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Carducci M.,University of Rome Tor Vergata | Perfetto L.,University of Rome Tor Vergata | Briganti L.,University of Rome Tor Vergata | Paoluzi S.,University of Rome Tor Vergata | And 5 more authors.
Biotechnology Advances | Year: 2012

Families of conserved protein domains, specialized in mediating interactions with short linear peptide motifs, are responsible for the formation of a variety of dynamic complexes in the cell. An important subclass of these motifs are characterized by a high proline content and play a pivotal role in biological processes requiring the coordinated assembly of multi-protein complexes. This is achieved via interaction of proteins containing modules such as Src Homology-3 (SH3) or WW domains and specific proline rich patterns. Here we make available via a publicly accessible database a synopsis of our current understanding of the interaction landscape of the human SH3 protein family. This is achieved by integrating an information extraction strategy with a new experimental approach. In a first approach we have used a text mining strategy to capture a large number of manuscripts reporting interactions between SH3 domains and target peptides. Relevant information was annotated in the MINT database. In a second experimental approach we have used a variant of the WISE (Whole Interactome Scanning Experiment) strategy to probe a large number of naturally occurring and chemically-synthesized peptides arrayed at high density on a glass surface. By this method we have tested 60 human SH3 domains for their ability to bind a collection of 9192 poly-proline containing peptides immobilized on a glass chip. To evaluate the quality of the resulting interaction dataset, we retested some of the interactions on a smaller scale and performed a series of pull down experiments on native proteins. Peptide chips, pull down assays, SPOT synthesis and phage display experiments have allowed us to further characterize the specificity and promiscuity of proline-rich binding domains and to map their interaction network. Both the information captured from the literature and the interactions inferred from the peptide chip experiments were collected and stored in the PepspotDB (http://mint.bio.uniroma2.it/PepspotDB/). © 2011 Elsevier Inc. Source


Tinti M.,University of Rome Tor Vergata | Tinti M.,University of Dundee | Kiemer L.,University of Rome Tor Vergata | Costa S.,University of Rome Tor Vergata | And 20 more authors.
Cell Reports | Year: 2013

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. © 2013 The Authors. Source


Maksimov P.,Friedrich Loeffler Institute | Zerweck J.,JPT Peptide Technologies Gmbh | Maksimov A.,Friedrich Loeffler Institute | Hotop A.,University of Gottingen | And 11 more authors.
Clinical and Vaccine Immunology | Year: 2012

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples (n = 184) were collected from patients presenting with acute toxoplasmosis (n = 21), latent T. gondii infection (n = 53), and inactive ocular toxoplasmosis (n = 10) and from seropositive forest workers (n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers (n = 75) and patients (n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source


Rauh D.,Martin Luther University of Halle Wittenberg | Fischer F.,University of Bayreuth | Gertz M.,University of Bayreuth | Lakshminarasimhan M.,University of Bayreuth | And 10 more authors.
Nature Communications | Year: 2013

Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins. © 2013 Macmillan Publishers Limited. Source


Maksimov P.,Federal Research Institute for Animal Health | Zerweck J.,JPT Peptide Technologies Gmbh | Maksimov A.,Federal Research Institute for Animal Health | Hotop A.,University of Gottingen | And 10 more authors.
PLoS ONE | Year: 2012

Background: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. Methodology: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). Findings: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. Conclusions: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution. © 2012 Maksimov et al. Source

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