Showing papers on "Protein–protein interaction published in 1994"

Split ubiquitin as a sensor of protein interactions in vivo

Abstract: We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub. However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.

Molecular identities of human sperm proteins that bind human zona pellucida: nature of sperm-zona interaction, tyrosine kinase activity, and involvement of FA-1.

Abstract: The present study was conducted to investigate the molecular identities, nature of interaction, and tyrosine phosphorylation activity of the spermzona pellucida binding proteins in humans. Sperm proteins belcnging to four major molecular regions, namely 95, 63, 51, and 14–18 kDa, reacted with zona pellucida proteins in the Western blot and immunoprecipitation procedures. In these procedures, zona pellucida protein that reacted strongest with the sperm proteins belonged to the molecular region of 55 kDa (ZP3), besides weakly reacting proteins in the 110-kDa (ZP1/ZP2) and 14–18-kDa molecular regions. The major forces involved in the sperm-zona protein interactions were of hydrophobic and ionic in nature. Three (95, 51, and 14–18 kDa) of the four molecular regions of sperm proteins that bound to the zona pellucida proteins also seem to involve o-phospho-L-tyrosine residues in their interaction, and these proteins demonstrated the presence of phosphotyrosine residues, and the 51-kDa protein also showed autophosphorylating activity in the in vitro kinase assay. The sperm binding zona protein of 55 kDa also demonstrated autophosphorylating activity. Using specific monoclonal antibody to the well characterized sperm-specific glycoprotein, designated FA-1, and the competitive inhibition in the immunoprecipitation procedure, it was found that the 51 kDa protein is indeed FA-1 antigen. Besides elucidating the molecular nature of the spermzona interaction, these antigens will find application in the development of a multivalent contraceptive vaccine, and may also help in specific diagnosis and treatment of infertility mediated through defective gamete (sperm or oocyte) function. © 1994 Wiley-Liss, Inc.

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114

Abstract: Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Periplasmic membrane-bound system for detecting protein-protein interactions

Abstract: The present invention relates to: (1) a fusion protein having a dimerizing domain with or without a ligand-binding region and tox R DNA-binding and hydrophobic transmembrane regions; (2) host cells comprising the fusion protein and a nucleic acid molecule having a reporter gene operatively linked to the ctx operon, wherein dimerization (ligand-dependent or -independent) is signaled by expression of the reporter gene; (3) a nucleic acid molecule coding for the fusion protein; (4) an expression vector comprising a coding region for the fusion protein; (5) a process for detecting dimer formation (ligand dependent or ligand independent) of the fusion protein, which comprises treating a culture of the host cells with a ligand, ligand mimetic, or dimerization inhibitor, and screening for expression of the reporter gene. The present invention can be used to generate a signal from a variety of ligand-binding domains, allowing ligand binding to be indicated by a simple colorimeteric test or antibiotic resistance. The fusion proteins could include therapeutically relevant domains, so that biologically pertinent interactions can be indicated by a readily measurable signal.

Method of detecting ligand interactions

Abstract: Provided by the present invention are novel methods of detecting ligand interactions, as well as regents useful in the method, including DNA and host cells; and more specifically relates to novel methods for the detection of protein/protein interactions and their application in epitope mapping and the study of ligand/receptor interactions. Also provided are vaccines and kits comprising the expression products and host cells of the invention.

Protein-protein interactions as therapeutic targets for cancer

Abstract: The recent elucidation of many of the pathways involved in the regulation of cellular proliferation provides new opportunities for therapeutic intervention in the treatment of cancer. The inherent specificity of many of these protein-protein interactions should provide great selectivity, and emerging models of cell cycle control suggest that disrupting growth factor signalling pathways might selective_ly induce programmed cell death in cancer cells. However, many of these protein-protein interactions appear to occur over fairly large surfaces, suggesting that the identification of small molecules with the necessary affinity and specificity to disrupt these interactions in vivo represents a challenging undertaking. In the current review, we highlight three particular targets in the growth factor signalling pathways : growth factor - receptor binding, using Gastrin Releasing Peptide as a specific example; SH2 I SH3 domain interactions; and viral oncoprotein interaction with cellular proteins in the nucleus, focusing on the binding of Human Papillomavirus proteins with cellular growth suppressor gene products. These studies highlight both the promise and the pitfalls of selectively targeting protein-protein interactions for the treatment of cancer.

Analysis of Two-Dimensional Differential Scanning Calorimetry Data: Elucidation of Complex Biomolecular Energetics.

Abstract: Publisher Summary Elucidation of the detailed energetics of protein folding-unfolding equilibria together with knowledge of specific structural information provides a powerful means for gaining a productive understanding of (1) the principles involved in regulating protein folding, (2) domain interactions within proteins, (3) multimolecular protein interactions and assembly into higher order macromolecular structures, (4) interactions of proteins with biological membranes, and (5) functional associations between proteins and small ligands. Direct access to protein energetics is possible by application of calorimetric techniques and, in particular, differential scanning calorimetry (DSC). DSC permits direct measurement of the enthalpy and heat capacity changes associated with thermally induced protein denaturation. Complex protein energetics can be elucidated by application of two-dimensional DSC methods in conjunction with the chemical protein structural destabilizing agent guanidinium hydrochloride (GuHCl). This chapter describes the thermodynamic linkage between denaturant binding to proteins and the effect on thermal stability with an emphasis on the developments and applications utilizing GuHCl as a chemical destabilizing agent of protein structure.

