Showing papers on "Protein–protein interaction published in 1993"
TL;DR: An improved version of the yeast two-hybrid system is developed and used to isolate human cDNAs encoding proteins able to bind p110RB to demonstrate that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110 RB.
Abstract: The retinoblastoma protein {pll0 nB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing [unction. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind pll0 RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-la2), which differs from the originally defined PP-lc~ by an amino-terminal l 1-amino-acid insert. In vitro-binding assays demonstrated that PP-lc~ isoforms preferentially bind the hypophosphorylated form of p ll0 RB. Moreover, similar pll0 RB sequences are required for binding PP-lc~2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early Gv The implications of these findings on the regulation of both proteins are discussed.
1,470 citations
335 citations
Journal Article•
TL;DR: High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase, which may represent a fundamental mechanism by which Pb exerts toxicity.
Abstract: The interaction of lead (Pb) with proteins may represent a fundamental mechanism by which Pb exerts toxicity. In this overview, various factors which influence the interaction of Pb with proteins will be discussed. Pb interacts with enzyme functional groups, and high-affinity metal-binding proteins, such as Pb-binding proteins and metallothioneins, can mediate this Pb-enzyme interaction. Many other factors influence Pb-protein interactions including ligand competition and binding affinities; protein folding and the nature of the metal-binding site; rates of protein synthesis and degradation; and intracellular localization of the ligand and metal. The remainder of this overview will focus on specific examples of important proteins known to be influenced by Pb or which hypothetically may be influenced by Pb. Gaps in knowledge and important research needs are emphasized. Many of the factors discussed play a role in the relative sensitivity of various enzymes in heme biosynthesis to Pb. Disruption of this critical pathway by Pb may result in neuropathologies and accumulation of neurotoxic heme precursors. High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase. Knowledge of regional localization in brain and the postnatal ontogeny of the high-affinity metal-binding proteins may be pivotal in understanding Pb neurotoxicity. Other specific examples related to or potentially related to Pb toxicity which are discussed include nucleic acid binding proteins, calmodulin, protein kinase C, and carbonic anhydrase. These proteins will serve as models to understand some basic principles and differences in Pb-protein interactions.
254 citations
TL;DR: This study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.
Abstract: CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.
132 citations
TL;DR: Fibrillar structure formation, nuclear disintegration and intermolecular p10 protein interactions seem to be three separate functions of the p10protein and these functions are located in distinct domains of the protein.
Abstract: Distinct functional domains in the Autographa californica nuclear polyhedrosis virus p10 protein were identified by analysis of p10 mutants. When up to 15 amino acids from the carboxy terminus were deleted, truncated p10 proteins were found in both the nucleus and the cytoplasm of infected cells, but formed no fibrillar structures. This suggested that the positively charged carboxy terminus is not required for nuclear or cytoplasmic localization of p10 protein, but is involved in protein-protein interactions leading to assembly of the p10 protein into fibrillar structures. Absence of the p10 protein prevented the release of polyhedra from infected cells, caused by impaired nuclear disintegration. This function of the p10 protein appears to be located between amino acid residues 52 and 79. The amino-terminal half of the p10 protein has already been implicated in the self-aggregation of this protein. Thus fibrillar structure formation, nuclear disintegration and intermolecular p10 protein interactions seem to be three separate functions of the p10 protein and these functions are located in distinct domains of the protein. The mutants expressing truncated p10 proteins were impaired in electron-dense spacer formation but polyhedron envelopes were still formed. This result suggested that the formation of electron-dense spacers is not a prerequisite for the formation of polyhedron envelopes.
74 citations
TL;DR: Using this system, it is shown that expression of activated rasH gene product does not inhibit and may stabilize HLH protein-protein interaction, and may be of general utility in studying the modulation of transcription factor interactions.
72 citations
TL;DR: The results suggest that the central region of the protein is necessary for multimerization and that the N-terminal zinc finger region is not important.
Abstract: The retroviral integrase protein (IN) is responsible for catalyzing a concerted integration reaction in which the two termini of linear viral DNA are joined to host DNA. To probe the potential for IN to form protein multimers, we used the yeast two-hybrid system. The coexpression of a GAL4 DNA binding domain-IN fusion and a GAL4 activation domain-IN fusion together resulted in the successful activation of a GAL4-responsive LacZ reporter gene. The system was used to examine a variety of IN deletion mutants. The results suggest that the central region of the protein is necessary for multimerization and that the N-terminal zinc finger region is not important.
63 citations
TL;DR: It is found that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32protein-covered DNA.
53 citations
TL;DR: These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CyTR-DNA interactions may primarily serve to stabilize the nucleo-protein complex.
