Showing papers on "Protein–protein interaction published in 2003"
TL;DR: A bimolecular fluorescence complementation approach for the simultaneous visualization of multiple protein interactions in the same cell based on complementation between fragments of fluorescent proteins with different spectral characteristics is described.
Abstract: The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
777 citations
TL;DR: Stable isotopic amino acids in cell culture is employed to differentially label proteins in EGF-stimulated versus unstimulated cells and SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
Abstract: Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
730 citations
TL;DR: Identification of the pharmacological sites has potential for exploitation in development of novel systems useful for 'delivering' specific proteins to a particular target tissue or organ and research in this field will provide a lot of exciting opportunities.
578 citations
TL;DR: It is shown that the protein–protein interaction networks of two distantly related species, Saccharomyces cerevisiae and Helicobacter pylori, harbor a large complement of evolutionarily conserved pathways, and that a large number of pathways appears to have duplicated and specialized within yeast.
Abstract: We implement a strategy for aligning two protein–protein interaction networks that combines interaction topology and protein sequence similarity to identify conserved interaction pathways and complexes. Using this approach we show that the protein–protein interaction networks of two distantly related species, Saccharomyces cerevisiae and Helicobacter pylori, harbor a large complement of evolutionarily conserved pathways, and that a large number of pathways appears to have duplicated and specialized within yeast. Analysis of these findings reveals many well characterized interaction pathways as well as many unanticipated pathways, the significance of which is reinforced by their presence in the networks of both species.
555 citations
TL;DR: Structural and binding studies provide the necessary framework for exploring how these molecular interactions initiate T cell activation.
Abstract: Over the past decade, key protein interactions contributing to T cell antigen recognition have been characterized in molecular detail. These have included interactions involving the T cell antigen receptor (TCR) itself, its coreceptors CD4 and CD8, the accessory molecule CD2, and the costimulatory receptors CD28 and CTLA-4. A clear view is emerging of how these molecules interact with their ligands at the cell-cell interface. Structural and binding studies have confirmed that the proteins span small but comparable distances and that, overall, they interact very weakly. However, there have been important surprises as well: that TCR interactions with peptide-MHC are topologically constrained and characterized by considerable conformational flexibility at the binding interface; that coreceptors engage peptide-MHC with extraordinarily fast kinetics and at angles apparently precluding direct interactions with the TCR bound to the same peptide-MHC; that the structural mechanisms allowing recognition by costimulatory and accessory molecules to be weak and yet specific are very heterogeneous; and that because of differences in both binding affinity and stoichiometry, there is enormous variation in the stability of the various costimulatory receptor/ligand complexes. These studies provide the necessary framework for exploring how these molecular interactions initiate T cell activation.
498 citations
TL;DR: BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells, and works efficiently in transgenic mice, raising the prospect of using biOTinylation tagging in protein complex purification directly from animal tissues.
Abstract: Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.
434 citations
TL;DR: Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed.
Abstract: Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilator molecule that plays important roles in diverse biological effects in the kidney, vasculature, and many other tissues. Because of its high biological reactivity and diffusibility, multiple tiers of regulation, ranging from transcriptional to posttranslational controls, tightly control NO biosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed. Dimerization of the NOS isozymes, required for their activity, exhibits distinguishing features among these proteins and may serve as a regulated process and target for therapeutic intervention. An increasingly wide array of proteins, ranging from scaffolding proteins to membrane receptors, has been shown to function as NOS-binding partners. Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins. Several resident and recruited proteins of plasmalemmal caveolae, including caveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOS in the endothelium. Inducible NOS (iNOS) interacts with the inhibitory molecules kalirin and NOS-associated protein 110 kDa, as well as activator proteins, the Rac GTPases. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. The calpain and ubiquitin-proteasome pathways are the major proteolytic systems responsible for the regulated degradation of NOS isozymes. The experimental basis for these protein-protein interactions, their functional importance, and potential implication for renal and vascular physiology and pathophysiology is reviewed.
290 citations
TL;DR: Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding, and an interface exchange hypothesis for coordinated protein interactions in homologous recombination is supported.
