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PubMed1996

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa.

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF-VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF-VIIa with macromolecular substrate.

Binding Sites↗

PubMed1998

Kinetics of T cell receptor beta, gamma, and delta rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes.

We performed a comprehensive analysis of T cell receptor (TCR) gamma rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRgamma rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes much earlier than expected. TCRgamma rearrangements increase significantly from the Pro-T to the CD44(-)CD25(+) Pre-T cell transition, and follow different patterns depending on each Vgamma gene segment, suggesting that ordered waves of TCRgamma rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRgamma genes occur concurrently with TCRdelta and D-Jbeta rearrangements, but before Vbeta gene assembly. Productive TCRgamma rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4(+)CD8(+) double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRdelta-/- mice display random proportions of TCRgamma rearranged alleles, supporting a role for functional TCRgamma/delta rearrangements in the gammadelta divergence process.

Animals↗

PubMed1999

RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

5' Untranslated Regions↗

PubMed2000

ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL(3), suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL(3)-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL(3)-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2000

APC mutations in sporadic colorectal tumors: A mutational "hotspot" and interdependence of the "two hits".

Although APC mutations occur at a high frequency in colorectal cancers, few studies have performed a comprehensive analysis by screening the whole gene for mutations and assessing allelic loss. APC seems to act as a tumor-suppressor gene in a "nonclassical" fashion: data from familial adenomatous polyposis (FAP) show that the site of the germ-line mutation determines the type of "second hit" in FAP tumors, and simple protein inactivation is selected weakly, if at all. In this study, we screened the entire coding region of APC for mutations and assessed allelic loss in a set of 41 colorectal cancer cell lines. Of 41 cancers, 32 (83%) showed evidence of APC mutation and/or allelic loss. We identified several APC mutations and found a "hotspot" for somatic mutation in sporadic colorectal tumors at codon 1,554. Our results suggest that APC mutations occur in the great majority of colorectal cancers, the exceptions almost all being RER+ tumors, which may substitute for altered APC function by mutations in beta-catenin and/or at other loci. When combined with previously published data, our results show that there is interdependence of the "two hits" at APC in sporadic colorectal tumors as well as in FAP. APC mutations in the "mutation cluster region," especially those close to codon 1,300, are associated with allelic loss, whereas tumors with mutations outside this region tend to harbor truncating mutations. The causes of this phenomenon are probably selection for retained N-terminal and lost C-terminal APC functions, effects on beta-catenin levels, and APC protein stability.

Base Sequence↗

PubMed1995

Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+.

Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of alpha 5 beta 1:Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within alpha 5 beta 1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn(2+)-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg(2+)-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca(2+)-binding site. Taken together, our results imply that the ligand-binding capacity of alpha 5 beta 1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.

Animals↗

PubMed1999

Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors.

Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3 receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3 formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis.

Amino Acid Sequence↗

PubMed2002

A novel ankyrin repeat-containing gene (Kank) located at 9p24 is a growth suppressor of renal cell carcinoma.

By a combination of genome subtraction and comprehensive analysis of loss of heterozygosity based on mapping hemizygous deletions for a potential tumor-related locus, a minimum overlapping region of deletions at 9p24 the size of 165 kb was identified and found to harbor a new potential tumor suppressor gene for renal cell carcinoma, the Kank gene. Kank (for kidney ankyrin repeat-containing protein) contains four ankyrin repeats at its C terminus. Expression of the gene was suppressed in 6 of 8 or 6 of 10 cancer tissues examined by reverse transcription-PCR or Western blotting, respectively, and in several kidney tumor cell lines due to methylation at CpG sites in the gene. Epigenetic methylation or imprinting seemed to be the first hit, which was followed by a second hit of deletion, resulting in loss of function in many of these deletion cases. Expression of this gene in expression-negative HEK293 cells induced growth retardation at G(0)/G(1) as well as morphological changes.

Actins↗

PubMed2003

Protein interactions within the N-end rule ubiquitin ligation pathway.

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.

Adenosine Triphosphate↗

PubMed2003

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration.

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.

Animals↗

PubMed2004

Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa: substrate specificity in udp-hexose 4-epimerases.