Bead-shift isolation of protein-DNA complexes on a 5S RNA gene

Abstract: Specific protein-DNA complexes formed on a Xenopus 5S RNA gene were isolated and characterized using a novel technique. A DNA template reversibly immobilized on paramagnetic beads was used to capture, affinity purify, and concentrate protein--DNA complexes formed in a whole cell extract. The complexes were then released from the beads in a soluble and transcriptionally active form via restriction enzyme digestion of the DNA. A band-shift gel was used to separate and obtain the DNase I footprints of five individual complexes. Three of the complexes resulted from the independent binding of two proteins, TFIIIA and an unidentified protein binding to a large region just downstream of the 3' end of the gene. Two more slowly migrating complexes contained an additional large central protected region covering most of the gene. The most slowly migrating complex displayed protein interactions over the 5' flanking sequences. The formation of two of these complexes was shown to be dependent on TFIIIC activity. The correlation between transcriptional activity and the formation of these complexes suggests that the observed protein--DNA interactions are important for transcription of 5S RNA genes.

A versatile expression vector for the in vitro study of protein-protein interactions: characterization of E47 mutant proteins.

Abstract: Several mutants of the E47 protein, a member of the family of basic/helix-loop-helix (b-HLH) transcriptional regulators, were examined for their ability to homo- and heterodimerize with the protein product of the T-cell oncogene tal-1/SCL. For this purpose, a novel bacterial expression system was developed in which proteins are expressed as fusions appended to glutathione-S-transferase via a thrombin cleavage site and either one or four protein kinase recognition sites embedded in a glycine-rich domain. Since the interaction domain can be purified away from the glutathione-S-transferase moiety and the radioactive label is located in a flexible N-terminal tag, protein folding should occur normally. Our studies with E47 proteins prepared in this system indicate that the ratio between E47 homodimers and E47/tal-1 heterodimers can dramatically shift upon subtle mutations in the loop region and the second helix of the E47 protein. This unexpected results suggests a novel mechanism to alter the equilibrium between different transactivating protein complexes of the b-HLH class.

Blot Overlay Assay: A Method to Detect Protein–Protein Interactions

Abstract: Publisher Summary This chapter focuses on the blot overlay assay method to detect protein–protein interactions. The blot overlay assay is a powerful method used to study the specificity of protein–protein interactions and provides an especially useful means that helps in identifying potential protein ligands. In this approach, a radiolabeled protein probe is used to overlay protein samples immobilized on nitrocellulose. Because this assay allows detection of protein–protein interactions within the context of a complex mixture of proteins, this method is also useful to investigate the specificity of an interaction between two ligands. For example, proteolytic fragments of proteins or nested deletion fragments may be probed to determine the portion of a molecule that contains the interactive site. However, the blot overlay assay is not a native assay and the interactions detected by this approach should be confirmed by employing solution binding assays under native conditions.

Analysis of mating type protein interactions in Coprinus cinereus

Abstract: The A mating type factor of the hymenomycete fungus Coprinus cinereus is a multi-allelic gene complex that controls mating compatibility and sexual development. It contains up to four pairs of specificity genes, the a , b , c , and d gene-pairs. Each gene-pair codes for two homeodomain transcription factors with distinct classes of homeodomain motifs. Mating compatibility between the A 42 and A 6 factors depends solely on the different alleles of the b gene-pair, b1-1 and b2-1 in A 42 and b1-3 and b2-3 in A 6. The b1-3 and b2-3 genes of A 6 were isolated and the complete DNA sequences of genomic and cDNA clones were determined. Construction of chimeric genes using the A 42 and A 6 b genes identified the N-terminal regions of the A proteins as being responsible for allele specificity. Analysis of protein-protein interactions showed that b1 and b2 proteins from different alleles of the same gene-pair can dimerise, whereas proteins from the same allele pair can not. It was shown that a region of 90 amino acids at the N-terminus of the b2-3 protein is sufficient for dimerisation with b1-1. This region is predicted to contain an amphipathic helix. A comparison with the equivalent region in the b2-1 protein identifies a similar helix. This suggests that a compatible A mating type reaction and thus allele specificity is recognised by the ability to dimerise through this domain. Polyclonal antibodies were raised against the b1-1 protein and a heterologous yeast expression system was established for testing potential DNA target sites of the b1-1 and b2-3 proteins, both techniques offering potentially useful tools for further molecular analysis of the A mating type proteins.

Eukaryotic Transcriptional Activation Mechanism: Protein-Protein Interactions

Abstract: Various in VlVO and in vitro assays have been employed to analyze how activators communicate with the general transcription machinery to stimulate transcription. As a first step, I analyzed the function of distinct kinds of activation domains in yeast and human. The results showed that the proline-rich activation domain of CTFl can, like acidic activation domains, activate transcription in yeast and human. Based on this, I compared the activation pathways by acidic and proline-rich activation domains in yeast and human. These detailed comparative approaches yielded clues to the fundamental aspects of transcriptional activation mechanism in eukaryotes: activators target TF11D(TBP)-TF11B­ promoter complex formation in the preinitiation complex assembly process by inducing (or stabilizing) qualitative or quantitative alterations within TF11D(TBP)-TF11B-promoter complexes that consequently enhance recruitment of downstream initiation factors. Consistent with this view, various activation domains have been demonstrated to have physical and functional interactions with TBP and/or TF11B. Given the central role of TBP in transcriptional regulation, I also identified distinct TBP domains (or residues) important for different regulatory interactions including those with acidic activators, TF11B, Drl (NC2) , Pol 1and Pol 111specific factors. These mutational analyses have provided an insight into how the interplay of many regulatory factors occurs on the surface of a target factor, TBP, to specify and regulate transcriptional activity. Furthermore, I investigated the