Abstract: To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full‐length RAD51 homolog from Pyrococcus furiosus . The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA‐bound filaments matching three‐dimensional electron microscopy reconstructions. A polymerization motif (RAD51‐PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric ‘switch’ promoting ATPase activity and DNA binding roles for the N‐terminal domain helix–hairpin–helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2‐dependent nuclear foci in human cells in response to γ‐irradiation‐induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51‐PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.
255 citations
TL;DR: It is suggested that ATP hydrolysis, is dependent on a complex rate-limiting step, involving N-terminal dimerization and association of the middle region, and therefore the catalytic loop, of Hsp90 with the N-Terminal domains.
Abstract: Understanding the mode of action of Hsp90 requires that molecular detail of its interactions with client proteins and co-chaperones are known. The structure determination of the N-terminal domain of Hsp90/Hsp90beta, proof that it is an ATPase, that this activity is regulated and the identification of co-chaperones that facilitate Hsp90 function were landmarks towards understanding conformational changes in Hsp90 brought about by ATP, co-chaperones and client proteins. Sti1 and Cdc37/p50, which associate with early Hsp90 complexes, were shown to be inhibitors of Hsp90 ATPase activity and therefore promote its 'open' state, whereas Sba1/p23, which associates with mature complexes, inhibits ATPase activity and stabilises the 'closed' state. The isolation and characterisation of Aha1, the only known strong activator of Hsp90 ATPase activity, which promotes the 'closed' state of Hsp90, will also be of major importance in understanding Hsp90 function. The structure determination of the middle region of Hsp90 has shed further light on the complex ATP-cycle of Hsp90, identifying a catalytic loop, with key residues that are essential for ATP hydrolysis. These studies, together with biochemical ones, suggest that ATP hydrolysis, is dependent on a complex rate-limiting step, involving N-terminal dimerization and association of the middle region, and therefore the catalytic loop, of Hsp90 with the N-terminal domains. The structure of the middle region of Hsp90 will also accelerate our understanding of client protein interactions since this region is implicated in their recognition and in particular their active-site openings.
253 citations
TL;DR: In this article, the authors argue that a systematic functional analysis of the human proteome requires technologies that are capable of time and spatially resolved, multiplexed analysis of protein interactions within cells.
219 citations
TL;DR: The promiscuous behavior of RAR1 and SGT1 might be crucial for the formation and activation of R protein-containing recognition complexes as well as for regulating downstream signaling processes.
TL;DR: Brightness analysis promises to develop into an important tool for determining protein complex formation in cells by analyzing the photon counting histogram (PCH) or its moments and demonstrating the use of molecular brightness in probing the oligomerization state of proteins.
Abstract: Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 μM by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.
TL;DR: The existence of a complex network of interactions implicating all six HCV NS proteins is demonstrated, which supports the idea of assembly of a highly ordered multisubunit protein complex probably involved in the replication of the viral genome.
Abstract: Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.
TL;DR: This analysis suggests that weak association energy may be a mechanism for in vivo regulation of assembly by, for example, dimerization factors and/or scaffold.
TL;DR: This review highlights the methods of bioinformatics and functional proteomics of protein‐protein interaction investigation, and the contact surfaces of temporary protein complexes have unique structure and properties and they represent prospective targets for a new generation of drugs.
Abstract: Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization.
TL;DR: The InterDom database focuses on providing supporting evidence for the detected protein interactions based on putative protein domain interactions, and derives potential domain interactions by combining data from multiple sources, ranging from domain fusions, protein interactions and complexes, to scientific literature.
Abstract: Advances in proteomics technology have enabled new proteins to be discovered at an unprecedented speed, and high throughput experimental methods have been developed to detect protein interactions and complexes en masse. Such bottom-up, data-driven approach has resulted in data that may be uninformative or potentially errorful, requiring further validation and annotation. The InterDom database focuses on providing supporting evidence for the detected protein interactions based on putative protein domain interactions. Using an integrative approach, InterDom derives potential domain interactions by combining data from multiple sources, ranging from domain fusions, protein interactions and complexes, to scientific literature. The InterDom database is available at http://InterDom.lit.org.sg.
