The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Winfried Weissenhorn - One of the best experts on this subject based on the ideXlab platform.
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ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and nedd4
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Scianimanico, T. Vernet, Rw Ruigrok, Sylvie Ricardblum, Winfried WeissenhornAbstract:Abstract The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
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Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Ricard-blum, S. Scianimanico, T. Vernet, Rw Ruigrok, Winfried WeissenhornAbstract:The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
Wei Xiao - One of the best experts on this subject based on the ideXlab platform.
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Uev1A, a ubiquitin conjugating Enzyme Variant, inhibits stress-induced apoptosis through NF-κB activation
Apoptosis : an international journal on programmed cell death, 2006Co-Authors: Noor A. Syed, Parker L. Andersen, Robert C. Warrington, Wei XiaoAbstract:We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating Enzyme Variant, which is required for Ubc13 (ubiquitin conjugating Enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKγ (nuclear factor-κB essential modulator/inhibitor of κB protein kinase), a regulatory subunit of IκB kinase in the NF-κB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-κB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-κB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.
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structure and interactions of the ubiquitin conjugating Enzyme Variant human uev1a implications for enzymatic synthesis of polyubiquitin chains
Biochemistry, 2006Co-Authors: D. Duong Hau, Michael J. Lewis, Linda F. Saltibus, Wei Xiao, Landon Pastushok, Leo SpyracopoulosAbstract:Lys(63)-linked polyubiquitination of TRAF2 or TRAF6 is an essential step within the signal transduction cascade responsible for activation of p38, c-Jun N-terminal kinase, and the transcription factor NF-kappaB. Attachment of ubiquitin (Ub) to a TRAF, and conjugation of Ub molecules to form a polyUb chain, is catalyzed by a heterodimer composed of a catalytically active E2 (hUbc13), involved in covalent bond transfer, and hUev1a, an E2-like protein involved in substrate Ub binding. Given the key biochemical processes in which hUev1a is involved, it is important to determine the molecular basis of the catalytic mechanism for Lys(63)-linked protein ubiquitination. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of hUev1a and its interactions with Ub and hUbc13. A structural model for the Ub-hUev1a-hUbc13-Ub tetramer was developed to gain chemical insight into the synthesis of Lys(63)-linked Ub chains. We propose that a network of hydrogen bonds involving hUbc13-Asp(81) and Ub-Glu(64) positions Ub-Lys(63) proximal to the active site. Interestingly, restrained molecular dynamics simulations in implicit solvent indicate that deprotonation of Ub-Lys(63) does not involve a general Asp or Glu base and may occur when the amino group approaches the thioester carbonyl carbon near the Burgi-Dunitz trajectory.
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Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2
Journal of biomolecular NMR, 2006Co-Authors: Michael J. Lewis, Linda F. Saltibus, D. Duong Hau, Wei Xiao, Leo SpyracopoulosAbstract:Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three Enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating Enzyme Variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.
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a homologue of croc 1 in a ciliated protist sterkiella histriomuscorum testifies to the ancient origin of the ubiquitin conjugating Enzyme Variant family
Molecular Biology and Evolution, 2002Co-Authors: Eduardo Villalobo, Wei Xiao, Loic Morin, Clara Moch, Rachel Lescasse, Michelle Hanna, Anne BarointourancheauAbstract:Resting cysts of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) have been shown to contain messenger RNA, one of which codes for a protein significantly similar to CROC-1. CROC-1 is a human regulatory protein capable of transactivating the promoter of c-fos and belongs to a newly characterized family of ubiquitin-conjugating Enzyme (E2) Variants (UEV). We have determined the corresponding macronuclear gene sequence, which is the first protistan UEV sequence available. The phylogenetic analysis indicates the deep separation and solid clustering of all the UEV sequences within the E2 tree showing the ancient origin of these regulatory genes and their high structural conservation during evolution. Furthermore, overexpression of the ciliate UEV is able to rescue the Saccharomyces cerevisiae mms2 null mutant from killing by DNA damaging agents, implying that the UEV family proteins are functionally conserved. In S. histriomuscorum, expression of UEV is correlated with the growth of the cells as transcripts are present in excysting and vegetative cells but are rapidly down-regulated during starvation. These data support the high conservation of the UEV family in eukaryotes, and a regulatory role of the gene is discussed in relation to known functions of UEVs. This analysis may promote the search for homologues of other regulatory genes (metazoan regulators of differentiation) in ciliates.
Joanna Timmins - One of the best experts on this subject based on the ideXlab platform.
