This study aims to gain deeper insight into HEV-induced innate immunity by characterizing the crosstalk between the virus and the host factor guanylate-binding protein 1 (GBP1). We observe that the ...amount of GBP1 is elevated upon infection, although number of transcripts is decreased, which is explained by a prolonged protein half-life. Modulation of GBP1 levels via overexpression significantly inhibits the viral life cycle. Use of various GBP-1 mutants revealed that the antiviral effect of GBP-1 on HEV is independent from the GTPase-activity, but depends on the capacity of GBP-1 to form GBP1 homodimers. This connects GBP-1 to the autophagosomal pathway. Indeed, dimerization competent GBP1 targets the viral capsid protein to the lysosomal compartment leading to inactivation of the viral particle. Most importantly, silencing of GBP1 abolishes the antiviral effect of IFNγ on HEV. In IFNγ treated cells the virus is targeted to lysosomal structures and destroyed therein. This process depends in part on GBP1. These observations about the relevance of GBP1 for type II interferon-mediated innate immunity against HEV could be a base for tailoring novel antivirals and improvement of disease management.
Although HEV represents a worldwide public health problem with 20 million infections and 44.000 death cases per year, there are still no specific antivirals available and many aspects of the viral life cycle are not well understood. Here we identify the guanylate binding protein 1 (GBP1) as a restriction factor affecting life cycle of HEV. Surprisingly, the antiviral effect of GBP1 does not depend on its GTPase function, but on its capacity to homodimerize. We revealed that GBP1 exerts its antiviral activity by targeting HEV to the lysosomal compartment where the virus is inactivated. Most importantly, we observed that the antiviral effect of interferon-γ on HEV strongly depends on GBP1. Our observation that GBP1 impairs HEV and is crucial for the antiviral effect of interferons on HEV extends understanding of host defense-mechanisms. As the interferon-system represents a universal defense-mechanism, our study could help to design novel antivirals targeting.
Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-γ-inducible human guanylate binding protein 1 (hGBP1) belongs to the family ...of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the β-phosphates in the γ-phosphate-binding site.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Guanylate-binding proteins (GBP) are a family of dynamin-related large GTPases which are expressed in response to interferons and other pro-inflammatory cytokines. GBPs mediate a broad spectrum of ...innate immune functions against intracellular pathogens ranging from viruses to bacteria and protozoa. Several binding partners for individual GBPs have been identified and several different mechanisms of action have been proposed depending on the organisms, the cell type and the pathogen used. Many of these anti-pathogenic functions of GBPs involve the recruitment to and the subsequent destruction of pathogen containing vacuolar compartments, the assembly of large oligomeric innate immune complexes such as the inflammasome, or the induction of autophagy. Furthermore, GBPs often cooperate with immunity-related GTPases (IRGs), another family of dynamin-related GTPases, to exert their anti-pathogenic function, but since most IRGs have been lost in the evolution of higher primates, the anti-pathogenic function of human GBPs seems to be IRG-independent. GBPs and IRGs share biochemical and structural properties with the other members of the dynamin superfamily such as low nucleotide affinity and a high intrinsic GTPase activity which can be further enhanced by oligomerisation. Furthermore, GBPs and IRGs can interact with lipid membranes. In the case of three human and murine GBP isoforms this interaction is mediated by C-terminal isoprenylation. Based on cell biological studies, and in analogy to the function of other dynamins in membrane scission events, it has been postulated that both GBPs and IRGs might actively disrupt the outer membrane of pathogen-containing vacuole leading to the detection and destruction of the pathogen by the cytosolic innate immune system of the host. Recent evidence, however, indicates that GBPs might rather function by mediating membrane tethering events similar to the dynamin-related atlastin and mitofusin proteins, which mediate fusion of the ER and mitochondria, respectively. The aim of this review is to highlight the current knowledge on the function of GBPs in innate immunity and to combine it with the recent progress in the biochemical characterisation of this protein family.
Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical ...relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.
Dynamins are large GTPases that belong to a protein superfamily that, in eukaryotic cells, includes classical dynamins, dynamin-like proteins, OPA1, Mx proteins, mitofusins and guanylate-binding ...proteins/atlastins. They are involved in many processes including budding of transport vesicles, division of organelles, cytokinesis and pathogen resistance. With sequenced genomes from Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, yeast species and Arabidopsis thaliana, we now have a complete picture of the members of the dynamin superfamily from different organisms. Here, we review the superfamily of dynamins and their related proteins, and propose that a common mechanism leading to membrane tubulation and/or fission could encompass their many varied functions.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
7.
SUMO playing tag with ubiquitin Praefcke, Gerrit J.K.; Hofmann, Kay; Dohmen, R. Jürgen
Trends in biochemical sciences (Amsterdam. Regular ed.),
2012, 2012-Jan, 2012-1-00, 20120101, Letnik:
37, Številka:
1
Journal Article
Recenzirano
In addition to being structurally related, the protein modifiers ubiquitin and SUMO (small ubiquitin-related modifier), share a multitude of functional interrelations. These include the targeting of ...the same attachment sites in certain substrates, and SUMO-dependent ubiquitylation in others. Notably, several cellular processes, including the targeting of repair machinery to DNA damage sites, require the sequential sumoylation and ubiquitylation of distinct substrates. Some proteins promote both modifications. By contrast, the activity of some enzymes that control either sumoylation or ubiquitylation is regulated by the respective other modification. In this review, we summarize recent findings regarding intersections between SUMO and ubiquitin that influence genome stability and cell growth and which are relevant in pathogen resistance and cancer treatment.
Modification with SUMO regulates many eukaryotic proteins. Down-regulation of sumoylated forms of proteins involves either their desumoylation, and hence recycling of the unmodified form, or their ...proteolytic targeting by ubiquitin ligases that recognize their SUMO modification (termed STUbL or ULS). STUbL enzymes such as Uls1 and Slx5-Slx8 in budding yeast or RNF4 and Arkadia/RNF111 in humans bear multiple SUMO interaction motifs to recognize substrates carrying poly-SUMO chains. Using yeast as experimental system and isothermal titration calorimetry, we here show that Arkadia specifically selects substrates carrying SUMO1-capped SUMO2/3 hybrid conjugates and targets them for proteasomal degradation. Our data suggest that a SUMO1-specific binding site in Arkadia with sequence similarity to a SUMO1-binding site in DPP9 is required for targeting endogenous hybrid SUMO conjugates and PML nuclear bodies in human cells. We thus characterize Arkadia as a STUbL with a preference for substrate proteins marked with distinct hybrid SUMO chains.
Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding ...of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of ...beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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