TL;DR: As predicted, family member specific interactions appear to be mediated by regions that exhibit the most divergence in amino acid sequence among family members, the linker or “hinge” region and the C terminus; and ionic interactions participate in the interaction of S100 family members with Ca2+‐independent target proteins.
Abstract: S100 proteins have no known enzymatic activity and exert their intracellular effects via interaction with and regulation of the activity of other proteins, termed target proteins, in both a Ca(2+)-dependent and Ca(2+)-independent manner. Structural studies have identified the linker region between the two EF-hand Ca(2+) binding domains and the C-terminus as Ca(2+)-dependent target protein binding sites in several S100 family members. In fact, C-terminal aromatic residues are obligatory for interaction of S100A1 with several of its Ca(2+)-dependent target proteins. Pharmacological studies suggest the presence of additional Ca(2+)-dependent binding motifs on some family members. A minimum of seven family members interact with and regulate the activity of aldolase A in a Ca(2+)-independent manner. In the case of S100A1, Ca(2+)-independent target protein interactions utilize a binding motif distinct from the C-terminal Ca(2+)-dependent target protein binding site. Several studies suggest that ionic interactions participate in the interaction of S100 family members with Ca(2+)-independent target proteins. While some target proteins are activated by multiple family members, other target proteins exhibit family member-specific activation, i.e., they are activated by a single family member. As predicted, family member specific interactions appear to be mediated by regions that exhibit the most divergence in amino acid sequence among family members, the linker or "hinge" region and the C terminus. Further specificity in S100-target protein interactions may arise from the different biochemical/biophysical properties of the individual family members, including affinity for metal ions (Ca(2+), Zn(2+), and Cu(2+)), oligomerization properties, heterodimerization, post-translational modifications, and lipid-binding. Delineation of the structural motifs that mediate S100-target protein interactions and determination of the in vivo relevance of these interactions are needed to fully understand the role of S100 proteins in normal and diseased cells.
TL;DR: It is shown that the apparent tendency for interactive proteins to evolve more slowly varies directly with the bias towards counting more interactions for abundant proteins, which could be important since abundant proteins are known to evolved more slowly.
Abstract: Several studies have suggested that proteins that interact with more partners evolve more slowly. The strength and validity of this association has been called into question. Here we investigate how biases in high-throughput protein–protein interaction studies could lead to a spurious correlation. We examined the correlation between evolutionary rate and the number of protein–protein interactions for sets of interactions determined by seven different high-throughput methods in Saccharomyces cerevisiae. Some methods have been shown to be biased towards counting more interactions for abundant proteins, a fact that could be important since abundant proteins are known to evolve more slowly. We show that the apparent tendency for interactive proteins to evolve more slowly varies directly with the bias towards counting more interactions for abundant proteins. Interactions studies with no bias show no correlation between evolutionary rate and the number of interactions, and the one study biased towards counting fewer interactions for abundant proteins actually suggests that interactive proteins evolve more rapidly. In all cases, controlling for protein abundance significantly decreases the observed correlation between interactions and evolutionary rate. Finally, we disprove the hypothesis that small data set size accounts for the failure of some interactions studies to show a correlation between evolutionary rate and the number of interactions. The only correlation supported by a careful analysis of the data is between evolutionary rate and protein abundance. The reported correlation between evolutionary rate and protein–protein interactions cannot be separated from the biases of some protein–protein interactions studies to count more interactions for abundant proteins.
TL;DR: This work analyzes the protein networks inferred from interaction databases to predict potential interactions, to validate the results of high-throughput interaction screens and to analyze theprotein networks inferredfrom interaction databases.
TL;DR: Nitrosylation may provide a broad-based mechanism for regulating interactions between proteins, and systematic proteomic analyses in which redox state and NO bioavailability are carefully controlled will reveal a large array of novel interactions.
