USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.

The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells.

USP7 or HAUSP is a ubiquitin-specific protease in human cells that regulates the turnover of p53 and is bound by at least two viral proteins, the ICP0 protein of herpes simplex type 1 and the EBNA1 protein of EpsteinBarr virus. We have overexpressed and purified USP7 and shown that the purified protein is monomeric and is active for cleaving both a linear ubiquitin substrate and conjugated ubiquitin on EBNA1. Using partial proteolysis of USP7 coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we showed that USP7 comprises four structural domains; an N-terminal domain known to bind p53, a catalytic domain, and two C-terminal domains. By passing a mixture of USP7 domains over EBNA1 and ICP0 affinity columns, we showed that the N-terminal p53 binding domain was also responsible for the EBNA1 interaction, while the ICP0 binding domain mapped to a C-terminal domain between amino acids 599 -801. Tryptophan fluorescence assays showed that an EBNA1 peptide mapping to residues 395-450 was sufficient to bind the USP7 N-terminal domain and did so with a dissociation constant of 0.9 -2 M, whereas p53 peptides spanning the USP7-binding region gave dissociation constants of 9 -17 M in the same assay. In keeping with these relative affinities, gel filtration analyses of the complexes showed that the EBNA1 peptide efficiently competed with the p53 peptide for USP7 binding, suggesting that EBNA1 could affect p53 function in vivo by competing for USP7.

BackgroundMany high-throughput experiments compare two phenotypes such as disease vs. healthy, with the goal of understanding the underlying biological phenomena characterizing the given phenotype. Because of the importance of this type of analysis, more than 70 pathway analysis methods have been proposed so far. These can be categorized into two main categories: non-topology-based (non-TB) and topology-based (TB). Although some review papers discuss this topic from different aspects, there is no systematic, large-scale assessment of such methods. Furthermore, the majority of the pathway analysis approaches rely on the assumption of uniformity of p values under the null hypothesis, which is often not true.ResultsThis article presents the most comprehensive comparative study on pathway analysis methods available to date. We compare the actual performance of 13 widely used pathway analysis methods in over 1085 analyses. These comparisons were performed using 2601 samples from 75 human disease data sets and 121 samples from 11 knockout mouse data sets. In addition, we investigate the extent to which each method is biased under the null hypothesis. Together, these data and results constitute a reliable benchmark against which future pathway analysis methods could and should be tested.ConclusionOverall, the result shows that no method is perfect. In general, TB methods appear to perform better than non-TB methods. This is somewhat expected since the TB methods take into consideration the structure of the pathway which is meant to describe the underlying phenomena. We also discover that most, if not all, listed approaches are biased and can produce skewed results under the null.Electronic supplementary materialThe online version of this article (10.1186/s13059-019-1790-4) contains supplementary material, which is available to authorized users.

Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G 1 but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.The initiation of DNA replication in eukaryotic cells is a carefully regulated process requiring the orchestrated assembly of many proteins at origin sites, including the origin recognition complex and minichromosome maintenance (MCM) complex. The MCM complex consists of six subunits, MCM2 through MCM7 (MCM2-7), which form a hexamer. Studies in Saccharomyces cerevisiae, where MCM proteins were first identified (25), showed that each of the MCM subunits performs an essential function in the initiation and elongation of DNA replication (20,21). Genetic and biochemical studies conducted in yeast, Xenopus, Drosophila, and mammals point to probable roles of the MCM proteins in melting origin DNA and in functioning as the replicative helicase at replication forks (8,27).Biochemical analyses of the MCM complex have shown that MCM4, -6, and -7 are the most stably associated subunits, referred to as the helicase core (MCM4/6/7), with MCM2 and a dimer of MCM3 and MCM5 being more loosely associated with the core (13,23,30,36). MCM4, MCM6, and MCM7 on their own can form hexamers with weak but measurable DNA helicase activity. The addition of MCM2 to the MCM4/6/7 core complex disrupts the hexamer and inhibits DNA helicase activity (12, 23). The complete MCM2-7 complex has no detectable helicase activity in vitro (12, 23), but helicase activity has been reported for a larger complex containing MCM2-7, cdc45, and GINS (29). As expected, MCM complexes exhibit ATPase activity (6,23,35). ATPase activity has not been observed in individual MCM subunits but occurs when certain pairs of MCM proteins interact (6).Each of the six MCM subunits shares a region of homology referre...

