Potential Protein Interactions Research Articles

Identification of potential key protein interaction networks of BK virus nephropathy in patients receiving kidney transplantation

We aim to identify the key protein interaction networks and implicated pathways of BK virus nephropathy (BKVN) via bioinformatic methods. The microarray data GSE75693 of 30 patients with stable kidney transplantation and 15 with BKVN were downloaded and analyzed by using the limma package to identify differentially expressed genes (DEGs). Then the gene ontology (GO) functional enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were done to investigate the molecular function (MF), biological process (BP), cellular components (CC) and pathways of DEGs. Finally, protein-protein interactions (PPIs) were constructed, and the hub proteins were identified. As a result, 249 up-regulated genes and 253 down-regulated genes of BKVN patients were selected based on criteria of P > 0.01 and fold change >2.0. GO and KEGG showed that DEGs were mainly located in nucleus and cytosol, and were implicated in the immune responses. In the PPI analysis, 26 up-regulated and 8 down-regulated proteins composed the pivotal interaction network. CXCL10, EGF and STAT1 were identified as hub proteins in BKVN. In conclusion, CXCL10, EGF and STAT1 may induce kidney injuries by promoting inflammation and prohibiting reparation of tissue damage in BKVN.

Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

Coding GPCR-G protein specificity.

G protein-coupled receptors (GPCRs) are ubiquitous gatekeepers of cellular response and signal predominantly by recruitment and activation of G proteins. In a recent paper in Nature, Flock et al. use large-scale bioinformatics to build a model of GPCR-G protein selectivity and an interactive database to interrogate potential receptor-G protein interactions.

The PRDM9 KRAB domain is required for meiosis and involved in protein interactions

PR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis.

Using structural knowledge in the protein data bank to inform the search for potential host-microbe protein interactions in sequence space: application to Mycobacterium tuberculosis

BackgroundA comprehensive map of the human-M. tuberculosis (MTB) protein interactome would help fill the gaps in our understanding of the disease, and computational prediction can aid and complement experimental studies towards this end. Several sequence-based in silico approaches tap the existing data on experimentally validated protein-protein interactions (PPIs); these PPIs serve as templates from which novel interactions between pathogen and host are inferred. Such comparative approaches typically make use of local sequence alignment, which, in the absence of structural details about the interfaces mediating the template interactions, could lead to incorrect inferences, particularly when multi-domain proteins are involved.ResultsWe propose leveraging the domain-domain interaction (DDI) information in PDB complexes to score and prioritize candidate PPIs between host and pathogen proteomes based on targeted sequence-level comparisons. Our method picks out a small set of human-MTB protein pairs as candidates for physical interactions, and the use of functional meta-data suggests that some of them could contribute to the in vivo molecular cross-talk between pathogen and host that regulates the course of the infection. Further, we present numerical data for Pfam domain families that highlights interaction specificity on the domain level. Not every instance of a pair of domains, for which interaction evidence has been found in a few instances (i.e. structures), is likely to functionally interact. Our sorting approach scores candidates according to how “distant” they are in sequence space from known examples of DDIs (templates). Thus, it provides a natural way to deal with the heterogeneity in domain-level interactions.ConclusionsOur method represents a more informed application of local alignment to the sequence-based search for potential human-microbial interactions that uses available PPI data as a prior. Our approach is somewhat limited in its sensitivity by the restricted size and diversity of the template dataset, but, given the rapid accumulation of solved protein complex structures, its scope and utility are expected to keep steadily improving.

Abstract P5-06-09: Cyclin d1 binding to chromatin and the induction of chromosomal instability requires the fuzzy domain