The O antigen of lipopolysaccharide in Gram-negative bacteria plays a critical role in bacterium-host interactions, and for pathogenic bacteria it is a major virulence factor. In Pseudomonas aeruginosa serotype O6 one of the initial steps in O-antigen biosynthesis is catalyzed by a saccharide epimerase, WbpP. WbpP is a member of the UDP-hexose 4-epimerase family of enzymes and exists as a homo-dimer. This enzyme preferentially catalyzes the conversion between UDP-GlcNAc and UDPGalNAc above UDP-Glc and UDP-Gal, using NAD(+) as a cofactor. The crystal structures of WbpP in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 A, respectively, which represents the first structural studies of a genuine UDP-GlcNAc 4-epimerase. These structures in combination with complementary mutagenesis studies suggest that the basis for the differential substrate specificity of WbpP is a consequence of the presence of a pliable solvent network in the active site. This information allows for a comprehensive analysis of the relationship between sequence and substrate specificity for UDP-hexose 4-epimerases and enables the formulation of consensus sequences that predict substrate specificity of UDP-hexose 4-epimerases yet to be biochemically characterized. Furthermore, the examination indicates that as little as one residue can dictate substrate specificity. Nonetheless, phylogenetic analysis suggests that this substrate specificity is an evolutionary and highly conserved property within UDP-hexose 4-epimerases.

Binding Sites↗

PubMed2004

Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors.

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.

Animals↗

PubMed2005

The second intracellular loop of the calcitonin gene-related peptide receptor provides molecular determinants for signal transduction and cell surface expression.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to G(s), but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general.

Alanine↗

PubMed2006

Eight amino acid residues in transmembrane segments of yeast glucose transporter Hxt2 are required for high affinity transport.

Hxt2 and Hxt1 are high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae, respectively, that differ at 75 amino acid positions in their 12 transmembrane segments (TMs). Comprehensive analysis of chimeras of these two proteins has previously revealed that TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity glucose transport activity and that leucine 201 in TM5 is the most important in this regard of the 20 amino acid residues in these regions that differ between Hxt2 and Hxt1. To evaluate the importance of the remaining residues, we systematically shuffled the amino acids at these positions and screened the resulting proteins for high affinity and high capacity glucose transport activity. In addition to leucine 201 (TM5), four residues of Hxt2 (leucine 59 and leucine 61 in TM1, asparagine 331 in TM7, and phenylalanine 366 in TM8) were found to be important for such activity. Furthermore, phenylalanine 198 (TM5), alanine 363 (TM8), and either valine 316 (TM7) or alanine 368 (TM8) were found to be supportive of maximal activity. Construction of a homology model suggested that asparagine 331 interacts directly with the substrate and that the other identified residues may contribute to maintenance of protein conformation.

Amino Acid Motifs↗

PubMed2007

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence.

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.

Cross-Linking Reagents↗

PubMed2006

Maturation of human neutrophil phagosomes includes incorporation of molecular chaperones and endoplasmic reticulum quality control machinery.

A Human neutrophils are an essential component of the innate immune response. Although significant progress has been made toward understanding mechanisms of phagocytosis and microbicidal activity, a comprehensive analysis of proteins comprising neutrophil phagosomes has not been conducted. To that end, we used subcellular proteomics to identify proteins associated with human neutrophil phagosomes following receptor-mediated phagocytosis. Proteins (n = 411 spots) resolved from neutrophil phagosome fractions were identified by MALDI-TOF MS and/or LC-MS/MS analysis. Those associated with phagocytic vacuoles originated from multiple subcellular compartments, including the cytosol, plasma membrane, specific and azurophilic granules, and cytoskeleton. Unexpectedly several enzymes typically associated with mitochondria were identified in phagosome fractions. Furthermore proteins characteristic of the endoplasmic reticulum, including 11 molecular chaperones, were resolved from phagosome preparations. Confocal microscopy confirmed that proteins representing these major subcellular compartments were enriched on phagosomes of intact neutrophils. Notably calnexin and glucose-regulated protein 78 co-localized with gp91(phox) in human neutrophils and were thus likely delivered to phagosomes by fusion of specific granules. We conclude that neutrophil phagosomes have heretofore unrecognized complexity and function, which includes potential for antigen processing events.

Blotting, Western↗

PubMed2006

A high efficiency strategy for binding property characterization of peptide-binding domains.

A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.

Animals↗

PubMed2002

Roles for the two-hybrid system in exploration of the yeast protein interactome.

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.

Computational Biology↗