Showing papers on "Protein–protein interaction published in 1995"

The BTB/POZ domain : a new protein-protein interaction motif common to DNA- and actin-binding proteins

Abstract: The BTB/POZ domain defines a newly characterized protein-protein interaction interface. It is highly conserved throughout metazoan evolution and generally found at the NH2 terminus of either actin-binding or, more commonly, nuclear DNA-binding proteins. By mediating protein binding in large aggregates, the BTB/POZ domain serves to organize higher order macromolecular complexes involved in ring canal formation or chromatin folding.

Analyzing protein-protein interactions using two-hybrid system.

Abstract: Publisher Summary This chapter presents the analysis of protein–protein interactions using two-hybrid system. Protein-protein interactions play a critical role in most biological processes. The studies defining domains of proteins that are responsible for specific interactions have contributed significantly to unraveling the mechanisms of tumorigenesis. The two-hybrid system is a yeast-based genetic assay for detecting protein– protein interactions in vivo. It can be used to establish interactions between two known proteins or to search genomic or cDNA libraries for proteins that interact with a target protein. For this latter application, the gene encoding the protein that interacts with a target protein is immediately available on a plasmid, which is not the case for many biochemical methods to detect interacting proteins. The two-hybrid system has also been used to define the protein domains that mediate an interaction and to identify specific residues that are involved in a protein–protein interaction. The chapter also discusses the basis for this method and presents the protocols that are necessary to use this system.

Defining protein interactions with yeast actin in vivo

Abstract: Using the two-hybrid protein interaction reporter system, actin, profilin, Srv2p and two SH3-containing proteins are found to bind yeast actin in vivo. When tested for ability to interact with 35 actin mutations distributed over the monomer surface, distinct subsets of mutations characteristic for each putative ligand are found to disrupt binding. In particular, the pattern of differential interactions for the actin-act in interaction is consistent with published structures for the actin filament. Despite functional similarities, the patterns of differential interaction for Srv2p and profilin are different. In contrast, the patterns for profilin and the SH3 domain proteins suggest a shared binding site and commonality in mechanism.

Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment.

Abstract: The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus-host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle.

HBx protein of hepatitis B virus interacts with the C-terminal portion of a novel human proteasome alpha-subunit

Abstract: Two-hybrid protein interaction screening in yeast was used to identify proteins that interact with the HBx nonstructural protein of hepatitis B virus (HBV). A new human member of the proteasome alpha-subunit family was obtained. Its protein sequence closely resembles the 28 kD subunits from other organisms. The interaction with HBx was abolished by a two amino-acid insertion behind position 128 in HBx, in a region previously found to be essential for its transcriptional transactivation function. These data support a model of HBx acting indirectly on transcriptional processes. By binding to a specific proteasome alpha-subunit, HBx might interfere with degradative processes, thereby enhancing the half-life of different transcription factors and other nuclear regulatory proteins. Interaction with the Hu 28k proteasome subunit could thus provide a unifying explanation for the markedly pleiotropic effects of HBx.

Method to detect protein-protein interactions

Abstract: Methods are provided for studying protein-protein interactions which require posttranslational modification of one of the proteins. The interaction is detected by reconstituting the activity of a transcriptional activator. This activity is dependent on the interactions between three different proteins. These include two chimeric proteins, one of which must be posttranslationally modified by the activity of the third protein in order for the chimeric proteins to interact. One of the chimeric proteins contains a transcriptional activation domain fused to a test protein. The second chimeric protein contains a DNA-binding domain of a transcriptional activator fused to the other test protein.

Functional interaction of ligands and receptors of the hematopoietic superfamily in yeast

Abstract: Circulating peptide hormones and growth factors interact with cell surface receptors to initiate specific cellular responses. These complexes can consist of a simple association between two proteins or a more elaborate association of multiple proteins. We describe the functional expression of ligands and corresponding receptors in a microbial system useful for the rapid dissection of these important protein interactions. GH or PRL and extracellular domains of their respective receptors were functionally expressed as fusion proteins in an extended two-hybrid protein-protein interaction system. Reversible and specific ligand-receptor interactions were demonstrated by concurrent expression of free ligand peptides (GH or PRL) as binding competitors. The versatility established by expressing three heterologous proteins allowed for the investigation of higher order structures. Ligand-dependent GH receptor dimerization was demonstrated but PRL receptor dimerization was not observed in an analogous assay, suggesting that these related growth factors may not engage receptors in a similar manner. Additionally, significant association of GH receptors was observed in the absence of ligand, suggesting that there may be substantial avidity between these receptor proteins before ligand binding. Ligand-dependent and ligand-independent receptor dimerization was demonstrated by vascular endothelial growth factor and receptor proteins in similar assays. These findings indicate that extracellular protein interactions such as ligand-receptor association, as well as the formation of higher order protein structures important for the activation of hematopoietic receptors, can be rapidly investigated in this microbial expression system.

