Protein-protein interactions of human viruses.

Viruses infect their human hosts by a series of interactions between viral and host proteins, indicating that detailed knowledge of such virus-host interaction interfaces are critical for our understanding of viral infection mechanisms, disease etiology and the development of new drugs. In this review, we primarily survey human host-virus interaction data that are available from public databases following the standardized PSI-MS format. Notably, available host-virus protein interaction information is strongly biased toward a small number of virus families including herpesviridae, papillomaviridae, orthomyxoviridae and retroviridae. While we explore the reliability and relevance of these protein interactions we also survey the current knowledge about viruses functional and topological targets. Furthermore, we assess emerging frontiers of host-virus protein interaction research, focusing on protein interaction interfaces of hosts that are infected by different viruses and viruses that infect multiple hosts. Finally, we cover the current status of research that investigates the relationships of virus-targeted host proteins to other comorbidities as well as the influence of host-virus protein interactions on human metabolism.

ZapG (YhcB/DUF1043), a novel cell division protein in gamma-proteobacteria linking the Z-ring to septal peptidoglycan synthesis.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

Proteome Data Improves Protein Function Prediction in the Interactome of Helicobacter pylori.

Helicobacter pylori is a common pathogen that is estimated to infect half of the human population, causing several diseases such as duodenal ulcer. Despite one of the first pathogens to be sequenced, its proteome remains poorly characterized as about one-third of its proteins have no functional annotation. Here, we integrate and analyze known protein interactions with proteomic and genomic data from different sources. We find that proteins with similar abundances tend to interact. Such an observation is accompanied by a trend of interactions to appear between proteins of similar functions, although some show marked cross-talk to others. Protein function prediction with protein interactions is significantly improved when interactions from other bacteria are included in our network, allowing us to obtain putative functions of more than 300 poorly or previously uncharacterized proteins. Proteins that are critical for the topological controllability of the underlying network are significantly enriched with genes that are up-regulated in the spiral compared with the coccoid form of H. pylori Determining their evolutionary conservation, we present evidence that 80 protein complexes are identical in composition with their counterparts in Escherichia coli, while 85 are partially conserved and 120 complexes are completely absent. Furthermore, we determine network clusters that coincide with related functions, gene essentiality, genetic context, cellular localization, and gene expression in different cellular states.

The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC.

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the Khalf of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

The Nitrogen Regulatory PII Protein (GlnB) and N-Acetylglucosamine 6-Phosphate Epimerase (NanE) Allosterically Activate Glucosamine 6-Phosphate Deaminase (NagB) in Escherichia coli.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli.

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF Khalf for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the Khalf for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins.

The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.

Bacterial protein meta-interactomes predict cross-species interactions and protein function.

BACKGROUND: Protein-protein interactions (PPIs) can offer compelling evidence for protein function, especially when viewed in the context of proteome-wide interactomes. Bacteria have been popular subjects of interactome studies: more than six different bacterial species have been the subjects of comprehensive interactome studies while several more have had substantial segments of their proteomes screened for interactions. The protein interactomes of several bacterial species have been completed, including several from prominent human pathogens. The availability of interactome data has brought challenges, as these large data sets are difficult to compare across species, limiting their usefulness for broad studies of microbial genetics and evolution. RESULTS: In this study, we use more than 52,000 unique protein-protein interactions (PPIs) across 349 different bacterial species and strains to determine their conservation across data sets and taxonomic groups. When proteins are collapsed into orthologous groups (OGs) the resulting meta-interactome still includes more than 43,000 interactions, about 14,000 of which involve proteins of unknown function. While conserved interactions provide support for protein function in their respective species data, we found only 429 PPIs (~1% of the available data) conserved in two or more species, rendering any cross-species interactome comparison immediately useful. The meta-interactome serves as a model for predicting interactions, protein functions, and even full interactome sizes for species with limited to no experimentally observed PPI, including Bacillus subtilis and Salmonella enterica which are predicted to have up to 18,000 and 31,000 PPIs, respectively. CONCLUSIONS: In the course of this work, we have assembled cross-species interactome comparisons that will allow interactomics researchers to anticipate the structures of yet-unexplored microbial interactomes and to focus on well-conserved yet uncharacterized interactors for further study. Such conserved interactions should provide evidence for important but yet-uncharacterized aspects of bacterial physiology and may provide targets for anti-microbial therapies.

Protein complexes in bacteria.

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 "gold standard" protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 "gold standard" protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial "model" species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies.

A second-generation protein-protein interaction network of Helicobacter pylori.

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins.

UNLABELLED: Mycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast two-hybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible protein-protein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis. IMPORTANCE: Mycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

RsfA (YbeB) proteins are conserved ribosomal silencing factors.

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.

The protein interaction network of bacteriophage lambda with its host, Escherichia coli.

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens.

An ontology for microbial phenotypes.

BACKGROUND: Phenotypic data are routinely used to elucidate gene function in organisms amenable to genetic manipulation. However, previous to this work, there was no generalizable system in place for the structured storage and retrieval of phenotypic information for bacteria. RESULTS: The Ontology of Microbial Phenotypes (OMP) has been created to standardize the capture of such phenotypic information from microbes. OMP has been built on the foundations of the Basic Formal Ontology and the Phenotype and Trait Ontology. Terms have logical definitions that can facilitate computational searching of phenotypes and their associated genes. OMP can be accessed via a wiki page as well as downloaded from SourceForge. Initial annotations with OMP are being made for Escherichia coli using a wiki-based annotation capture system. New OMP terms are being concurrently developed as annotation proceeds. CONCLUSIONS: We anticipate that diverse groups studying microbial genetics and associated phenotypes will employ OMP for standardizing microbial phenotype annotation, much as the Gene Ontology has standardized gene product annotation. The resulting OMP resource and associated annotations will facilitate prediction of phenotypes for unknown genes and result in new experimental characterization of phenotypes and functions.

A comparison and optimization of yeast two-hybrid systems.

Two-hybrid (Y2H) assays are available in a variety of different versions, including bacterial, yeast, and mammalian systems. However, even when done exclusively in yeast, multiple different host strains, vectors, reporter genes, or protocols can be used. Here we systematically compare protein-protein interactions (PPIs) from several previously published Y2H datasets. PPIs of a human gold-standard dataset were generated by Y2H assays as well as other methods such as LUMIER or protein fragment complementation assays (PCAs). Different Y2H methods detect substantially different subsets of these PPIs, even when protocols are standardized. In order to maximize the number of interactions found and to minimize the number of false positive interactions we recommend to combine multiple vectors and protocols. While the combined results of all 18 methods detected about 92% of a gold-standard interaction set, a combination of just three Y2H assays detected up to 78% of these protein pairs, or up to 83% when a fourth assay was included. These findings indicate that three or four separate assays may be sufficient to detect the majority of protein-protein interactions in many systems.

Studying protein complexes by the yeast two-hybrid system.

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.