The Experts below are selected from a list of 188088 Experts worldwide ranked by ideXlab platform
Cecile M Pickart - One of the best experts on this subject based on the ideXlab platform.
-
Molecular Insights into PolyUbiquitin Chain Assembly: Crystal Structure of the Mms2/Ubc13 Heterodimer
Cell, 2001Co-Authors: Andrew P. Vandemark, Cecile M Pickart, Roseanne M. Hofmann, Colleen Tsui, Cynthia WolbergerAbstract:While the signaling properties of Ubiquitin depend on the topology of polyUbiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyUbiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate Ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyUbiquitin signals.
-
molecular insights into polyUbiquitin chain assembly crystal structure of the mms2 ubc13 heterodimer
Cell, 2001Co-Authors: Andrew P. Vandemark, Cecile M Pickart, Roseanne M. Hofmann, Colleen Tsui, Cynthia WolbergerAbstract:While the signaling properties of Ubiquitin depend on the topology of polyUbiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyUbiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate Ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyUbiquitin signals.
-
Crystal structure of the human Ubiquitin-like protein NEDD8 and interactions with Ubiquitin pathway enzymes.
The Journal of biological chemistry, 1998Co-Authors: Frank G. Whitby, Cecile M Pickart, Gang Xia, Christopher P. HillAbstract:The NEDD8/Rub1 class of Ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of Ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for Ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the Ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the Ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyUbiquitin chain with an efficiency similar to that of Ubiquitin. A chimeric tetramer composed of three Ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraUbiquitin. Seven residues that differ from the corresponding residues in Ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in Ubiquitin), is shown to perform a key role in selecting against reaction with the Ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into Ubiquitin-specific pathways.
-
INHIBITION OF THE 26 S PROTEASOME BY POLYUbiquitin CHAINS SYNTHESIZED TO HAVE DEFINED LENGTHS
The Journal of biological chemistry, 1997Co-Authors: Julia S. Piotrowski, Keith D. Wilkinson, Robert E. Cohen, Richard K. Beal, Laura M. Hoffman, Cecile M PickartAbstract:Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple Ubiquitins can be ligated together through the formation of isopeptide bonds between Lys48 and Gly76 of successive Ubiquitins. Such a polyUbiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyUbiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys48-linked polyUbiquitin chains of defined length. In the first round of synthesis, two Ubiquitin derivatives (K48C-Ubiquitin and Asp77-Ubiquitin) were used as substrates for the well characterized Ubiquitin-conjugating enzyme E2-25K. DiUbiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyUbiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyUbiquitin chains as inhibitors of Ubiquitin-conjugate degradation. K0.5 was found to decrease approximately 90-fold, from 430 to 4.8 microM, as the chain was lengthened from two to eight Ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.
-
inhibition of the 26 s proteasome by polyUbiquitin chains synthesized to have defined lengths
Journal of Biological Chemistry, 1997Co-Authors: Julia S. Piotrowski, Keith D. Wilkinson, Robert E. Cohen, Richard K. Beal, Laura M. Hoffman, Cecile M PickartAbstract:Abstract Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple Ubiquitins can be ligated together through the formation of isopeptide bonds between Lys48 and Gly76 of successive Ubiquitins. Such a polyUbiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyUbiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys48-linked polyUbiquitin chains of defined length. In the first round of synthesis, two Ubiquitin derivatives (K48C-Ubiquitin and Asp77-Ubiquitin) were used as substrates for the well characterized Ubiquitin-conjugating enzyme E2-25K. DiUbiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyUbiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyUbiquitin chains as inhibitors of Ubiquitin-conjugate degradation.K 0.5 was found to decrease ∼90-fold, from 430 to 4.8 μm, as the chain was lengthened from two to eight Ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.
David Fushman - One of the best experts on this subject based on the ideXlab platform.
-
Nonenzymatic assembly of branched polyUbiquitin chains for structural and biochemical studies.