Abstract The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma (Rb) and the nuclear respiratory factor 1 (NRF1) proteins to regulate nuclear DNA synthesis and mitochondrial biogenesis. Cyclin D1 is required for oncogene-dependent growth and genetic ablation of the murine cyclin D1 gene resulted in resistance to Ras or ErbB2-induced mammary tumorigenesis and APC-induced gastrointestinal tumorigenesis. Cyclin D1 overexpression occurs in human breast, prostate, lung, and gastrointestinal malignancies and its abundance is induced at the level of transcription, translation and through post-translational modifications. Cyclin D1 plays a key role in transcriptional regulation inducing gene expression governing chromosomal instability (CIN) and cell-cycle progression. Cyclin D1 is also known to bind TF regulatory regions in chromatin immuno-precipitation (ChIP) assays. Genome wide analysis of cyclin D1 occupancy using ChIP-Seq identified binding sites including both the coding and non-coding genome with enrichment for genes regulating CIN and the G2/M phase (Top2A, AurkB, Cenpp, Mlf1ip, Zw10, Ckap2) consistent with enrichment of cyclin D1 at G2/M and the finding that cyclin D1 induces CIN. We sought to identify the molecular mechanisms governing the recruitment of cyclin D1 in the context of local chromatin to promote CIN. In order to define the domain of cyclin D1 involved in aneuploidy and tumorigenesis, we transduced and assessed the induction of aneuploidy in MEF cells using cyclin D1 wt (wt), cyclin D1 C-terminus domain (C4), cyclin D1 mutant lacking of the E-box motif (ΔE) or ctrl. We also searched for potential histone protein interaction motifs in cyclin D1 and determined the epigenetic motif recognized by cyclin D1 using a histone peptide array. The recognition of an epigenetic code by cyclin D1 may facilitate genome wide expression changes during cell-cycle progression and tumorigenesis. We finally identified a “fuzzy” domain of cyclin D1 which is required to local chromatin access for regulatory promoter regions governing and promoting CIN. Citation Format: Pestell RG, Di Sante G, Di Rocco A, Pupo C, Crosariol M, Tompa P, Tantos A, Wang C, Yu Z, Vadlamudi R, Mann M, Casimiro MC. Cyclin d1 binding to chromatin and the induction of chromosomal instability requires the fuzzy domain [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-09.

Abstract B33: Understanding resistance mechanisms to PI3K/mTOR inhibitors in ovarian cancer model systems

Abstract The combined use of computational models and experimental data is an emerging tool in systems biology to help explain the mechanism of resistance to various protein inhibitors in oncological medicine. Improved understanding of the signaling network dynamics may offer novel approaches to overcoming drug resistance. The aim of this study was to analyze the signaling dynamics of the PI3K/Akt/mTOR pathway in ovarian cancer cells after treatment with different PI3K/mTOR inhibitors. Systems computational approach was applied to unwind the role of multiple feedbacks in PI3K/Akt/mTOR pathway in the responses of cancer cells bearing mutations in PTEN, PIK3CA and HER2 overexpression. In this study, a set of PI3K (LY294002), mTOR (Rapamycin) and dual PI3K/mTOR (BEZ235) inhibitors were applied to a panel of ovarian cancer cell lines under heregulin driven conditions over 10 point time-course treatment, in order to assess the association of growth inhibition with pathway activation. The evaluation of expression levels of various proteins and phospho-proteins was assessed using reverse phase protein array (RPPA) as a high throughput proteomic analysis to investigate the dynamic changes in response to the inhibitors. In part, PTEN expression status was analyzed as biomarker of the feedback-dependent pAkt activation in response to mTOR inhibition by rapamycin in different cell lines. The three inhibitors displayed a significant inhibition in cell proliferation on all the ovarian cancer cell lines following a 5-day treatment. Our results have shown a significant positive correlation between the signaling dynamics of phosphorylated Src (Tyr527) and phosphorylated PTEN (Ser380/Thr382/383) upon treatment with the drugs, consistent with the role of Src in the inactivation of PTEN. Additionally, we demonstrate that PI3K plays a role in the activation of RAS pathway, as phosphorylated cRaf (Ser259 and Ser338) and phosphorylated mTOR (Ser2481) were upregulated and showed a significant positive correlation in their signaling dynamics. Although, all the cell lines have shown a significant inhibition to cell proliferation following a 5-day treatment with the inhibitors, the signaling expression of 12 different phospho-proteins within the PI3K/mTOR and MAPK pathways differ. Rapamycin has been shown to downregulate the expression of phospho-mTOR (Ser2448) and its substrates, phospho-4E-BP1 and phospho-S6K1 but has shown an upregulation in phospho-Akt (Ser473), confirming the existence of the negative feedback loop, S6K1-Akt, upon treatment. BEZ235 presented the highest efficacy in inhibiting cell proliferation in all the ovarian cancer cell lines but failed to downregulate the phosphorylation of ERK. Due to the high overexpression of erbB2 that may cause HER2 homodimerization, the signaling expression of the ovarian cancer cell line, SKOV3, showed upregulation in all the measured phospho-proteins. We propose an extended model of PI3K/Akt/mTOR pathway in ovarian cancer, based on theoretical modeling and experimental data, highlighting possible resistance compensatory mechanisms within the pathway and with its parallel cross-talk pathway MAPK. The model highlights potential novel protein interactions and feedback loops, offering strategies for combinatorial anti-cancer therapy. Citation Format: Ghassan Tashkandi, Alexey Goltsov, Peter Mullen, Jim Bown, David Harrison, Simon Langdon. Understanding resistance mechanisms to PI3K/mTOR inhibitors in ovarian cancer model systems. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B33.

Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis.

Pathological angiogenesis is a hallmark of many diseases. We demonstrated that TR3/Nur77 is an excellent target for pro-angiogenesis and anti-angiogenesis therapies. Here, we report that TR3 transcriptionally regulates endothelial cell migration, permeability and the formation of actin stress fibers that is independent of RhoA GTPase. 1) Amino acid residues 344-GRR-346 and de-phosphorylation of amino acid residue serine 351 in the DNA binding domain, and 2) phosphorylation of amino acid residues in the 41-61 amino acid fragment of the transactivation domain, of TR3 are required for its induction of the formation of actin stress fibers, cell proliferation, migration and permeability. The 41-61 amino acid fragment contains one of the three potential protein interaction motifs in the transactivation domain of TR3, predicted by computational modeling and analysis. These studies further our understanding of the molecular mechanism, by which TR3 regulates angiogenesis, identify novel therapeutic targeted sites of TR3, and set the foundation for the development of high-throughput screening assays to identify compounds targeting TR3/Nur77 for pro-angiogenesis and anti-angiogenesis therapies.

Design and evaluation of antimalarial peptides derived from prediction of short linear motifs in proteins related to erythrocyte invasion.

The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs) in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds.

Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal “domain present in USPs” (DUSP) and “ubiquitin-like” (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys63-, Lys6-, Lys33-, and Lys11-linked chains over Lys27-, Lys29-, and Lys48-linked and linear chains consistent with USP11’s function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Proteomics based identification of cell migration related proteins in HBV expressing HepG2 cells.

Proteomics study was performed to investigate the specific protein expression profiles of HepG2 cells transfected with mutant HBV compared with wildtype HBV genome, aiming to identify the specific functions of SH3 binding domain (proline rich region) located in HBx. In addition to the cell movement and kinetics changes due to the expression of HBV genome we have observed previously, here we further targeted to explore the specific changes of cellular proteins and potential intracellular protein interactions, which might provide more information of the potential cellular mechanism of the differentiated cell movements. Specific changes of a number of proteins were shown in global protein profiling in HepG2 cells expressing wildtype HBV, including cell migration related proteins, and interestingly the changes were found recovered by SH3 binding domain mutated HBV. The distinctive expressions of proteins were validated by Western blot analysis.

Semantically assessing the reliability of protein interactions

Protein–protein interactions (PPIs) play very important roles in many cellular processes, and provide rich information for discovering biological facts and knowledge. Although various experimental approaches have been developed to generate large amounts of PPI data for different organisms, high-throughput experimental data usually suffers from high error rates, and as a consequence, the biological knowledge discovered from this data is distorted or incorrect. Therefore, it is vital to assess the quality of protein interaction data and extract reliable protein interactions from the high-throughput experimental data. In this paper, we propose a new Semantic Reliability (SR) method to assess the reliability of each protein interaction and identify potential false-positive protein interactions in a dataset. For each pair of target interacting proteins, the SR method takes into account the semantic influence between proteins that interact with the target proteins, and the semantic influence between the target proteins themselves when assessing the interaction reliability. Evaluations on real protein interaction datasets demonstrated that our method outperformed other existing methods in terms of extracting more reliable interactions from original protein interaction datasets.

Mycobacterium tuberculosis RNA Polymerase-binding Protein A (RbpA) and Its Interactions with Sigma Factors

RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the β subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σ(A) and the stress-induced σ(B) as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σ(B) via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell.

Expressed in high metastatic cells (Ehm2) is a positive regulator of keratinocyte adhesion and motility: The implication for wound healing

BackgroundMultiple factors have been shown to delay dermal wound healing. These resultant wounds pose a significant problem in terms of morbidity and healthcare spend. Recently, an increasing volume of research has focused on the molecular perturbations underlying non-healing wounds. ObjectivesThis study investigates the effect of a novel cancer promoter, Ehm2, in wound healing. Ehm2 belongs to the FERM family of proteins, known to be involved in membrane–cytoskeletal interactions, and has been shown to promote cancer metastasis in melanoma, prostate cancer and breast cancer. MethodsEhm2 mRNA levels were analysed using qRT-PCR, standardised to GAPDH, from either acute or chronic wounds, and normal skin. IHC analysis was also undertaken from wound edge biopsies. An anti-Ehm2 transgene was created and transfected into the HaCaT cell line. The effect of Ehm2 knockdown on migration, adhesion, growth, cell cycle progression and apoptosis was analysed using standard laboratory methods. Western Blot analysis was used to investigate potential downstream protein interactions. ResultsEhm2 is expressed nearly three times higher in acute wound tissues, compared to chronic wound tissues. Increased Ehm2 expression is found in wounds undergoing healing, especially at the leading wound edge. In vitro, Ehm2 knockdown reduces cellular adhesion, migration and motility, without affecting growth, cell cycle and apoptosis. Finally, Ehm2 knockdown results in reduced NWasp protein expression. ConclusionThese results suggest Ehm2 may be an important player in the wound healing process, and show that Ehm2 knockdown downregulates the expression of NWasp, through which it may have its effect on cellular migration.

