The Experts below are selected from a list of 12810 Experts worldwide ranked by ideXlab platform
Cecile M. Pickart - One of the best experts on this subject based on the ideXlab platform.
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mms2 ubc13 covalently bound to ubiquitin reveals the structural basis of linkage specific Polyubiquitin chain formation
Nature Structural & Molecular Biology, 2006Co-Authors: Michael J Eddins, Cecile M. Pickart, Candice M Carlile, Kamila M Gomez, Cynthia WolbergerAbstract:Lys63-linked Polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-κB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked Polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2–Ubc13-ubiquitin (UEV–E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2–Ubc13-Ub complex to the acceptor-binding site of Mms2–Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for Polyubiquitin chain elongation.
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different hect domain ubiquitin ligases employ distinct mechanisms of Polyubiquitin chain synthesis
The EMBO Journal, 2005Co-Authors: Min Wang, Cecile M. PickartAbstract:Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with Polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of Polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a Polyubiquitin chain and the mechanism of its assembly.
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Diverse Polyubiquitin interaction properties of ubiquitin-associated domains.
Nature structural & molecular biology, 2005Co-Authors: Shahri Raasi, David Fushman, Ranjani Varadan, Cecile M. PickartAbstract:The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although Polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the Polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective Polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class (∼30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.
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Binding of Polyubiquitin Chains to Ubiquitin-associated (UBA) Domains of HHR23A
Journal of molecular biology, 2004Co-Authors: Shahri Raasi, Irina Orlov, Karen G. Fleming, Cecile M. PickartAbstract:Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/Polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/Polyubiquitin complexes, even though Polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked Polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates Polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into Polyubiquitin chain recognition and set the stage for future structural investigations of UBA/Polyubiquitin complexes.
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binding of Polyubiquitin chains to ubiquitin associated uba domains of hhr23a
Journal of Molecular Biology, 2004Co-Authors: Shahri Raasi, Irina Orlov, Karen G. Fleming, Cecile M. PickartAbstract:Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/Polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/Polyubiquitin complexes, even though Polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked Polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates Polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of nZ4‐6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into Polyubiquitin chain recognition and set the stage for
Zhijian J. Chen - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of the rig i pathway reveals a signaling role of unanchored Polyubiquitin chains in innate immunity
Cell, 2010Co-Authors: Wenwen Zeng, Lijun Sun, Anirban Adhikari, Xiang Chen, Xiaomo Jiang, Fajian Hou, Zhijian J. ChenAbstract:RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also Polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-Polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-Polyubiquitin chains are signaling molecules in antiviral innate immunity.
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Direct activation of protein kinases by unanchored Polyubiquitin chains
Nature, 2009Co-Authors: Zongping Xia, Lijun Sun, Anirban Adhikari, Xiang Chen, Gabriel Pineda, Xiaomo Jiang, Wenwen Zeng, Zhijian J. ChenAbstract:TRAF6 is a ubiquitin ligase that is essential for the activation of NF-kappaB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of UBC13 (also known as UBE2N) and UEV1A (UBE2V1) to catalyse Lys 63-linked Polyubiquitination, which activates the TAK1 (also known as MAP3K7) kinase complex. TAK1 in turn phosphorylates and activates IkappaB kinase (IKK), leading to the activation of NF-kappaB. Although several proteins are known to be Polyubiquitinated in the IL1R and Toll-like receptor pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. By reconstituting TAK1 activation in vitro using purified proteins, here we show that free Lys 63 Polyubiquitin chains, which are not conjugated to any target protein, directly activate TAK1 by binding to the ubiquitin receptor TAB2 (also known as MAP3K7IP2). This binding leads to autophosphorylation and activation of TAK1. Furthermore, we found that unanchored Polyubiquitin chains synthesized by TRAF6 and UBCH5C (also known as UBE2D3) activate the IKK complex. Disassembly of the Polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored Polyubiquitin chains directly activate TAK1 and IKK, suggesting a new mechanism of protein kinase regulation.
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Diversity of Polyubiquitin chains.
Developmental cell, 2009Co-Authors: Anirban Adhikari, Zhijian J. ChenAbstract:Polyubiquitin chains linked through different lysines of ubiquitin may exert both proteasome-dependent and -independent functions. In a recent Cell issue, Xu et al. employ quantitative proteomics to profile Polyubiquitin linkages in yeast. They find that linkages through all lysines of ubiquitin, except lysine-63, can target proteasomal degradation in vivo, and that lysine-11 Polyubiquitination is important for endoplasmic reticulum-associated degradation (ERAD).