Bioorganic & medicinal chemistry, 2013Co-Authors: Emma K. Dixon, Carlos A Castaneda, Tanuja R. Kashyap, Yan Wang, David FushmanAbstract:Polymeric chains of a small protein Ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyUbiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific Ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per Ubiquitin is conjugated to another Ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyUbiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one Ubiquitin and a specific ε-amine on another Ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the Ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-Ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal Ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-Ubiquitins as well as control di-Ubiquitins by the yeast proteasome-associated deUbiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyUbiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyUbiquitin, these developments now provide tools for making fully natural polyUbiquitin chains of essentially any type of linkage and length.
-
Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System
Molecular & cellular proteomics : MCP, 2012Co-Authors: Rajesh Singh, Sylvia Zerath, Oded Kleifeld, Martin Scheffner, Michael H. Glickman, David FushmanAbstract:Of all Ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of Ubiquitin. We show via NMR that structurally, Rub1 and Ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of Ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and Ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or Ubiquitin or both, we examined whether Ubiquitin receptors can differentiate between Rub1 and Ubiquitin. Surprisingly, Rub1 interacts with proteasome Ubiquitin-shuttle proteins comparably to Ubiquitin but binds more weakly to a proteasomal Ubiquitin receptor Rpn10. We identified Rub1-Ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with Ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-Ubiquitin heterodimers are recognized by various proteasomal Ubiquitin shuttles and receptors comparably to K48-linked Ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deUbiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into Ubiquitin pathways, it is recognized essentially like Ubiquitin.
-
nonenzymatic assembly of natural polyUbiquitin chains of any linkage composition and isotopic labeling scheme
Journal of the American Chemical Society, 2011Co-Authors: Carlos A Castaneda, Jia Liu, Apurva Chaturvedi, Urszula Nowicka, Ashton T Cropp, David FushmanAbstract:Polymeric chains made of a small protein Ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyUbiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the Ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyUbiquitin chains using recombinant Ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one Ubiquitin and the e-amine of a specific lysine on the other Ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed Ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyUbiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-Ubiquitins, Lys33-linked diUbiquitin, and a mixed-linkage Lys33,Lys11-linked triUbiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on Ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyUbiquitin signals.
-
affinity makes the difference nonselective interaction of the uba domain of ubiquilin 1 with monomeric Ubiquitin and polyUbiquitin chains
Journal of Molecular Biology, 2008Co-Authors: Daoning Zhang, Shahri Raasi, David FushmanAbstract:Abstract Ubiquilin/PLIC proteins belong to the family of UBL–UBA proteins implicated in the regulation of the Ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyUbiquitins are mediated by its UBA domain, which is one of the tightest Ubiquitin binders among known Ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with Ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on Ubiquitin with a K d of 20 μM. To gain structural insights into UQ1-UBA's interactions with polyUbiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-Ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual Ubiquitin units in both chains in a mode similar to its interaction with mono-Ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two Ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyUbiquitin chain recognition by UBA domains.
Kylie J Walters - One of the best experts on this subject based on the ideXlab platform.
-
myosin vi contains a compact structural motif that binds to Ubiquitin chains
Cell Reports, 2016Co-Authors: Hanspeter Wollscheid, Urszula Nowicka, Matteo Biancospino, Eleonora Valentini, Aaron Ehlinger, Filippo Acconcia, Elisa Magistrati, Simona Polo, Kylie J WaltersAbstract:Summary Myosin VI is critical for cargo trafficking and sorting during early endocytosis and autophagosome maturation, and abnormalities in these processes are linked to cancers, neurodegeneration, deafness, and hypertropic cardiomyopathy. We identify a structured domain in myosin VI, myosin VI Ubiquitin-binding domain (MyUb), that binds to Ubiquitin chains, especially those linked via K63, K11, and K29. Herein, we solve the solution structure of MyUb and MyUb:K63-linked diUbiquitin. MyUb folds as a compact helix-turn-helix-like motif and nestles between the Ubiquitins of K63-linked diUbiquitin, interacting with distinct surfaces of each. A nine-amino-acid extension at the C-terminal helix (Helix2) of MyUb is required for myosin VI interaction with endocytic and autophagic adaptors. Structure-guided mutations revealed that a functional MyUb is necessary for optineurin interaction. In addition, we found that an isoform-specific helix restricts MyUb binding to Ubiquitin chains. This work provides fundamental insights into myosin VI interaction with Ubiquitinated cargo and functional adaptors.