Abstract: Because nitric oxide (NO) may be a ubiquitous regulator of cellular signaling, we have modified the yeast two-hybrid system to explore the possibility of NO-dependent protein-protein interactions.We screened for binding partners of procaspase-3, a protein implicated in apoptotic signaling pathways, and identified multiple NO-dependent interactions.Two such interactions, with acid sphingomyelinase and NO synthase, were shown to occur in mammalian cells dependent on endogenous NO.Nitrosylation may thus provide a broad-based mechanism for regulating interactions between proteins.If so, systematic proteomic analyses in which redox state and NO bioavailability are carefully controlled will reveal a large array of novel interactions.
TL;DR: It is demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach and providing an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.
TL;DR: It is found that the Dam1p-Ndc80p and Dam1 p-Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.
Abstract: Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein–protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p–Ndc80p and Dam1p–Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.
TL;DR: A new 'interaction generality' measure (IG2) is developed to assess the reliability of protein-protein interactions using only the topological properties of their interaction-network structure, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein- protein interaction networks.
Abstract: Motivation: Recent screening techniques have made large amounts of protein–protein interaction data available, from which biologically important information such as the function of uncharacterized proteins, the existence of novel protein complexes, and novel signal-transduction pathways can be discovered. However, experimental data on protein interactions contain many false positives, making these discoveries difficult. Therefore computational methods of assessing the reliability of each candidate protein–protein interaction are urgently needed. Results: We developed a new ‘interaction generality’ measure (IG2) to assess the reliability of protein–protein interactions using only the topological properties of their interaction-network structure. Using yeast protein–protein interaction data, we showed that reliable protein–protein interactions had significantly lower IG2 values than lessreliable interactions, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein–protein interaction networks. Availability: The protein–protein interaction data used in this study along with the associated IG2 values are available at http://genome.gsc.riken.go.jp.
TL;DR: Evidence is presented that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes, which offer a new mechanism for the functional diversity of proteins.
Abstract: Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo α-lactalbumin and form HAMLET (human α-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein–fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo α-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid–protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.
TL;DR: It is demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers, and these likely provide the basis for PRRSV nucleocapsid assembly.
Abstract: As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.
TL;DR: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits.
Abstract: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.
TL;DR: Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting.
TL;DR: Using the adenylate cyclase bacterial two‐hybrid (BTH) system, in vivo evidence is presented that the IncHI1 plasmid R27‐encoded proteins TraG and TrhB interact, supporting a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane M pf complex.
Abstract: Summary
Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135–173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77–124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.
TL;DR: An in vitro protein microarray was developed and tested, in which both arraying and probing proteins were prepared by cell‐free translation, and fluorescently labeled proteins were confirmed to be useful for probing protein‐protein interactions on protein microarrays in model experiments.
Abstract: Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.
TL;DR: This review describes several databases that store, query, and visualize protein-protein interaction data and shows that each high-throughput technique such as yeast two-hybrid assay or protein complex identification through mass spectrometry has its limitations in detecting certain types of interactions.
Abstract: Protein-protein interactions play important roles in nearly all events that take place in a cell. High-throughput experimental techniques enable the study of protein-protein interactions at the proteome scale through systematic identification of physical interactions among all proteins in an organism. High-throughput protein-protein interaction data, with ever-increasing volume, are becoming the foundation for new biological discoveries. A great challenge to bioinformatics is to manage, analyze, and model these data. In this review, we describe several databases that store, query, and visualize protein-protein interaction data. Comparison between experimental techniques shows that each high-throughput technique such as yeast two-hybrid assay or protein complex identification through mass spectrometry has its limitations in detecting certain types of interactions and they are complementary to each other. In silico methods using protein/DNA sequences, domain and structure information to predict protein-protein interaction can expand the scope of experimental data and increase the confidence of certain protein-protein interaction pairs. Protein-protein interaction data correlate with other types of data, including protein function, subcellular location, and gene expression profile. Highly connected proteins are more likely to be essential based on the analyses of the global architecture of large-scale interaction network in yeast. Use of protein-protein interaction networks, preferably in conjunction with other types of data, allows assignment of cellular functions to novel proteins and derivation of new biological pathways. As demonstrated in our study on the yeast signal transduction pathway for amino acid transport, integration of high-throughput data with traditional biology resources can transform the protein-protein interaction data from noisy information into knowledge of cellular mechanisms.