR experiments, performed on two custom-generated, non-redundant nucleotide databases, each containing the complete collection of deposited human or murine cDNAs. The simulation approach and experimental protocol proposed here permit the efficient isolation of coding cDNA fragments from differentially expressed genes. AVAILABILITY: Freely available on request from the authors. CONTACT: fesce.riccardo@hsr.it

Kinesins are microtubule-dependent molecular motors involved in intracellular transport and mitosis. Here, we report the cloning, sequencing, mapping, and expression of a novel member of the kinesin superfamily. The sequence of this newly identified human cDNA reveals an open reading frame encoding a putative protein of 792 residues. Based on its high sequence similarity to the kinesin-like molecule KIF3B, we named this protein KIF3C. KIF3C is encoded by transcripts that are distinct from the KIF3B mRNA in human, rat, and mouse and is preferentially expressed in the brain. Fluorescence in situ hybridization reveals that, in the human genome, the KIF3C gene maps to chromosome 2 at 2p23. The sequence of KIF3C predicts an unusually long insertion in the proximity of L11, a region thought to mediate microtubule binding. Taken together, these findings suggest that KIF3C is a novel kinesin-like protein that might be specifically involved in microtubule-based transport in neuronal cells.

The growth of hepatoma cells can be inhibited by treatment with TGFbeta1 or with exogenous reducing agents. To gain information on the molecular mechanisms underlying growth arrest, we visualized and compared gene expression profiles of proliferating versus non proliferating HepG2 cells by computer-assisted gene fishing, an improved technique of RNA fingerprinting that allows the selective amplification of coding regions within transcripts. While many transcripts are selectively regulated by either treatment, a set of bands appear to be coordinately regulated by 2ME and TGFbeta1, suggesting their possible involvement in the mechanisms of growth arrest. Display tags corresponding to 18 differentially expressed genes were cloned and, in most cases, identified as known genes or, more frequently, as their homospecific/cross-specific homologues. A novel member of the kinesin superfamily was identified amongst the genes induced by both 2ME and TGFbeta1. This gene, KIF3C, is upregulated in several cell lines undergoing growth arrest. Taken together, our findings show that computer-assisted gene fishing is a powerful tool for the identification and cloning of genes involved in the control of cell proliferation and indicate that extracellular reducing agents can regulate cell growth through modulation of gene expression.

Protein folding in the endoplasmic reticulum (ER) often involves the formation of disulfide bonds. The oxidizing conditions required within this organelle were shown to be maintained through the release of small thiols, mainly cysteine and glutathione. Thiol secretion was stimulated when proteins rich in disulfide bonds were translocated into the ER, and secretion was prevented by the inhibition of protein synthesis. Endogenously generated cysteine and glutathione counteracted thiol-mediated retention in the ER and altered the extracellular redox. The secretion of thiols might link disulfide bond formation in the ER to intra- and intercellular redox signaling.

Luciano Giacò,1 Massimo Amicosante,2 Maurizio Fraziano,1 Pier Federico Gherardini,1 Gabriele Ausiello,1 Manuela Helmer-Citterich,1 Vittorio Colizzi,1 Andrea Cabibbo11Department of Biology, 2Department of Internal Medicine, University of Rome "Tor Vergata", Rome, ItalyAbstract: The ability to predict immunogenic regions in selected proteins by in-silico methods has broad implications, such as allowing a quick selection of potential reagents to be used as diagnostics, vaccines, immunotherapeutics, or research tools in several branches of biological and biotechnological research. However, the prediction of antibody target sites in proteins using computational methodologies has proven to be a highly challenging task, which is likely due to the somewhat elusive nature of B-cell epitopes. This paper proposes a web-based platform for scoring potential immunological reagents based on the structures or 3D models of the proteins of interest. The method scores a protein's peptides set, which is derived from a sliding window, based on the average solvent exposure, with a filter on the average local model quality for each peptide. The platform was validated on a custom-assembled database of 1336 experimentally determined epitopes from 106 proteins for which a reliable 3D model could be obtained through standard modeling techniques. Despite showing poor sensitivity, this method can achieve a specificity of 0.70 and a positive predictive value of 0.29 by combining these two simple parameters. These values are slightly higher than those obtained with other established sequence-based or structure-based methods that have been evaluated using the same epitopes dataset. This method is implemented in a web server called B-Pred, which is accessible at http://immuno.bio.uniroma2.it/bpred. The server contains a number of original features that allow users to perform personalized reagent searches by manipulating the sliding window's width and sliding step, changing the exposure and model quality thresholds, and running sequential queries with different parameters. The B-Pred server should assist experimentalists in the rational selection of epitope antigens for a wide range of applications.Keywords: B-cell epitopes, immunoinformatics, bioinformatics, web server, epitope prediction

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

The multifunctional cytokine interleukin-6 (IL-6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse. Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL-6 shows species specificity. Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL-6 molecule are involved in human receptor binding. In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding. Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL-6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.

In our report “Cysteine and glutathione secretion in response to protein disulfide bond formation in the ER” (12 Sept., [p. 1681][1]) ([1][2]), we describe data suggesting that cysteine and glutathione are secreted through the exocytic pathway in response to disulfide bond formation in the endoplasmic reticulum (ER). However, we have been unable to replicate some of the experiments conducted by the first author of that report. This calls into question our results and conclusions. We are now in the process of repeating most of the experiments, and are ready to communicate the available results on request. Meanwhile, we wish to alert readers that the original conclusion that cysteine and glutathione are secreted through the exocytic pathway is not supported unless further experimental evidence becomes available. We apologize for any difficulties that may have been experienced by the scientific community. 1. [↵][3]1. S. Carelli, 2. A. Ceriotti, 3. A. Cabibbo, 4. G. Fassina, 5. M. Ruvo, 6. R. Sitia , Science 277, 1681 (1997). [OpenUrl][4][Abstract/FREE Full Text][5] [1]: /lookup/doi/10.1126/science.277.5332.1681 [2]: #ref-1 [3]: #xref-ref-1-1 "View reference 1 in text" [4]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.issn%253D0036-8075%26rft.aulast%253DCarelli%26rft.auinit1%253DS.%26rft.volume%253D277%26rft.issue%253D5332%26rft.spage%253D1681%26rft.epage%253D1684%26rft.atitle%253DCysteine%2Band%2BGlutathione%2BSecretion%2Bin%2BResponse%2Bto%2BProtein%2BDisulfide%2BBond%2BFormation%2Bin%2Bthe%2BER%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.277.5332.1681%26rft_id%253Dinfo%253Apmid%252F9287224%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [5]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEzOiIyNzcvNTMzMi8xNjgxIjtzOjQ6ImF0b20iO3M6MjY6Ii9zY2kvMjc5LzUzNTUvMTI4My4xMC5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30=

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.