162 research outputs found

    Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na<sup>+</sup>/K<sup>+</sup> transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

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    <div><p>Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na<sup>+</sup>/K<sup>+</sup> transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Jun├şn virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.</p></div

    SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy

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    <div><p>HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.</p></div

    The GAGA factor regulatory network: Identification of GAGA factor associated proteins

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    <div><p>The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.</p></div

    Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

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    <div><p>Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of <i>mps3-nc</i> in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.</p></div

    The IRE1╬▒/XBP1s Pathway Is Essential for the Glucose Response and Protection of ╬▓ Cells

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    <div><p>Although glucose uniquely stimulates proinsulin biosynthesis in ╬▓ cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1╬▒) to initiate X-box-binding protein 1 (<i>Xbp1</i>) mRNA splicing in adult primary ╬▓ cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional <i>Xbp1</i> mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated <i>Ire1╬▒</i> deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective ╬▓ cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1╬▒ degrades insulin mRNAs, <i>Ire1╬▒</i> deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, ╬▓ cell failure upon <i>Ire1╬▒</i> deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, <i>Ire1╬▒</i> deletion in ╬▓ cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with ╬▓ cell-specific <i>Ire1╬▒</i> deletion. The results demonstrate that glucose activates IRE1╬▒-mediated <i>Xbp1</i> splicing to expand the secretory capacity of the ╬▓ cell for increased proinsulin synthesis and to limit oxidative stress that leads to ╬▓ cell failure.</p></div

    Curation of the Mammalian Palmitoylome Indicates a Pivotal Role for Palmitoylation in Diseases and Disorders of the Nervous System and Cancers

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    <div><p>Palmitoylation involves the reversible posttranslational addition of palmitate to cysteines and promotes membrane binding and subcellular localization. Recent advancements in the detection and identification of palmitoylated proteins have led to multiple palmitoylation proteomics studies but these datasets are contained within large supplemental tables, making downstream analysis and data mining time-consuming and difficult. Consequently, we curated the data from 15 palmitoylation proteomics studies into one compendium containing 1,838 genes encoding palmitoylated proteins; representing approximately 10% of the genome. Enrichment analysis revealed highly significant enrichments for Gene Ontology biological processes, pathway maps, and process networks related to the nervous system. Strikingly, 41% of synaptic genes encode a palmitoylated protein in the compendium. The top disease associations included cancers and diseases and disorders of the nervous system, with Schizophrenia, HD, and pancreatic ductal carcinoma among the top five, suggesting that aberrant palmitoylation may play a pivotal role in the balance of cell death and survival. This compendium provides a much-needed resource for cell biologists and the palmitoylation field, providing new perspectives for cancer and neurodegeneration.</p></div

    Mass Spectrometry Data.Evf2++ Evf2TSTS

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    Mass spectrometry sequencing of DLX1 complexes isolated by affinity purification of mouse E13.5 embryonic brain ganglionic eminence nuclear extracts. Complexes are analyzed from Evf2+/+ (wildtype) and Evf2TSTS (lacking the Evf2 long noncoding RNA) mice, and shown in two Excel sheets

    Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

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    <div><p>The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.</p></div

    The FEAR Protein Slk19 Restricts Cdc14 Phosphatase to the Nucleus until the End of Anaphase, Regulating Its Participation in Mitotic Exit in <i>Saccharomyces cerevisiae</i>

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    <div><p>In <i>Saccharomyces cerevisiae</i> mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of <i>SLK19</i>, <i>slk19<sup>3R</sup></i> (three lysine-to-arginine mutations), strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to <i>slk19Δ</i> cells, <i>slk19<sup>3R</sup></i> cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike <i>slk19Δ</i>cells, however, <i>slk19<sup>3R</sup></i> cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, <i>slk19<sup>3R</sup></i> cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: <i>slk19<sup>3R</sup></i> causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that <i>slk19<sup>3R</sup></i> partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.</p></div

    The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HRÔÇôMediated Double-Strand Break Repair

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    <div><p>CtIP plays an important role in homologous recombination (HR)ÔÇômediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (<i>NBS</i>) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.</p> </div
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