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TAB2 and TAB3 Activate the NF-κB Pathway through Binding to Polyubiquitin Chains
Molecular cell, 2004Co-Authors: Atsuhiro Kanayama, Rashu B. Seth, Lijun Sun, Mei Hong, Abdullah Shaito, Yu Hsin Chiu, Li Deng, Zhijian J. ChenAbstract:The activation of NF-kappaB and IKK requires an upstream kinase complex consisting of TAK1 and adaptor proteins such as TAB1, TAB2, or TAB3. TAK1 is in turn activated by TRAF6, a RING domain ubiquitin ligase that facilitates the synthesis of lysine 63-linked Polyubiquitin chains. Here we present evidence that TAB2 and TAB3 are receptors that bind preferentially to lysine 63-linked Polyubiquitin chains through a highly conserved zinc finger (ZnF) domain. Mutations of the ZnF domain abolish the ability of TAB2 and TAB3 to bind Polyubiquitin chains, as well as their ability to activate TAK1 and IKK. Significantly, replacement of the ZnF domain with a heterologous ubiquitin binding domain restored the ability of TAB2 and TAB3 to activate TAK1 and IKK. We also show that TAB2 binds to Polyubiquitinated RIP following TNFalpha stimulation. These results indicate that Polyubiquitin binding domains represent a new class of signaling domains that regulate protein kinase activity through a nonproteolytic mechanism.
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activation of the iκb kinase complex by traf6 requires a dimeric ubiquitin conjugating enzyme complex and a unique Polyubiquitin chain
Cell, 2000Co-Authors: Li Deng, Cecile M. Pickart, Chen Wang, Erika Spencer, Liyong Yang, Amy Braun, Jianxin You, Clive A Slaughter, Zhijian J. ChenAbstract:TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique Polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this Polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked Polyubiquitin chains.
Shahri Raasi - One of the best experts on this subject based on the ideXlab platform.
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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.
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Diverse Polyubiquitin interaction properties of ubiquitin-associated domains.
Nature structural & molecular biology, 2005Co-Authors: Shahri Raasi, David Fushman, Ranjani Varadan, Cecile M. PickartAbstract:The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although Polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the Polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective Polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class (∼30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.
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Binding of Polyubiquitin Chains to Ubiquitin-associated (UBA) Domains of HHR23A
Journal of molecular biology, 2004Co-Authors: Shahri Raasi, Irina Orlov, Karen G. Fleming, Cecile M. PickartAbstract:Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/Polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/Polyubiquitin complexes, even though Polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked Polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates Polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into Polyubiquitin chain recognition and set the stage for future structural investigations of UBA/Polyubiquitin complexes.
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binding of Polyubiquitin chains to ubiquitin associated uba domains of hhr23a
Journal of Molecular Biology, 2004Co-Authors: Shahri Raasi, Irina Orlov, Karen G. Fleming, Cecile M. PickartAbstract:Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/Polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/Polyubiquitin complexes, even though Polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked Polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates Polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of nZ4‐6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into Polyubiquitin chain recognition and set the stage for
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rad23 ubiquitin associated domains uba inhibit 26 s proteasome catalyzed proteolysis by sequestering lysine 48 linked Polyubiquitin chains
Journal of Biological Chemistry, 2003Co-Authors: Shahri Raasi, Cecile M. PickartAbstract:Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked Polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind Polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle Polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of Polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked Polyubiquitin chains. The UBA domains bind Lys48-linked Polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.
David Fushman - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk between Lys63- and Lys11-Polyubiquitin signaling at DNA damage sites is driven by Cezanne
Genes & development, 2019Co-Authors: Shichang Liu, Tanuja R. Kashyap, David Fushman, Cari A. Sagum, Jianji Chen, Rajesh Singh, Apurva Chaturvedi, John R. Horton, Xiaodong ChengAbstract:The establishment of Polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-Polyubiquitin and targeting Lys11-Polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked Polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage Polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.
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Unique Structural, Dynamical, and Functional Properties of K11-Linked Polyubiquitin Chains
Structure (London England : 1993), 2013Co-Authors: Carlos A. Castañeda, Tanuja R. Kashyap, Mark A. Nakasone, Susan Krueger, David FushmanAbstract:K11-linked Polyubiquitin chains play important signaling and regulatory roles in both degradative and nonproteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other Polyubiquitins, we determined solution structures of K11-linked diubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and nonproteasomal pathways but with intermediate affinity and different binding modes than either K48-linked or K63-linked diubiquitin. Our data support the hypothesis that Polyubiquitin chains of different linkages possess unique conformational and dynamical properties, allowing them to be recognized differently by downstream receptor proteins.
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Nonenzymatic assembly of branched Polyubiquitin chains for structural and biochemical studies.
Bioorganic & medicinal chemistry, 2013Co-Authors: Emma K. Dixon, Carlos A. Castañeda, 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.