Yeast two-hybrid system to detect protein-protein interactions with Rho GTPases.

Abstract: Publisher Summary This chapter describes the use of RhoA, Rac1, and G25K in the two-hybrid system and highlights that each is capable of interacting with Rho–GTPase-activating protein (GAP). The experiments discussed in the chapter suggest that the strength of an interaction must be in the nanomolar range for detection. For the two-hybrid system to be successful, it is vital that fusion proteins enter the yeast nucleus, otherwise they are unable to function as transcription activators. For the correct interpretation of a negative result, it is essential to establish that the proteins of interest are expressed. This can be done by analyzing yeast cell extracts by western blotting using antibodies against the protein of interest. Alternatively, the hemagglutinin epitope (HA) present on the pAS vector can be used as a tag to detect protein production using an anti-HA antibody. The yeast two-hybrid system has been used successfully to detect interactions between small GTPases and their target proteins. In particular, a Ras effector, c- raf , has been identified in this way.

Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8α in yeast

Abstract: The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.

Detection of protein-protein interactions by coimmunoprecipitation and dimerization.

Abstract: Publisher Summary This chapter describes the detection of protein-protein interactions by coimmunoprecipitation and dimerization Regulation of transcription is controlled both by general transcription factors and by sequence-specific binding proteins A number of oncogene products can act as transcription factors or cofactors These proteins may act directly by binding to specific DNA regulatory sequences as exhibited by Jun and ErbA Alternatively, they may act as cofactors like the Fos family members which alone do not bind to DNA, but in association with any of the Jun family members can activate and augment transcription Many of these proteins can be classified into specific families based on common structural motifs, such as the leucine zipper family, the helix-loop helix family, the homeodomain family, and zinc finger binding proteins Association between proteins from different classes not only increases the repertoire and complexity of regulatory transcriptional pathways, but also allows for coordination and cross-coupling of these pathways The challenge then is to develop methods to identify and characterize these specific protein-protein interactions Several methods have been used to successfully detect the physical associations

Chapter 24. SH2 and SH3 Domains: Choreographers of Multiple Signaling Pathways

Abstract: Publisher Summary To a large degree cellular signal transduction pathways are choreographed by modular Src homology 2 (SH 2 ) and Src homology 3 (SH 3 ) domains that mediate well-specific protein: protein interactions. SH 2 domains are modules of ∼100 amino acids that specifically bind phosphotyrosine-containing proteins and peptides. SH 3 domains are modules of ∼60 amino acids that bind to the proline-rich sequences. This chapter includes a list of selected therapeutic targets, possessing SH 2 or SH 3 domains. The design of specific antagonists to these domains holds the promise of targeted treatment of a broad range of pathologies. The role of SH 2 and SH 3 domains in signal transduction has been extensively studied. In growth factor, cytokine and antigen signaling, occupancy of a receptor by agonist results in receptor dimerization and the phosphorylation of regulatory tyrosines on the cytoplasmic surface. Phosphorylation is catalyzed by kinases that are a part of the receptor (receptor tyrosine kinases) or recruited to the receptor from the cytoplasm (non-receptor tyrosine kinases). The resulting phosphotyrosines permit binding of specific SH 2 -containing proteins and initiate a cascade of the sequential protein interactions. SH 3 domains are independently folded protein modules of 55-70 amino acids that selectively bind proline-rich protein sequences. Although generally of lower affinity than SH 2 -mediated interactions, SH 3 interactions are essential for multiple signaling cascades.