Augmented transitive relationships with high impact protein distillation in protein interaction prediction

Predicting new protein–protein interactions is important for discovering novel functions of various biological pathways. Predicting these interactions is a crucial and challenging task. Moreover, discovering new protein–protein interactions through biological experiments is still difficult. Therefore, it is increasingly important to discover new protein interactions. Many studies have predicted protein–protein interactions, using biological features such as Gene Ontology (GO) functional annotations and structural domains of two proteins. In this paper, we propose an augmented transitive relationships predictor (ATRP), a new method of predicting potential protein interactions using transitive relationships and annotations of protein interactions. In addition, a distillation of virtual direct protein–protein interactions is proposed to deal with unbalanced distribution of different types of interactions in the existing protein–protein interaction databases. Our results demonstrate that ATRP can effectively predict protein–protein interactions. ATRP achieves an 81% precision, a 74% recall and a 77% F-measure in average rate in the prediction of direct protein–protein interactions. Using the generated benchmark datasets from KUPS to evaluate of all types of the protein–protein interaction, ATRP achieved a 93% precision, a 49% recall and a 64% F-measure in average rate. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.

A Novel Biclustering Approach to Association Rule Mining for Predicting HIV-1–Human Protein Interactions

Identification of potential viral-host protein interactions is a vital and useful approach towards development of new drugs targeting those interactions. In recent days, computational tools are being utilized for predicting viral-host interactions. Recently a database containing records of experimentally validated interactions between a set of HIV-1 proteins and a set of human proteins has been published. The problem of predicting new interactions based on this database is usually posed as a classification problem. However, posing the problem as a classification one suffers from the lack of biologically validated negative interactions. Therefore it will be beneficial to use the existing database for predicting new viral-host interactions without the need of negative samples. Motivated by this, in this article, the HIV-1–human protein interaction database has been analyzed using association rule mining. The main objective is to identify a set of association rules both among the HIV-1 proteins and among the human proteins, and use these rules for predicting new interactions. In this regard, a novel association rule mining technique based on biclustering has been proposed for discovering frequent closed itemsets followed by the association rules from the adjacency matrix of the HIV-1–human interaction network. Novel HIV-1–human interactions have been predicted based on the discovered association rules and tested for biological significance. For validation of the predicted new interactions, gene ontology-based and pathway-based studies have been performed. These studies show that the human proteins which are predicted to interact with a particular viral protein share many common biological activities. Moreover, literature survey has been used for validation purpose to identify some predicted interactions that are already validated experimentally but not present in the database. Comparison with other prediction methods is also discussed.

Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets

We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not necessarily imply that one detection methodology was better or worse, but rather that, to a large extent, the insights reflected the methodological biases themselves. Consequently, interpreting the protein interaction data within their experimental or cellular context provided the best avenue for overcoming biases and inferring biological knowledge.

Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

BackgroundFoamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation.ResultsSeveral Prototype FV (PFV) Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85PR-RT and p40IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71Gag resulted in a significant copackaging of these proteins.ConclusionsNon-particle associated PFV Pol appears to be naturally released from infected cells by a yet unknown mechanism. The absence of particle-associated Pol precursor suggests its rapid processing upon particle incorporation. Analysis of different PFV Gag-Pol fusion constructs demonstrates that orthoretroviral-like Pol expression is compatible with FV replication in principal as long as fusion protein processing is possible. Furthermore, unlike orthoretroviruses, PFV particle release and infectivity tolerate larger differences in relative cellular Gag/Pol levels.

Solution Structure of the Mycobacterium tuberculosis EsxG·EsxH Complex: FUNCTIONAL IMPLICATIONS AND COMPARISONS WITH OTHER M. TUBERCULOSIS Esx FAMILY COMPLEXES

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn2+ binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn2+-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.