Advances in high-throughput technologies allow for measurements of many types of omics data, yet the meaningful integration of several different data types remains a significant challenge. Another important and difficult problem is the discovery of molecular disease subtypes characterized by relevant clinical differences, such as survival. Here we present a novel approach, called erturbation clustering for datategration and disease ubtyping (PINS), which is able to address both challenges. The framework has been validated on thousands of cancer samples, using gene expression, DNA methylation, noncoding microRNA, and copy number variation data available from the Gene Expression Omnibus, the Broad Institute, The Cancer Genome Atlas (TCGA), and the European Genome-Phenome Archive. This simultaneous subtyping approach accurately identifies known cancer subtypes and novel subgroups of patients with significantly different survival profiles. The results were obtained from genome-scale molecular data without any other type of prior knowledge. The approach is sufficiently general to replace existing unsupervised clustering approaches outside the scope of bio-medical research, with the additional ability to integrate multiple types of data.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

The EBNA1 protein of Epstein-Barr virus (EBV) is essential for EBV latent infection in ensuring the replication and stable segregation of the EBV genomes and in activating the transcription of other EBV latency genes. We have tested the ability of four host proteins (Brd2, Brd4, DEK, and MeCP2) implicated in the segregation of papillomavirus and Kaposi's sarcoma-associated herpesvirus to support EBNA1-mediated segregation of EBV-based plasmids in Saccharomyces cerevisiae. We found that Brd4 enabled EBNA1-mediated segregation while Brd2 and MeCP2 had a general stimulatory effect on plasmid maintenance. EBNA1 interacted with Brd4 in both yeast and human cells through N-terminal sequences previously shown to mediate transcriptional activation but not segregation. In keeping with this interaction site, silencing of Brd4 in human cells decreased transcriptional activation by EBNA1 but not the mitotic chromosome attachment of EBNA1 that is required for segregation. In addition, Brd4 was found to be preferentially localized to the FR enhancer element regulated by EBNA1, over other EBV sequences, in latently EBV-infected cells. The results indicate that EBNA1 can functionally interact with Brd4 in native and heterologous systems and that this interaction facilitates transcriptional activation by EBNA1 from the FR element.As part of their life cycle, gammaherpesviruses and papillomaviruses establish persistent infections in proliferating cells in which their double-stranded circular DNA genomes are maintained at a constant copy number. Maintenance of copy number involves the doubling of the population of viral genomes each cell cycle and a segregation mechanism to ensure equal delivery of the genomes to the daughter cells during cell division. The mechanism of mitotic segregation is conserved in the gammaherpesviruses and papillomaviruses in that, in all cases, the viral genomes are tethered to the host mitotic chromosomes through the viral origin DNA binding protein, which binds directly to the viral segregation element and interacts with one or more host chromosomal proteins.The mechanism of papillomavirus genome segregation has been studied most extensively using the bovine papillomavirus (BPV). The BPV E2 protein tethers the viral genomes to host chromosomes through interactions with multiple E2 recognition sites in the minichromosome maintenance element (MME) (22,30,38,47). E2 binds the MME through its DNA binding and dimerization domain and interacts with mitotic chromosomes through the domain responsible for transcriptional activation (6, 47). The mechanism by which the E2 transactivation domain contacts mitotic chromosomes to mediate segregation has been the subject of several studies, and considerable evidence has implicated an interaction with the bromodomain protein Brd4 in this process. Brd4 was identified as a binding partner of E2 that colocalized with E2 on host mitotic chromosomes and was shown to enable E2 to maintain plasmids containing the MME in budding yeast (Saccharomyces cerevisiae) (9,34,62). Inte...