A hybrid method for extraction of protein-protein interactions from literature

In this work, a hybrid method is proposed to eliminate the limitations of traditional protein-pro- tein interactions (PPIs) extraction methods, such as pattern learning and machine learning. Each sentence from the biomedical literature containing a protein pair describes a PPI which is predicted by first learning syntax patterns typical of PPIs from training corpus and then using their presence as features, along with bag-of-word features in a maximum entropy model. Tested on the BioCreAtIve corpus, the PPIs extraction method, which achieved a precision rate of 64%, recall rate of 60%, improved the performance in terms of F1 value by 11% compared with the component pure pattern- based and bag-of-word methods. The results on this test set were also compared with other three ex- traction methods and found to improve the performance remarkably.     

Structure-based design and synthesis of small molecule protein-tyrosine phosphatase 1B inhibitors

Protein-tyrosine phosphatase (PTP) inhibitors are attractive as potential signal transduction-directed therapeutics which may be useful in the treatment of a variety of diseases. We have previously reported the X-ray structure of 1,1-difluoro-1-(2-naphthalenyl)methyl] phosphonic acid (4) complexed with the human the protein-tyrosine phosphatase 1B (PTP1B) and its use in the design of an analogue which binds with higher affinity within the catalytic site (Burke, T. R., Jr. et al. Biochemistry 1996, 35, 15989). In the current study, new naphthyldifluoromethyl phosphonic acids were designed bearing acidic functionality intended to interact with the PTP1B Arg47, which is situated just outside the catalytic pocket. This residue has been shown previously to provide key interactions with acidic residues of phosphotyrosyl-containing peptide substrates. Consistent with trends predicted by molecular dynamics calculations, the new analogues bound with 7- to 14-fold higher affinity than the parent 4, in principal validating the design rationale.     

Peptide modulators of protein-protein interactions in intracellular signaling

Signal transduction cascades involve multiple enzymes and are orchestrated by selective protein-protein interactions that are essential for the progression of intracellular signaling events. Modulators of these protein-protein interactions have been used to dissect the role of individual components of each signaling cascade. We describe several methods that have been developed for the identification of peptides that inhibit the interaction between signaling proteins and hence selectively modulate their functions. Such peptide modulators provide important tools for basic research and have great potential as leads for the development of new classes of therapeutic drugs.     

The G beta gamma complex of the yeast pheromone response pathway. Subcellular fractionation and protein-protein interactions

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction. A slower-migrating, presumably phosphorylated, form of Ste4p is enriched in the non-membrane fraction. The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to co-sediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum. Under the conditions of our assay, Gpa1p was not associated with either particle. The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G betagamma complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes. The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of the cell-surface receptors. These results indicate that at least 40% of Ste4p and Ste18p are part of a G betagamma complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G alpha.     

Structure-based engineering of environmentally sensitive fluorophores for monitoring protein-protein interactions

Single, extrinsic, environmentally sensitive fluorophores can be used to quantitate formation of protein-protein complexes. These can be prepared semi-synthetically by covalent coupling to single cysteine mutations introduced at positions where the fluorophore is predicted to respond to formation of the complex without adversely affecting the interaction. The three-dimensional structure of a protein-protein interface can be used to select such locations by identifying residues that are located at the edge of a buried interfacial region, and are in partial steric contact with both partners as indicated by a change in their static solvent-accessible surface area upon complex formation. Using this design approach, cysteine mutations were introduced into the B1 domain of protein G, which successfully monitor complex formation with minimal interference. Such constructs have great utility in the analysis of solution properties of interface mutants.     

Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions

Mechanisms underlying stimulation of platelet-derived growth factor (PDGF) beta-receptors expressed on connective tissue cells in human colorectal adenocarcinoma were investigated in this study. PDGF-AB/BB, but not PDGF receptors, was expressed by tumor cells in situ, as well as in tumor cell isolates of low passage from human colorectal adenocarcinoma. In an experimental co-culture system, conditioned medium from tumor cells only marginally activated PDGF beta-receptors expressed on fibroblasts. In contrast, co-culturing of the two cell types led to a marked PDGF beta-receptor activation. Functional PDGF-AB/BB was found to be associated with heparinase-I-sensitive components on the tumor cell surface. PDGF-AB/BB, isolated from heparinase-I-sensitive cell surface components, induced a marked activation of PDGF beta-receptors. Furthermore, co-culturing tumor cells together with fibroblasts led to a sustained activation of PDGF beta-receptors expressed on fibroblasts. Double immunofluorescence staining of tissue sections from human colorectal adenocarcinoma, combined with computer-aided image analysis, revealed that nonproliferating tumor cells were the predominant cellular source of PDGF-AB/BB in the tumor stroma. In addition, PDGF-AB/BB-expressing tumor cells were found juxtapositioned to microvascular cells expressing activated PDGF beta-receptors. Confocal microscopy revealed a cytoplasmic and cell-membrane-associated expression of PDGF-AB/BB in tumor cells situated in the stroma. In contrast, epithelial cells situated in normal or tumorous acinar structures revealed only a cell-membrane-associated PDGF-AB/BB expression. The is vitro and in situ results demonstrate that tumor cells not only facilitate but also have the ability to modulate connective tissue cell responsiveness to PDGF-AB/BB in a paracrine fashion, through direct cell-cell interactions in human colorectal adenocarcinoma.     

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors

BACKGROUND: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets. RESULTS: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors. CONCLUSIONS: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.     

The ras recruitment system, a novel approach to the study of protein-protein interactions

The yeast two-hybrid system represents one of the most efficient approaches currently available for identifying and characterizing protein-protein interactions [1-4]. Although very powerful, this procedure exhibits several problems and inherent limitations [5]. A new system, the Sos recruitment system (SRS), was developed recently [6] based on a different readout from that of the two-hybrid system [6-8]. SRS overcomes several of the limitations of the two-hybrid system and thus serves as an attractive alternative for studying protein-protein interactions between known and novel proteins. Nevertheless, we encountered a number of problems using SRS and so have developed an improved protein recruitment system, designated the Ras recruitment system (RRS), based on the absolute requirement that Ras be localized to the plasma membrane for its function [9-10]. Ras membrane localization and activation can be achieved through interaction between two hybrid proteins. We have demonstrated the effectiveness of the novel RRS system using five different known protein-protein interactions and have identified two previously unknown protein-protein interactions through a library screening protocol. The first interaction (detailed here) is between JDP2, a member of the basic leucine zipper (bZIP) family, and C/EBPgamma, a member of the CCAAT/enhancer-binding protein (C/EBP) family. The second interaction is between the p21-activated protein kinase Pak65 and a small G protein (described in the accompanying paper by Aronheim et al. [11]). The RRS system significantly extends the usefulness of the previously described SRS system and overcomes several of its limitations.     

Molecular mechanisms for focal adhesion assembly through regulation of protein-protein interactions

Focal adhesions provide a useful model for studying cell/extracellular matrix interactions and the subsequent cytoskeletal reorganization. Recent advances have suggested potential mechanisms by which cells may regulate focal adhesion assembly following integrin-mediated cell adhesion.     

Probing the role of homomeric and heteromeric receptor interactions in TGF-beta signaling using small molecule dimerizers

BACKGROUND: Transforming growth factor Beta (TGF-Beta) arrests many cell types in the G1 phase of the cell and upregulates plasminogen activator inhibitor 1 (PAI-1). The type 1 (TGF-Beta RI) an II (TGF-Beta RII) TGF-Beta receptors mediate these and other effects of TGF-Beta on target cells. TGF-Beta initially binds to TGF-Beta RII and subsequently TGF-Beta RI is recruited to form a heteromeric complex. TGF-Beta RI phosphorylates the downstream effectors Smad2 and Smad3, leading to their translocation into the nucleus. Here, we explored the role of receptor oligomerization in TGF-Beta signaling. RESULTS: We constructed fusion proteins containing receptor cytoplasmic tails linked to binding domains for small-molecule dimerizers. In COS-1 cells, recruitment of a soluble TGF-Beta RII tail to a myristoylated TGF-Beta RI tail promoted Smad2 nuclear translocation. In mink lung cells, homo-oligomerization of a myristoylated TGF-Beta Ri tail in presence of a myristoylated TGF-Beta RII tail activated the PAI-1 promoter. Oligomerization of an acidic mutant of the TGF-Beta RI tail in absence of TGF-Beta RII activated the PAI-A promoter and inhibited the growth of mink lung cells. CONCLUSIONS: Non-toxic, small molecules designed to oligomerize cytoplasmic tails of TGF-Beta receptors at the plasma membrane can activate TGF-Beta signaling. Although TGF-Beta normally signals through two receptors that are both necessary for signaling, in one small-molecule system, a dimerizer activates signaling through a single type of receptor that is sufficient to induce TGF-Beta signaling. These methods of activating TGF-Beta signaling could be extended to signaling pathways of other TGF-Beta superfamily members such as activin and the bone morphogenetic proteins.     

Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors

The exact mechanism of hemoglobin (Hb) associated vasoconstriction has not been elucidated. We investigated this problem using isolated superfused rat aortic rings with intact endothelium. Human stroma-free hemoglobin solution (SFH) at 2uM reversed vaso relaxation induced by 33uM acetylcholine (Ach). Further, pretreatment with 4uM SFH inhibited Ach(333uM) induced dilation. The SFH induced contraction was reversible by glyceryltrinitrate (GTN), a nitric oxide (NO) donor. Preincubation with a NO synthase inhibitor nitro-L-arginine-methyl ester (NAME, 0.4nM) caused almost complete inhibition of the Hb vasoactivity. Unlike SFH (ferrous oxyHb), prenitrosylated SFH (HbNO) or ferric Hb derivatives (e.g., metHb, HbCN) did not elicit vasoconstriction. The presence of 2uM SFH did not significantly reduce the vasodilatory effectiveness of endothelium independent vasodilators isoproteranol (ISO) and papaverine (PPV). These results suggest that a primary mechanism for Hb vasoconstrictor activity is ferrous Hb scavenging of endothelium derived NO, a signal for guanylate cyclase-cGMP mediated smooth muscle relaxation. Additionally, it appears that the Hb induced vasoactivities may be modulated with NO independent vasodilators.     

Structurally similar small molecule photoaffinity CCK-A agonists and antagonists as novel tools for directly probing 7TM receptors-ligand interactions

Incorporation of photolabile benzoyl (2a-d) or trifluoromethyl-3H-diazirine (3a-d) substituents into 1,5-benzodiazepine ligands did not significantly impair the rat CCK-A binding affinity of either agonists or antagonists. The modified agonist ligands also retained functional potency and efficacy in the rat amylase assay. Despite their strong structural similarity, the SAR of this limited set of compounds suggests that these small molecule antagonists and agonists might differ in their mode of binding to the CCK-A receptor. Preliminary affinity results show that representative agonists and antagonists from these series can be used to efficiently covalently label the CCK-A receptor.     

Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system

In the adult axon, the neurofilaments (NFs) are heteropolymers formed from the low (NFL), middle (NFM), and high (NFH) molecular weight neurofilament triplet proteins (NFTPs). All three proteins have the basic intermediate filament protein tripartite structure, which consists of a short amino-terminal head region, an alpha-helical rod region of approximately310 amino acids, and a carboxyl-terminal tail region of variable length. In vitro polymerization studies have shown that only NFL can assemble into homopolymeric 10-nm filaments. The assembly of intermediate filaments, including the NFs, begins with the formation of a coiled-coil dimer involving the alpha-helical rod domains of two molecules. In order to determine whether homodimers or heterodimers of NFTPs are the preferred intermediates in the assembly of NFs, we have used the yeast two-hybrid system to study the interactions between the different NFTPs. By monitoring the activity of the lacZ reporter gene product, we are able to show that the interactions of NFL with NFL, NFM, or NFH are stronger than the interactions of NFM with NFM or NFH and the interaction of NFH with NFH. These results imply that NFM and NFH are more likely to form heterodimers with NFL than homodimers and are consistent with the inability of NFM and NFH to self-polymerize in vitro and in vivo.     

A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, gamma-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and gamma-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.