Identification Of Protein-protein Interactions Research Articles

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting.

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

4] Analysis and identification of protein-protein interactions using protein recruitment systems

Publisher Summary The immense output of various genome-sequencing projects has provided the scientific community with numerous proteins with no assigned function. There is no single protein that functions in isolation within the cell, and protein–protein interactions serve as a general means to provide functional specificity and to control the activity of enzymes and signaling molecules. To achieve a better functional understanding of known and novel gene products, much current research activity in molecular cell biology is focused on the identification of interacting proteins and characterization of these interactions. To date, multiple methods exist to identify and characterize protein–protein interactions; however, few of them employ a genetic selection system in vivo such as the yeast two-hybrid system. The two-hybrid system is an excellent research tool and is commonly used to identify and characterize novel and known interaction partners for proteins of interest. Although powerful, the two-hybrid system, which is based on a transcriptional readout, exhibits several limitations and inherent problems. To overcome these problems and limitations, a cytoplasmic protein recruitment system designated the son of sevenless “(Sos) recruitment system” (SRS) has been developed. This and the related Ras recruitment system (RRS) take advantage of a general phenomenon in signal transduction—namely, that the generation of local high concentrations of a signaling intermediate can result in a dramatic increase in signaling activity.

Macromolecular assemblage of aminoacyl-tRNA synthetases: identification of protein-protein interactions and characterization of a core protein

In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of defined and conserved structural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the monospecific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the complex. Twenty-one protein pairs have been unambiguously identified, leading to a global view of the topological arrangement of the subunits of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GlnRS, with the class II synthetases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein partners. These findings suggest that p38, for which no homologous protein has been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex.

A Model of Elegance

GenBank, http://www.ncbi.nlm.nih.gov/Web/GenbankGenome Sequencing Center, University of Washington School of Medicine, http://genome.wustl.edu/gsc/gschmpg.htmlThe Sanger Center, http://www.sanger.ac.ukXREFdb, http://www.ncbi.nlm.nih.gov/Bassett/modelorgs

Identification of Protein-Protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans

In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal β strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal β strands are directly involved in interactions required for MSP filament assembly.

Preparative two-dimensional gel electrophoresis of membrane proteins.

Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 microg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).

Protein-protein interactions in the yeast PKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade.

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with the Saccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in the PKC1-mediated signalling pathway. Subsequently, Pkc1p and the other PKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of the PKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.

15] Identification of protein-protein interactions by λgt11 expression cloning

This chapter discusses the identification of protein–protein interactions by λgt11 expression cloning. Protein–protein interactions govern a wide variety of fundamental biological processes, ranging from the control of enzymatic activity through subunit association and enzyme-substrate recognition, to the specific interactions of protein ligands with their receptors. Intermolecular associations are also central to the manner in which oncogene-encoded proteins regulate cell proliferation. The realization that highly specific protein–protein interactions mediate cell behavior at almost every level has led to attempts to develop general methods with which to identify molecules that physically associate with a given protein. These approaches fall roughly into two classes: biochemical and genetic. When correct processing and post-translational modification are a prerequisite for molecular interaction, a modified genetic strategy that employs transfection of a cDNA expression library into mammalian cells can be used. Such a technique has been used quite successfully to identify several cell surface receptors based on their ligand binding activities. A frequently used biochemical approach to identify specific molecular interactions has been to employ an immobilized target protein to adsorb unknown binding partners from cell extracts. One advantage of the expression cloning method is that the actual screening is carried out ex vivo. This permits a greater degree of control over the preparation and labeling of the probe than in the two-hybrid system.

A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation.

The yeast RAP1 protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAP1 is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(C1-3A) sequences. Previous studies have indicated that the regulatory function of RAP1 is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAP1-interacting factor (RIF1). Strains carrying gene disruptions of RIF1 grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rap1s mutants. Furthermore, hybrid proteins containing rap1s missense mutations are defective in an interaction with RIF1 in the two-hybrid system. Taken together, these data support the idea that the rap1s phenotypes are attributable to a failure to recruit RIF1 to silencers and telomeres and suggest that RIF1 is a cofactor or mediator for RAP1 in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIF1 that partially restores the interaction with rap1s mutant proteins.