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Results for “Protein Interaction Mapping”

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At least 145 records · Page 8Linked to original sources

PubMed2007

A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria.

Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

Bacterial Proteins↗

PubMed2003

Pathway studio--the analysis and navigation of molecular networks.

SUMMARY: PathwayAssist is a software application developed for navigation and analysis of biological pathways, gene regulation networks and protein interaction maps. It comes with the built-in natural language processing module MedScan and the comprehensive database describing more than 100 000 events of regulation, interaction and modification between proteins, cell processes and small molecules. AVAILABILITY: PathwayAssist is available for commercial licensing from Ariadne Genomics, Inc. The light version with limited functionality will be available for free for academic users at www.ariadnegenomics.com/downloads/.

Database Management Systems↗

PubMed2005

The modular structure of SIP facilitates its role in stabilizing multiprotein assemblies.

Siah-interacting protein (SIP) was identified as a novel adaptor that physically links the E3 ubiquitin ligase activity of Siah-1 with Skp1 and Ebi F-Box protein in the degradation of beta-catenin, a transcriptional activator of TCF/LEF genes. In this study, we have used solution NMR spectroscopy to characterize the domain structure of SIP, which includes a novel helical hairpin domain at the N-terminus flexibly linked to a CS domain and an unstructured carboxy terminal SGS domain. These studies have been complemented by mapping the sites of functionally important protein-protein interactions involving Siah-1 and Skp1 to individual domains of SIP. NMR-based chemical shift perturbation assays show that Siah-1 interacts with the flexible linker between SIP N and CS domains. This site for interaction in the linker does not perturb residues in the structured region at the N-terminus but does appear to restrict the rotational freedom of the SIP CS domain in the context of the full-length protein. In contrast, Skp1 engages the SIP CS domain exclusively through weak interactions that are not coupled to the other domains. The principal role of the modular structure of SIP appears to be in bringing these two proteins into physical proximity and orchestrating the orientation required for polyubiquitination of beta-catenin in the intact SCF-type complex.

Amino Acid Motifs↗

PubMed2003

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein.

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.

Amino Acid Sequence↗

PubMed2005

Plastid division is mediated by combinatorial assembly of plastid division proteins.

Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.

Arabidopsis Proteins↗

PubMed2004

NMR mapping of the HIV-1 Tat interaction surface of the KIX domain of the human coactivator CBP.

Tat is required for the expression of the HIV-1 genome. HIV-1 Tat interacts with the human transcriptional coactivator and acetyltransferase CREB-binding protein (CBP) via the KIX domain of CBP. Chemical shift perturbation mapping with nuclear magnetic resonance spectroscopy was used to identify the surface of human KIX that interacts with Tat. It was found that Tat binds to the c-Jun/MLL/Tax binding surface of KIX, as opposed to the CREB binding site. The results provide new insight into the molecular basis of the assembly of protein complexes involving p300/CBP and Tat during HIV gene expression.

Acetyltransferases↗

PubMed2004

LGL: creating a map of protein function with an algorithm for visualizing very large biological networks.

Networks are proving to be central to the study of gene function, protein-protein interaction, and biochemical pathway data. Visualization of networks is important for their study, but visualization tools are often inadequate for working with very large biological networks. Here, we present an algorithm, called large graph layout (LGL), which can be used to dynamically visualize large networks on the order of hundreds of thousands of vertices and millions of edges. LGL applies a force-directed iterative layout guided by a minimal spanning tree of the network in order to generate coordinates for the vertices in two or three dimensions, which are subsequently visualized and interactively navigated with companion programs. We demonstrate the use of LGL in visualizing an extensive protein map summarizing the results of approximately 21 billion sequence comparisons between 145579 proteins from 50 genomes. Proteins are positioned in the map according to sequence homology and gene fusions, with the map ultimately serving as a theoretical framework that integrates inferences about gene function derived from sequence homology, remote homology, gene fusions, and higher-order fusions. We confirm that protein neighbors in the resulting map are functionally related, and that distinct map regions correspond to distinct cellular systems, enabling a computational strategy for discovering proteins' functions on the basis of the proteins' map positions. Using the map produced by LGL, we infer general functions for 23 uncharacterized protein families.

Algorithms↗

PubMed2002

Large scale statistical prediction of protein-protein interaction by potentially interacting domain (PID) pair.

Protein-protein interaction plays a critical role in biological processes. The identification of interacting proteins by computational methods can provide new leads in functional studies of uncharacterized proteins without performing extensive experiments. We developed a database for the potentially interacting domain pairs (PID) extracted from a dataset of experimentally identified interacting protein pairs (DIP: database of interacting proteins) with InterPro, an integrated database of protein families, domains and functional sites. In developing protein interaction databases and predictive methods, sensitive statistical scoring systems is critical to provide a reliability index for accurate functional analysis of interaction networks. We present a statistical scoring system, named "PID matrix score" as a measure of the interaction probability (interactability) between domains. This system provided a valuable tool for functional prediction of unknown proteins. For the evaluation of PID matrix, cross validation was performed with subsets of DIP data. The prediction system gives about 50% sensitivity and more than 98% specificity, which implies that the information for interacting proteins pairs could be enriched about 30 fold with the PID matrix. It is demonstrated that mapping of the genome-wide interaction network can be achieved by using the PID matrix.

Algorithms↗

PubMed2003

Identification of proteins that interact with the central coiled-coil region of the human protein kinase NEK1.