51 citations
TL;DR: It is found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1) and this composition is maintained also after in vitro reconstitution of the particle.
Abstract: Xenopus r-protein mRNAs are known to be coordinately regulated at the translational level. To find out if RNA/protein interactions are involved in this control mechanism, we have characterized the particles containing the translationally repressed rp-mRNA and we have investigated the proteins that specifically bind to this type of mRNA. By sedimentation analysis and isopycnic centrifugation we have found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1). This composition is maintained also after in vitro reconstitution of the particle. We carried out also a detailed analysis of in vitro RNA/protein complex formation by focusing our attention on the 5'UTR, very similar in different rp-mRNAs and important in the translational regulation. We describe specific interactions of L1 mRNA with four proteins. The binding site of two of them, 57 kD and 47 kD, is in the typical pyrimidine sequence at the 5' end and is position dependent. Proteins of the same size interact also with the analogous region of r-protein S1 and L14 mRNA, not with unrelated RNAs. Binding of two other proteins, 31 kD and 24 kD, in the downstream region of the 5'UTR was also observed. The most evident 57 kD protein has been partially purified. Although the binding of these proteins to the r-protein mRNA 5'UTR is specific, their involvement in the translation regulation remains to be proved.
TL;DR: Results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament.
TL;DR: The discovery, properties, and classification of activation domains, the sequences known as activation domains or activating regions that are required for an activator protein to stimulate transcription of a target gene are reviewed.
Abstract: Changes in gene expression are central to nearly every developmental or adaptive response that eukaryotic cells are known to undergo. Gene regulation usually involves changes in the rate of transcription, and this has led to intense interest in the regulatory proteins that control the transcription of specific genes. The past decade has seen striking progress in the identification and characterization of these factors. 1-4 It is now well established that regulated transcription requires the binding of one or more proteins to a set of DNA sequence motifs (c/s-regulatory sequences) in the flanking regions of the gene. These sequences collectively specify a program of transcriptional activity that is elicited by developmental, physiological, and other signals. The factors that recognize cis-regulatory sequences usually function positively to activate transcription, in which case they are termed activator proteins. The DNA-binding domains of activator proteins have been studied extensively, primarily because straightforward assays to measure DNA:protein interactions have been available, and they have been the subject of numerous reviews.~.5 In the past few years, increased attention has been focused on other functional domains within transcriptional regulatory proteins, especially the sequences known as activation domains or activating regions that are required for an activator protein to stimulate transcription of a target gene. In this article we review the discovery, properties, and classification of activation domains. We use specific examples to illustrate how these elements are used to modulate the activity of regu-
TL;DR: It is shown that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti- biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety.
Abstract: We describe an in vivo approach for the isolation of proteins interacting with a protein of interest. The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo. The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted. These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein. The infection lyses the cells and exposes the protein complexes. The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters. The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters. Positive plaques can be plaque-purified for DNA sequencing. We have tested this approach by using c-Fos and c-Jun as our model system. We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety. Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety. We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.
TL;DR: Using a human transcription factor, upstream stimulatory factor, as a model system, a method for the rapid and efficient purification of proteins that interact with a cloned polypeptide expressed as a fusion with an oligohistidine domain is developed.
TL;DR: The high-molecular-weight rhoptry proteins of Plasmodium falciparum exist in a multiprotein complex consisting of proteins of 140, 130, and 110 kDa, and a 120-kDa protein was identified as the major proteolytic fragment of the 140-k da protein.
TL;DR: A gene expression vector is developed that adds three features to the C-terminus of the putative synthesized protein: a protein kinase A recognition domain allowing the protein to be radio-labeled in vitro, an epitope marker for immunocharacterization of theprotein with a commercial monoclonal antibody, and a (His)6 block facilitating purification of the protein by metal chelating affinity chromatography.
TL;DR: In the dimeric proteins comprised of regular motifs, interface interactions are similar to those that stabilize monomers, and additional stability is gained by converting loops within motifs or domains to linkers across interfaces.
Abstract: Interactions between proteins are extremely variable. However, in the dimeric proteins comprised of regular motifs, interface interactions are similar to those that stabilize monomers. Additional stability is gained by converting loops within motifs or domains to linkers across interfaces. In multi-domain proteins, interactions can be greatly effected by the conformation of linkers between domains. Complex association of subunits, involving higher rotational symmetry or cubic symmetry, frequently involves motif sharing across interfaces.
TL;DR: The replacement of specific amino acid residues in proteins using site-directed mutagenesis allows for a rationally designed, systematic, and quantitative analysis of the interactions that govern molecular recognition among proteins.