Specificity of lipid-protein interactions

Abstract: Publisher Summary This chapter discusses the specificity of lipid–protein interactions. The lipid–protein interactions can be divided into two broad types: the highly specific and the obligatory. The highly specific class are those exhibiting an absolute requirement for a particular lipid for activity, either as a substrate in the case of the phospholipases and other lipid-metabolizing enzymes, or as an activator as in the case—for example, of protein kinase C, or β-hydroxybutyrate dehydrogenase. The nature of the specific phospholipid interaction and the likely mode of binding of the protein at the membrane interface have been deduced from the crystal structure of the PLA 2 enzyme complex with a phosphonate-containing analog of phosphatidylethanolamine, a specific inhibitor designed to be a transition state analog. The lipid selectivity is additionally affected by pH titration, if the pKas of the groups involved in the specificity of interaction are in the accessible range. The pH dependences of the fraction of various spin-labeled lipids that are associated with the myelin proteolipid protein in recombinants with dimyristoyl-phosphatidylcholine of identical lipid/protein ratios, as determined by ESR experiments. Besides changing the electrostatics by neutralizing the negative charge of the lipid polar headgroup, pH titration may also have other effects such as changing the degree of polar group hydration on protonation.

Protein--protein interactions within the endoplasmic reticulum.

Abstract: Publisher Summary This chapter discusses protein–protein interactions within the endoplasmic reticulum. The translocation of proteins into the endoplasmic reticulum (ER) is the initial transport step for proteins with ER, Golgi, lysosomal, vacuolar, or extracellular destinations. The process of protein translocation into the ER involves a complex series of events and interactions among numerous proteins, including the nascent polypeptide, signal recognition particle, signal-sequence receptor, signal peptidase, and several ER-resident proteins. Recent work in yeast, animal, and plant systems has implicated ER-resident proteins in the folding, assembly, and modification of proteins translocated into the ER. Most seed storage proteins are transported through the ER en route to specialized storage organelles. This chapter focuses on the identification of protein–protein interactions within the ER and ER-derived protein bodies. Successful detection of the interaction between two polypeptides requires a relatively stable protein–protein complex, steric availability of recognizable epitopes upon protein association, and no cross-reactivity between antibodies and associated proteins.

[20] NMR and nucleic acid-protein interactions: The Lac repressor-operator system

Abstract: Publisher Summary This chapter discusses the lac repressor-operator system as an example to illustrate the nuclear magnetic resonance (NMR) approach to protein-DNA recognition. The recognition of DNA sequences, by proteins, lies at the heart of many cellular processes, such as gene regulation, replication and DNA repair. Therefore, the mechanism of protein-DNA interaction constitutes a major problem in molecular biology. Approaches to this problem range from biochemical and genetic studies (mutagenesis of DNA-binding proteins) to the application of the methods of structural biology (X-ray crystallography and NMR spectroscopy). NMR spectroscopy started to contribute around 1985, with the structure elucidation of the lac repressor headpiece and a low-resolution structure of the headpiece-operator complex in 1987, whereas in the first structures all contained a helix-turn-helix motif as the essential DNA-binding sub domain, later a plethora of DNA-binding motifs were characterized, including zinc-fingers, leucine zippers, helix-loop-helix proteins, and even B-sheet DNA-binding proteins. NMR has significantly contributed to this, as often DNA-binding domains of proteins are relatively small, independently folded domains that can be expressed and studied separately. For example, for the various subclasses of zinc-fingers, the first structural information came from NMR. The chapter concludes that the structure of the lac headpiece-operator complex agrees very well with the genetic data, and indeed provides a basis for a detailed interpretation of these data in structural terms.

Identification of protein-protein interactions by lambda gt11 expression cloning.

Abstract: Publisher Summary 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.

[22] Protein-protein interactions of DNA-binding proteins: Studies on replication initiator protein, RepA, of plasmid P1

Abstract: Publisher Summary Site-specific DNA-binding proteins often engage in protein–protein interactions, which are crucial to the function of the protein. Such interactions can occur among identical or functionally related proteins and often influence the DNA binding process itself. Protein–protein interactions are likely to occur when multiple DNA binding sites for the same protein or functionally related proteins are present in the region of interest: the origins and terminators of replication, centromere-like sites for plasmid partition, the promoters and enhancers of transcription, and the sites of recombination. DNA-binding proteins also interact with proteins that do not interact with DNA themselves. Because protein–protein interactions can alter the affinity of DNA-protein interactions (cooperative DNA binding), initial evidence for the presence of protein–protein interactions may be obtained from the measurements of DNA–protein affinity. This chapter describes such approaches to the study of protein–protein interactions involving the replication initiator protein, RepA, of plasmid P1.