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ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and nedd4
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Scianimanico, T. Vernet, Rw Ruigrok, Sylvie Ricardblum, Winfried WeissenhornAbstract:Abstract The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
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Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Ricard-blum, S. Scianimanico, T. Vernet, Rw Ruigrok, Winfried WeissenhornAbstract:The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin Enzyme Variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
Leo Spyracopoulos - One of the best experts on this subject based on the ideXlab platform.
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structure and interactions of the ubiquitin conjugating Enzyme Variant human uev1a implications for enzymatic synthesis of polyubiquitin chains
Biochemistry, 2006Co-Authors: D. Duong Hau, Michael J. Lewis, Linda F. Saltibus, Wei Xiao, Landon Pastushok, Leo SpyracopoulosAbstract:Lys(63)-linked polyubiquitination of TRAF2 or TRAF6 is an essential step within the signal transduction cascade responsible for activation of p38, c-Jun N-terminal kinase, and the transcription factor NF-kappaB. Attachment of ubiquitin (Ub) to a TRAF, and conjugation of Ub molecules to form a polyUb chain, is catalyzed by a heterodimer composed of a catalytically active E2 (hUbc13), involved in covalent bond transfer, and hUev1a, an E2-like protein involved in substrate Ub binding. Given the key biochemical processes in which hUev1a is involved, it is important to determine the molecular basis of the catalytic mechanism for Lys(63)-linked protein ubiquitination. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of hUev1a and its interactions with Ub and hUbc13. A structural model for the Ub-hUev1a-hUbc13-Ub tetramer was developed to gain chemical insight into the synthesis of Lys(63)-linked Ub chains. We propose that a network of hydrogen bonds involving hUbc13-Asp(81) and Ub-Glu(64) positions Ub-Lys(63) proximal to the active site. Interestingly, restrained molecular dynamics simulations in implicit solvent indicate that deprotonation of Ub-Lys(63) does not involve a general Asp or Glu base and may occur when the amino group approaches the thioester carbonyl carbon near the Burgi-Dunitz trajectory.
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Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2
Journal of biomolecular NMR, 2006Co-Authors: Michael J. Lewis, Linda F. Saltibus, D. Duong Hau, Wei Xiao, Leo SpyracopoulosAbstract:Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three Enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating Enzyme Variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.
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main chain and side chain dynamics of the ubiquitin conjugating Enzyme Variant human mms2 in the free and ubiquitin bound states
Biochemistry, 2005Co-Authors: Leo Spyracopoulos, Michael J. Lewis, Linda F. SaltibusAbstract:Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 Enzyme to an E2 Enzyme and finally to the protein target with the help of a Ub−protein ligase. Protein ubiquitination brought about by the Ubc13−Mms2 (E2−E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2−Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state 15N and 2H nuclear magnetic resonance relaxation measureme...
Kay Hofmann - One of the best experts on this subject based on the ideXlab platform.
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The GI-UEV domain, a catalytically inactive ubiquitin-conjugating Enzyme Variant with a role in translational regulation
Israel Journal of Chemistry, 2006Co-Authors: Hartmut Scheel, Kay HofmannAbstract:Ubiquitin-conjugating Enzymes (UBCs) form the second step in the Enzyme cascade required for protein ubiquitylation. Most eukaryotic genomes contain a multitude of different catalytically active UBCs. In addition, several proteins contain homology domains related to UBCs that have lost their catalytic activity and are referred to as "ubiquitin-conjugating Enzyme Variants" or UEV-domains. A common property of those domains is a role in ubiquitin binding and recognition. We report here on a novel class of more distantly related UEV domains, which forms a superset of the previously described GI-homology region found in Gcn2 and IMPACT proteins. In the Gcn2 and IMPACT protein families, the GI-UEV domain binds to proteins of the Gcnl family and thus regulates translation levels via eIF2α phosphorylation. Bioinformatical analysis shows that GI-UEV domains occur in a large number of proteins, many of them without an established role in translational regulation. Residue conservation and domain context predict that GI-UEV domains might also have a role in the ubiquitin/proteasome system, suggesting a possible cross-talk between ubiquitylation and translational regulation.
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Ubiquitin-binding proteins: similar, but different.
Essays in biochemistry, 2005Co-Authors: Katrine M. Andersen, Kay Hofmann, Rasmus Hartmann-petersenAbstract:Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases and phosphatases, specific sets of ubiquitinating/deubiquitinating Enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating Enzyme) Variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of ubiquitin-binding proteins.