-
Ubiquitin and its binding domains.
Frontiers in bioscience (Landmark edition), 2012Co-Authors: Leah Randles, Kylie J WaltersAbstract:Post-translational modification by Ubiquitin (Ubiquitination, ubiquitylation, Ubiquitinylation) is used as a robust signaling mechanism in a variety of processes that are essential for cell homeostasis. Its signaling specificity is conferred by the inherent dynamics of Ubiquitin, the multivalency of Ubiquitin chains, and its subcellular context, often defined by Ubiquitin receptors and the substrate. Greater than 150 Ubiquitin receptors have been found and their Ubiquitin-binding domains (UBDs) are structurally diverse and include alpha-helical motifs, zinc fingers (ZnF), pleckstrin-homology (PH) domains, Ubiquitin conjugating (Ubc)-related structures and src homology 3 (SH3) domains. New UBD structural motifs continue to be identified expanding the Ubiquitin-signaling map to proteins and structural families not previously associated with Ubiquitin trafficking. In this manuscript, we highlight several Ubiquitin receptors from the multiple UBD folds with a focus on the structural characteristics of their interaction with Ubiquitin.
-
Ubiquitin binding domains from structures to functions
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Soichi Wakatsuki, Ivan Dikic, Kylie J WaltersAbstract:Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the protein modifier Ubiquitin. Recent atomic-level resolution structures of Ubiquitin-UBD complexes have revealed some of the mechanisms that underlie the versatile functions of Ubiquitin in vivo. The preferences of UBDs for Ubiquitin chains of specific length and linkage are central to these functions. These preferences originate from multimeric interactions, whereby UBDs synergistically bind multiple Ubiquitin molecules, and from contacts with regions that link Ubiquitin molecules into a polymer. The sequence context of UBDs and the conformational changes that follow their binding to Ubiquitin also contribute to Ubiquitin signalling. These new structure-based insights provide strategies for controlling cellular processes by targeting Ubiquitin-UBD interfaces.
Jean-pierre Julien - One of the best experts on this subject based on the ideXlab platform.
-
Neuronal Expression of UBQLN2^P497H Exacerbates TDP-43 Pathology in TDP-43^G348C Mice through Interaction with Ubiquitin
Molecular Neurobiology, 2019Co-Authors: Vincent Picher-martel, Laurence Renaud, Christine Bareil, Jean-pierre JulienAbstract:Mutations in the gene encoding ubiquilin-2 (UBQLN2) are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). UBQLN2 plays a central role in Ubiquitin proteasome system (UPS) and UBQLN2 up-regulation exacerbates TDP-43 cytoplasmic aggregates. To analyze interaction between UBQLN2 and TDP-43 and to produce a relevant ALS animal model, we have generated a new transgenic mouse expressing UBQLN2^P497H under the neurofilament heavy (NFH) gene promoter. The UBQLN2^P497H mice were then bred with our previously described TDP-43^G348C mice to generate double-transgenic UBQLN2^P497H; TDP-43^G348C mice. With low-expression levels of UBQLN2, the double-transgenic mice developed TDP-43 cytosolic accumulations in motor neurons starting at 5 months of age. These double-transgenic mice exhibited motor neuron loss, muscle atrophy, as well as motor and cognitive deficits during aging. The microglia from double-transgenic mice were hyperresponsive to intraperitoneal injection of lipopolysaccharide (LPS). In vivo and in vitro analyses suggested that extra UBQLN2 proteins can exacerbate cytoplasmic TDP-43 accumulations by competing with the UPS for binding to Ubiquitin. Thus, increasing the pool of Ubiquitin promoted the UPS function with ensuing reduction of TDP-43 cytosolic accumulations. In conclusion, the double-transgenic UBQLN2^P497H; TDP-43^G348C mice provides a unique mouse model of ALS/FTD with enhanced TDP-43 pathology that can be exploited for drug testing.
