218 research outputs found
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An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication
Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis, B cell lymphomas, and nasopharyngeal carcinoma. Many of the genes required for EBV virion morphogenesis are found in all herpesviruses, but some are specific to gammaherpesviruses. One of these gamma-specific genes, BLRF2, encodes a tegument protein that has been shown to be essential for replication in other gammaherpesviruses. In this study, we identify BLRF2 interacting proteins using binary and co-complex protein assays. Serine/Arginine-rich Protein Kinase 2 (SRPK2) was identified by both assays and was further shown to phosphorylate an RS motif in the BLRF2 C-terminus. Mutation of this RS motif (S148A+S150A) abrogated the ability of BLRF2 to support replication of a murine gammaherpesvirus 68 genome lacking the BLRF2 homolog (ORF52). We conclude that the BLRF2 RS motif is phosphorylated by SRPK2 and is important for viral replication
Weight gain reveals dramatic increases in skeletal muscle extracellular matrix remodeling
Context: In animal models of obesity, chronic inflammation and dysregulated extracellular matrix remodeling in adipose tissue leads to insulin resistance. Whether similar pathophysiology occurs in humans is not clear. Objective: The aim of this study was to test whether 10% weight gain induced by overfeeding triggers inflammation and extracellular matrix remodeling (gene expression, protein, histology) in skeletal muscleandsc adipose tissue in humans.Wealso investigated whether such remodelingwas associated with an impaired metabolic response (hyperinsulinemic-euglycemic clamp). Design, Setting, Participants, and Intervention: Twenty-nine free-living males were fed 40% over their baseline energy requirements for 8 weeks. Results: Ten percent body weight gain prompted dramatic up-regulation of a repertoire of extracellular matrix remodeling genes in muscle and to a lesser degree in adipose tissue. The amount of extracellular matrix genes in the muscle were directly associated with the amount of lean tissue deposited during overfeeding. Despite weight gain and impaired insulin sensitivity, there was no change in local adipose tissue or systemic inflammation, but there was a slight increase in skeletal muscle inflammation. Conclusion:Wepropose that skeletal muscle extracellular matrix remodeling is another feature of the pathogenic milieu associated with energy excess and obesity, which, if disrupted, may contribute to the development of metabolic dysfunction. © 2014 by the Endocrine Society
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The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth
The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation
Determinants of the changes in glycemic control with exercise training in type 2 diabetes:A randomized trial
To assess the determinants of exercise training-induced improvements in glucose control (HbA1C) including changes in serum total adiponectin and FFA concentrations, and skeletal muscle peroxisome proliferator-activated receptor-Îł coactivator-1α (PGC-1α) protein content.A sub-cohort (nâ=â35; 48% men; 74% Caucasian) from the HART-D study undertaking muscle biopsies before and after 9 months of aerobic (AT), resistance (RT), or combination training (ATRT).Changes in HbA1C were associated with changes in adiponectin (râ=â-0.45, Pâ=â0.007). Participants diagnosed with type 2 diabetes for a longer duration had the largest increase in PGC-1α (râ=â0.44, Pâ=â0.008). Statistical modeling examining changes in HbA1C suggested that male sex (Pâ=â0.05), non-Caucasian ethnicity (Pâ=â0.02), duration of type 2 diabetes (râ=â0.40; P<0.002) and changes in FFA (râ=â0.36; P<0.004), adiponectin (râ=â-0.26; P<0.03), and PGC-1α (râ=â-0.28; Pâ=â0.02) explain âŒ65% of the variability in the changes in HbA1C.Decreases in HbA1C after 9 months of exercise were associated with shorter duration of diabetes, lowering of serum FFA concentrations, increasing serum adiponectin concentrations and increasing skeletal muscle PGC-1α protein expression.ClinicalTrials.gov NCT00458133
Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.
In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action
Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening
In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action
Synthesis and structural characterization of new oxorhenium and oxotechnetium complexes with XN2S-tetradentate semi-rigid ligands(X = O, S, N)
Twelve novel oxo-technetium and oxo-rhenium complexes based on N2S2-, N2SO- or N3S-tetradentate semi-rigid
ligands have been synthesised and studied herein. By reacting the ligands with a slight excess of suitable [MO]3+
precursor (ReOCl3(PPh3)2 or [NBu4][99gTcOCl4]), the monoanionic complexes of general formula [MO(PhâXN2S)]â
could be easily produced in high yield. The complexes have been characterized by means of IR, electrospray mass
spectrometry, elemental analysis, NMR and conductimetry. The crystal structures of [PPh4][ReO(PhâON2S)] 1b and
[NBu4][99gTcO(PhâON2S)] 1c have been established. The [MO]3+ moiety was coordinated via the two deprotonated
amide nitrogens, the oxygen and the terminal sulfur atoms in 1b and 1c. In both compounds, the ON2S coordination
set is in the equatorial plane, and the complexes adopted a distorted square-pyramidal geometry with an axial
oxo-group. The chemical and structural identity of the different prototypic complexes (rhenium, 99gTc complexes and
their corresponding 99mTc radiocomplexes) have been also established by a comparative HPLC study
Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection
Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection
Abstract Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFkB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFkB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis
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