Simona Panni

A novel peptide–SH3 interaction

SH3 domains constitute a family of protein–protein interaction modules that bind to peptides displaying an X‐proline‐X‐X‐proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non‐receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage‐displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross‐linking, we demonstrate that a proline‐X‐X‐aspartate‐tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8‐related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3‐containing proteins that do not bind to canonical XPXXP‐containing peptides, and that establish distinct interactions in the signaling network.

Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins.

A genome-scale specificity and interaction map for yeast SH3 domain-containing proteins reveal how family members show selective binding to target proteins and predicts the dynamic localization of new candidate endocytosis proteins.

VirusMINT: a viral protein interaction database

Understanding the consequences on host physiology induced by viral infection requires complete understanding of the perturbations caused by virus proteins on the cellular protein interaction network. The VirusMINT database (http://mint.bio.uniroma2.it/virusmint/) aims at collecting all protein interactions between viral and human proteins reported in the literature. VirusMINT currently stores over 5000 interactions involving more than 490 unique viral proteins from more than 110 different viral strains. The whole data set can be easily queried through the search pages and the results can be displayed with a graphical viewer. The curation effort has focused on manuscripts reporting interactions between human proteins and proteins encoded by some of the most medically relevant viruses: papilloma viruses, human immunodeficiency virus 1, Epstein–Barr virus, hepatitis B virus, hepatitis C virus, herpes viruses and Simian virus 40.

Can we infer peptide recognition specificity mediated by SH3 domains

Protein interaction domain families that modulate the formation of macromolecular complexes recognize specific sequence or structural motifs. For instance SH3 and WW domains bind to polyproline peptides while SH2 and FHA domains bind to peptides phosphorylated in Tyr and Thr respectively. Within each family, variations in the chemical characteristics of the domain binding pocket modulate a finer peptide recognition specificity and, as a consequence, determine the selection of functional protein partners in vivo. In the proteomic era there is the need for reliable inference methods to help restricting the sequence space of the putative targets to be confirmed experimentally by more laborious experimental approaches. Here we will review the published data about the peptide recognition specificity of the SH3 domain family and we will propose a classification of SH3 domains into eight classes. Finally, we will discuss whether the available information is sufficient to infer the recognition specificity of any uncharacterized SH3 domain.

Selectivity and promiscuity in the interaction network mediated by protein recognition modules

A substantial fraction of protein interactions in the cell is mediated by families of protein modules binding to relatively short linear peptides. Many of these interactions have a high dissociation constant and are therefore suitable for supporting the formation of dynamic complexes that are assembled and disassembled during signal transduction. Extensive work in the past decade has shown that, although member domains within a family have some degree of intrinsic peptide recognition specificity, the derived interaction networks display substantial promiscuity. We review here recent advances in the methods for deriving the portion of the protein network mediated by these domain families and discuss how specific biological outputs could emerge in vivo despite the observed promiscuity in peptide recognition in vitro.

Alternative Bacteriophage Display Systems

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm. Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis. This review describes and compares the properties of these alternative display systems.

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na+-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K+ gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na+. Internal Na+ exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.

HuPho: the human phosphatase portal

Phosphatases and kinases contribute to the regulation of protein phosphorylation homeostasis in the cell. Phosphorylation is a key post‐translational modification underlying the regulation of many cellular processes. Thus, a comprehensive picture of phosphatase function and the identification of their target substrates would aid a systematic approach to a mechanistic description of cell signalling. Here we present a website designed to facilitate the retrieval of information about human protein phosphatases. To this end we developed a search engine to recover and integrate information annotated in several publicly available web resources. In addition we present a text‐mining‐assisted annotation effort aimed at extracting phosphatase related data reported in the scientific literature. The HuPho (human phosphatases) website can be accessed at http://hupho.uniroma2.it.

Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens†

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.

Domain repertoires as a tool to derive protein recognition rules.

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.