Abstract: Publisher Summary The replacement of specific amino acid residues in proteins using site-directed mutagenesis allows for a rationally designed, systematic, and quantitative analysis of the interactions that govern molecular recognition among proteins. The strategy involves analyzing the interface of a structurally defined protein–protein complex and examining the effects of specific side-chain substitutions on the free energy of the interaction. It is a unique system in that one can mutate both the antibody and the antigen, which increases the power of this strategy. Serological analysis and the X-ray crystal structure of the lysozyme–antibody complex has made it possible to predict the importance of specific contact residues at the interface of the two proteins. From there, site-directed mutagenesis methods are applied to cloned sequences of antibody and antigen using general methods that is improved to attain the highest efficiencies. Immunochemical and inhibition assays are used to quantitate the effect of specific mutations on antibody–antigen binding.
TL;DR: X-ray crystallographic work on eukaryotic transcription factors traverses the entire length of a typical class II nuclear gene promoter, including the TATA-box-binding protein, two b/HLH/Z promoter proximal binding factors, and an enhancer-binding factor.
Abstract: Our X-ray crystallographic work on eukaryotic transcription factors traverses the entire length of a typical class II nuclear gene promoter, including the TATA-box-binding protein, two b/HLH/Z promoter proximal binding factors (Max and USF), and an enhancer-binding factor (HNF-3 gamma). These high-resolution studies of specific protein/DNA interactions will be extended to include homologous proteins complexed with similar oligonucleotides and a systematic examination of the roles of individual amino acids and bases implicated in specific DNA binding. In the longer term, we will turn our attention to the myriad of protein/protein interactions occurring within TFIID and the PIC, and between the PIC and factors binding to promoter proximal and distal enhancer elements.
TL;DR: In this paper, a number of enzymes which function by interaction with other proteins, most notably electron transfer proteins, are membrane bound, and they serve the electron transfer pathways of the inner mitochondrial membrane.
Abstract: Proteins exist in the cellular aqueous environment containing carbohydrate, lipid, nucleic acids and other proteins. While soluble proteins interact with their substrate in a three-dimensional matrix, a number of the cellular proteins exist in a membranous structure, located as components of one or another of the cellular membranes. Movement of these proteins is more restricted, and involves two-dimensional or translational mobility along the membrane. As one might expect, restriction of some proteins to a two-dimensional matrix would serve the purpose of facilitating their interactions. Indeed, a number of enzymes which function by interaction with other proteins, most notably electron transfer proteins, are membrane bound. One example of such enzymes are those serving the electron transfer pathways of the inner mitochondrial membrane. A second example would be the proteins localized to the endoplasmic reticulum and comprising a number of electron transfer oxidative pathways. Such proteins and their enzymatic activities are usually studied in the membrane fragments, e.g., the endoplasmic reticulum, the vesicular particles called microsomes (Claude 1943). The microsomes contain several electron transfer chains, all of which, parenthetically, may interact with the microsomal electron transfer hemoprotein cytochrome b 5 (Schenkman et al. 1976). These enzymes include the cytochrome P450 monooxygenases (Jansson et al. 1985 and references therein), the stearoyl CoA Δ9 desaturase (Strittmatter et al. 1974), the linolenoyl CoA Δ6 desaturase (Okayasu et al. 1981), the γ-linolenoyl CoA Δ5 desaturase (Do and Sprecher 1975), and the fatty acid elongase (Keyes et al. 1979).
TL;DR: A model assay has been developed in which MHC molecules have been substituted by a monoclonal antibody to the CD4 amino-terminal domain-specific epitope, Leu3a, enabling this assay format to be used for the detection of proteins or small organic compounds which interfere with a wide range of therapeutically-relevant macromolecular interactions.
01 Jan 1993
TL;DR: Non-covalent molecular interactions in biological systems are ubiquitous and finely tuned, and many complexes involving proteins are large and apparently intractable for structural studies.
Abstract: Non-covalent molecular interactions in biological systems are ubiquitous and finely tuned. While enzyme substrate complexes are relatively well understood and considerable knowledge has been gained recently about the way proteins interact with DNA (Harrison, 1991), understanding of protein-protein interactions is relatively poor. Many complexes involving proteins are large and apparently intractable for structural studies. Examples of interest include: cell-cell interactions mediated by different adhesion molecules; a cytokine bound to a membrane receptor; a tyrosine kinase bound to a protein substrate; a blood clotting complex. In cases where there is information available about protein-protein interactions, e.g. in antibody/protein antigen complexes, the interacting protein surface patches seem to involve more than one peptide loop from different parts of the protein sequence (Chothia, 1991).