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Effect of Different Lysine Linkages on Polyubiquitin Chain Structure and Function
Biophysical Journal, 2013Co-Authors: Carlos A. Castañeda, Susan Krueger, Apurva Chaturvedi, Emma Dixon, T. Ashton Cropp, David FushmanAbstract:Polyubiquitination is a critical post-translational modification of proteins. Polyubiquitination signals for a wide variety of cellular events including proteasomal degradation, DNA repair, cell cycle regulation, T-cell activation, etc. The molecular basis of the diversity of Polyubiquitin signaling lies in the different structural and dynamical properties of Polyubiquitin chains, linked between the C-terminus of one ubiquitin (Ub) and the epsilon-amine of a lysine side chain (K6, K11, K27, K29, K33, K48 or K63) on a second Ub. The canonical K48-linked and K63-linked chains have been well characterized with biophysical techniques. Owing to a lack of linkage-specific Ub-conjugating enzymes, little is known about the structural or functional properties of the other Polyubiquitin chains. We devised a chemical ligation method to assemble Polyubiquitin chains comprised of every lysine linkage, of specific length, and with selective isotopic labeling. Solution NMR measurements, including chemical shift perturbations, 15N relaxation measurements and residual dipolar couplings, in conjunction with small angle neutron scattering (SANS) measurements, have enabled us to describe the structural and dynamical properties of free Polyubiquitin chains of every lysine linkage for the first time. We observed that a few of these chains (particularly K6-linked chains and K11-linked chains at high salt) can adopt compact conformations. Furthermore, binding studies demonstrated that K11-linked chains bind to Ub receptors with differential affinities than either K48-linked or K63-linked chains. In any case, these studies highlight the importance of determining the conformational ensemble of each of these Polyubiquitin chains. Our analyses will provide a foundation for future work with Polyubiquitin chains of any desirable length and linkage composition.
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Exploring the Linkage Dependence of Polyubiquitin Conformations Using Molecular Modeling
Journal of molecular biology, 2009Co-Authors: David Fushman, Olivier WalkerAbstract:Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called Polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different Polyubiquitin signals are recognized is central to understanding the specificity of various types of Polyubiquitination. In Polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of Polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of Polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called "noncanonical" chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub(2)). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked Polyubiquitin. Our results show that the eight possible Ub(2) chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub(2) chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical Polyubiquitin signals by various receptors are discussed.
Robert Layfield - One of the best experts on this subject based on the ideXlab platform.
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Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Daniel Scott, Jo Strachan, David Tooth, Mark S. Searle, Neil J. Oldham, Barry Shaw, Varun Gopala Krishna, Robert LayfieldAbstract:Unanchored Polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored Polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored Polyubiquitin chains in purified fractions.
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Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored Polyubiquitin.
Proteomics, 2016Co-Authors: Daniel Scott, Thomas P. Garner, Jed Long, Jo Strachan, Sharad Mistry, Andrew R. Bottrill, David Tooth, Mark S. Searle, Neil J. Oldham, Robert LayfieldAbstract:Unanchored Polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated Polyubiquitin. However tools able to discriminate unanchored Polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked Polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked Polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of Polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored Polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).
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Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains
Journal of proteome research, 2012Co-Authors: Joanna Strachan, Thomas P. Garner, Jed Long, David Tooth, Mark S. Searle, Neil J. Oldham, Lucy V. Roach, Kleitos Sokratous, Robert LayfieldAbstract:The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) Polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored Polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored Polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous Polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored Polyubiquitin chains, this work also represents the first definition of Polyubiquitin chain length in vivo.
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Diverse Polyubiquitin chains accumulate following 26S proteasomal dysfunction in mammalian neurones.
Neuroscience letters, 2011Co-Authors: Lynn Bedford, Robert Layfield, R. John Mayer, Junmin PengAbstract:A generality has been that Polyubiquitin chain linkage can differentially address proteins for various physiological processes. 26S proteasomal degradation is the most established function of ubiquitin signalling, classically linked to Lys48 Polyubiquitin chains. The other well-characterised Polyubiquitin linkage, via Lys63, mediates nonproteolytic functions. However, there are five other lysine residues and ubiquitin's amino terminus which can participate in Polyubiquitination. Our 26S proteasome knockout mouse provides a unique opportunity to comprehensively investigate the ubiquitin signals in their physiological context in neurones following genetic inhibition of the proteasome, using quantitative mass spectrometry of ubiquitin linkage-specific signature peptides. We provide the first evidence for diverse Polyubiquitin chains in mammalian neurones in vivo and show that Polyubiquitin linked via Lys6, Lys11, Lys29 and Lys48, but not Lys63, accumulates upon 26S proteasome dysfunction. This adaptable nature of ubiquitin signals for proteasomal targeting could reflect the extensive cellular processes which are regulated by proteasome proteolysis and/or may involve specific ubiquitin linkage preferences for subsets of proteins in mammalian neurones. Our molecular pathological findings make a significant contribution to the understanding of ubiquitin signalling in ubiquitin-proteasome function.