NEK protein kinases are evolutionarily conserved kinases structurally related to the Aspergillus nidulans mitotic regulator NIMA. At least nine members of the NEK family in vertebrates have been described to date, but for most of them the interacting protein partners are unknown. The pleiotropic deleterious effects and the formation of kidney cysts caused by NEK1 mutation in mice emphasize its involvement in the regulation of diverse cellular processes and in the etiology of polycystic kidney disease (PKD), respectively. Here we report the identification of proteins that interacted with the human NEK1 protein kinase in a yeast two-hybrid screen of a human fetal brain cDNA library, using the catalytic and regulatory domains of NEK1 separately as baits. These proteins are known to take part either in the development of PKD, in the double-strand DNA break repair at the G2/M transition phase of the cell cycle, or in neural cell development. The proteins involved in PKD include the motor protein KIF3A and the proteins tuberin and alpha-catulin. Mapping studies of the human NEK1 regulatory domain (NRD) indicated a strong interaction of most of the proteins retrieved from the library with putative coiled coils located in the central region of NRD. Our results give further support to the previous observation that NEK1 is of functional importance for the etiology of PKD.

Animals↗

PubMed2003

Putative hAPN receptor binding sites in SARS_CoV spike protein.

AIM: To obtain the information of ligand-receptor binding between the S protein of SARS-CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites in the S protein of SARS-CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS-CoV in validating the bioinformatics predictions. RESULTS: Possible binding sites in the SARS-CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS-CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION: CD13 may be a possible receptor of the SARS-CoV S protein, which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

Amino Acid Sequence↗

PubMed2002

The jury is out on "guilt by association" trials.

The availability of comprehensive protein-protein interaction maps will significantly enhance medical research and aid the functional characterisation of novel genes. To date, the largest scale studies of protein-protein interactions have used the yeast two hybrid method. In this review we take a closer look at the different approaches used in these studies and discuss some key considerations that should be taken into account when designing high throughput interaction mapping projects.

Genome↗

PubMed2006

Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages.

The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.

Amino Acids↗

PubMed2004

ChIC and ChEC; genomic mapping of chromatin proteins.

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.

Antibodies↗

PubMed2005

Induced alpha-helix structure in the aryl hydrocarbon receptor transactivation domain modulates protein-protein interactions.

The aryl hydrocarbon receptor (AhR) is an intracellular receptor protein that regulates gene transcription in response to both man-made and natural ligands. A modular transactivaton domain (TAD) has been mapped to the 304 C-terminal amino acids and consists of acidic, Q-rich, and P/S/T-rich subdomains. We have used steady-state intrinsic tryptophan fluorescence and circular dichroism spectroscopy to investigate the conformation of the acidic Q-rich region. The results reveal that this region of the protein is structurally flexible but adopts a more folded conformation in the presence of the natural osmolyte trimethylamine N-oxide (TMAO) and the solvent trifluoroethanol (TFE). In protein-protein interaction studies, the acidic Q-rich region bound to components of the general transcription machinery [TATA-binding protein (TBP), TAF4, and TAF6] as well as the coactivator proteins SRC-1a and TIF2. The binding site for TBP mapped to the acidic subdomain, while SRC-1a bound preferentially to the Q-rich sequence. Significantly, the binding of TBP was modulated by induced folding of the TAD with TMAO. The results indicate that the AhR TAD makes multiple interactions with the transcriptional machinery and protein conformation plays a critical role in receptor function. Taken together, these findings support a role for protein folding in AhR action and suggest possible mechanisms of receptor-dependent gene activation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Promoters can contribute to the elucidation of protein function.

Protein interaction maps are valuable tools in the understanding of the actions of genes and proteins in a real-life context. However, many proteins and genes interact functionally - without physical contact - via transcriptional coregulation of the genes, which is partially manifested in functional promoter structures called promoter modules. Here, the example of interleukin 9 (IL-9) induction by IL-1 and tumour necrosis factor (TNF) is used to demonstrate how comparative promoter analysis can identify a shared promoter module and thus indicate the crucial partners. This strategy can be used for genes from a small initial set of coregulated genes, such as can be derived from expression array data. The combination of expression and promoter analysis with proteomics should be considered as an important link in functional genomics.

Animals↗

PubMed2004

Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system.

The yeast two-hybrid assay has proven to be a powerful method to detect protein-protein interactions as well as to derive genome-wide protein interaction maps. More recently, three-hybrid assays have emerged as a means to detect both protein-RNA and protein-small molecule interactions. Despite the routine use of the two-hybrid assay and the potential of three-hybrid systems, there has been little quantitative characterization to understand how the strength of the protein interaction correlates with transcription activation. It is not known if the additional interaction in three-hybrid systems compromises the sensitivity of the system. Thus, here, we set out to determine the K(D) cutoff of a small molecule three-hybrid system and to determine if there is a correlation between the K(D) and the levels of transcription activation. A series of mutations to FK506-binding protein 12 (FKBP12) were designed to vary the affinity of this protein for the small molecule synthetic ligand for FK506-binding protein 12 (SLF). These FKBP12 variants were overexpressed and purified, and their K(D)'s for SLF were measured using a fluorescence polarization assay. Then the levels of transcription activation in a Mtx-DHFR yeast three-hybrid system were determined for these variants using a lacZ reporter gene. The K(D) cutoff of the Mtx yeast three-hybrid system is found to be ca. 50 nM. Further, the levels of transcription activation correlate with the strength of the binding interaction, though the dynamic range is only 1 order of magnitude. These results establish that the three-hybrid assay has the requisite sensitivity for drug discovery. However, the small dynamic range highlights a limitation to equilibrium-based assays for discriminating interactions based on affinity.

Escherichia coli↗

PubMed1999

A novel in vivo assay for the analysis of protein-protein interaction.

The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner.

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.

Anthracenes↗