-
Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells
Molecular Brain, 2015Co-Authors: Vincent Picher-martel, Kallol Dutta, Daniel Phaneuf, Gen Sobue, Jean-pierre JulienAbstract:Background Mutations in the gene encoding Ubiquilin-2 (UBQLN2) are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). UBQLN2 plays a central role in Ubiquitin proteasome system (UPS) and UBQLN2 mutants can form cytoplasmic aggregates in vitro and in vivo. Results Here, we report that overexpression of WT or mutant UBQLN2 species enhanced nuclear factor κB (NF-κB) activation in Neuro2A cells. The inhibition of NF-κB stress-mediated activation with SB203580, a p38 MAPK inhibitor, demonstrated a role for MAPK in NF-κB activation by UBQLN2 species. Live cell imaging and microscopy showed that UBQLN2 aggregates are dynamic structures that promote cytoplasmic accumulation of TAR DNA-binding protein (TDP-43), a major component of ALS inclusion bodies. Furthermore, up-regulation of UBQLN2 species in neurons caused an ER-stress response and increased their vulnerability to death by toxic mediator TNF-α. Withaferin A, a known NF-κB inhibitor, reduced mortality of Neuro2A cells overexpressing UBQLN2 species. Conclusions These results suggest that UBQLN2 dysregulation in neurons can drive NF-κB activation and cytosolic TDP-43 aggregation, supporting the concept of pathway convergence in ALS pathogenesis. These Ubiquilin-2 pathogenic pathways might represent suitable therapeutic targets for future ALS treatment.
Nicole Burton - One of the best experts on this subject based on the ideXlab platform.
-
linkage specific Ubiquitin chain formation depends on a lysine hydrocarbon ruler
Nature Chemical Biology, 2021Co-Authors: Joanna Liwocha, David T. Krist, Fynn M. Hansen, Vinh H. Truong, Ozge Karayel, Nicholas Purser, Daniel Houston, Gerbrand J Van Der Heden Van Noort, Nicole BurtonAbstract:Virtually all aspects of cell biology are regulated by a Ubiquitin code where distinct Ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor Ubiquitin and a specific nucleophilic amino acid on the acceptor Ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic Ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the Ubiquitin code, through unanticipated and complex reliance of many distinct Ubiquitin-carrying enzymes on a canonical acceptor lysine.
-
Linkage-specific Ubiquitin chain formation depends on a lysine hydrocarbon ruler
Nature Chemical Biology, 2020Co-Authors: Joanna Liwocha, David T. Krist, Gerbrand J. Heden Van Noort, Fynn M. Hansen, Vinh H. Truong, Ozge Karayel, Nicholas Purser, Daniel Houston, Nicole Burton, Mark J. BostockAbstract:Virtually all aspects of cell biology are regulated by a Ubiquitin code where distinct Ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor Ubiquitin and a specific nucleophilic amino acid on the acceptor Ubiquitin, yet it is unknown whether the fundamental feature of most acceptors—the lysine side chain—affects catalysis. Here, use of synthetic Ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the Ubiquitin code, through unanticipated and complex reliance of many distinct Ubiquitin-carrying enzymes on a canonical acceptor lysine. Using synthetic Ubiquitins with non-natural acceptor site, the authors revealed that the length of lysine side chain in acceptor Ubiquitins affects Ubiquitin chain linkage specificity with native lysine as the preferred geometry.
