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Sample records for enzyme ubiquitination state

  1. UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.

    PubMed

    Hans, Friederike; Fiesel, Fabienne C; Strong, Jennifer C; Jäckel, Sandra; Rasse, Tobias M; Geisler, Sven; Springer, Wolfdieter; Schulz, Jörg B; Voigt, Aaron; Kahle, Philipp J

    2014-07-01

    Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration. PMID:24825905

  2. Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme~Ubiquitin Conjugate

    SciTech Connect

    Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.; Brzovic, Peter S.; Klevit, Rachel E.

    2011-03-15

    Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c~Ub conjugate populates an array of extended conformations, and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. Finally, we propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly with regard to interaction with Ub ligases.

  3. Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

    PubMed

    Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

    2016-08-23

    Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists. PMID:27501351

  4. Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

    SciTech Connect

    Ke Qingdong; Ellen, Thomas P.; Costa, Max

    2008-04-15

    Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis.

  5. E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

    PubMed Central

    St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

    2014-01-01

    Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

  6. In silico analysis and developmental expression of ubiquitin-conjugating enzymes in Schistosoma mansoni.

    PubMed

    Costa, Marcela P; Oliveira, Victor F; Pereira, Roberta V; de Abreu, Fabiano C P; Jannotti-Passos, Liana K; Borges, William C; Guerra-Sá, Renata

    2015-05-01

    Ubiquitin-conjugating enzymes (Ub-E2) perform the second step of ubiquitination and, consequently, are essential for regulating proteolysis and for modulating protein function, interactions and trafficking. Previously, our group demonstrated the crucial role of ubiquitination and the Ub-proteasome pathway during the Schistosoma mansoni life cycle. In the present investigation, we used a homology-based genome-wide bioinformatics approach to identify and molecularly characterise the Ub-E2 enzymes in S. mansoni. The putative functions were further investigated through molecular phylogenetic and expression profile analyses using cercariae, adult worms, eggs and mechanically transformed schistosomula (MTS) cultured in vitro for 3.5 h or 1 or 3 days. We identified, via in silico analysis, 17 Ub-E2 enzymes with conserved structural characteristics: the beta-sheet and the helix-2 form a central core bordered by helix-1 at one side and helix-3 and helix-4 at the other. The observed quantitative differences in the steady-state transcript levels between the cercariae and adult worms may contribute to the differential protein ubiquitination observed during the parasite's life cycle. This study is the first to identify and characterise the E2 ubiquitin conjugation family in S. mansoni and provides fundamental information regarding their molecular phylogenetics and developmental expression during intra-mammalian stages. PMID:25663106

  7. Functional interactions between ubiquitin E2 enzymes and TRIM proteins.

    PubMed

    Napolitano, Luisa M; Jaffray, Ellis G; Hay, Ronald T; Meroni, Germana

    2011-03-01

    The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets. PMID:21143188

  8. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    PubMed

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  9. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    PubMed

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP. PMID:27049943

  10. Expression and distribution of the class III ubiquitin-conjugating enzymes in the retina

    PubMed Central

    Mirza, Saima; Plafker, Kendra S.; Aston, Christopher

    2010-01-01

    Purpose Mounting evidence implicates chronic oxidative stress as a significant pathogenic factor in the development and progression of retinopathies, including age-related macular degeneration (AMD). The age-dependent toxic accumulation of oxidatively damaged proteins, lipids, and DNA in susceptible cells of the retina arises, at least in part, from a decreased capacity to eliminate these damaged biomolecules. The goal of this study was to determine the expression patterns and function of class III ubiquitin-conjugating enzymes (UbcM3, UBE2E2, and UbcM2) in the retina. These enzymes have been implicated in the ubiquitin-dependent degradation of oxidatively damaged and misfolded proteins. Methods Complementary western blotting and immunohistochemistry was performed with specific antibodies to determine the retinal cell expression pattern of each enzyme. Additional analyses using antibodies raised against UbcM2 were performed to determine the relative levels of the enzyme in lysates derived from various mouse organs as compared to the retina. An established light-damage model of oxidative stress-induced retinal degeneration was used to determine alterations in the susceptibility of mice harboring a single intact allele of UbcM2. Ubiquitin charging and auto-ubiquitylation assays were done to assess the catalytic state of UbcM2 following photo-oxidative stress. Results Expression of the class III ubiquitin-conjugating enzymes in the retina, from highest to lowest, is UbcM2>UbcM3>UBE2E2. In addition to being the most robustly expressed, UbcM2 is further distinguished by its expression in photoreceptors and retinal pigment epithelial cells. UbcM2 is expressed in most mouse tissues analyzed and is most abundant in the retina. Studies using a bright-light-damage model of acute oxidative stress in mice harboring a single disrupted allele of UbcM2 revealed that a 58% reduction in enzyme levels did not increase the susceptibility of photoreceptors to acute photo

  11. OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

    SciTech Connect

    Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario; Vittal, Vinayak; Leung, Charles C.Y.; Ceccarelli, Derek F.; Mateo, Abigail-Rachele F.; Pruneda, Jonathan N.; Mao, Daniel Y.L.; Szilard, Rachel K.; Orlicky, Stephen; Munro, Meagan; Brzovic, Peter S.; Klevit, Rachel E.; Sicheri, Frank; Durocher, Daniel

    2012-03-26

    Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

  12. UBIQUITIN-CONJUGATING ENZYME 3 DELAYS HUMAN LENS EPITHELIAL CELLS IN METAPHASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubc3/Cdc34 is a ubiquitin-conjugating enzyme (Ubc) with well established functions in the G1-to-S-phase transition. Expecting to find similar effects in human lens epithelial cells (HLECs), the authors explored roles for this ubiquitin-conjugating enzyme in regulation of the HLEC cycle. Catalytical...

  13. RING E3-Catalyzed E2 Self-Ubiquitination Attenuates the Activity of Ube2E Ubiquitin-Conjugating Enzymes.

    PubMed

    Banka, Prerana Agarwal; Behera, Adaitya Prasad; Sarkar, Sayani; Datta, Ajit B

    2015-07-01

    Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s. PMID:25960396

  14. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.

    PubMed

    Jin, Jianping; Li, Xue; Gygi, Steven P; Harper, J Wade

    2007-06-28

    Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s. PMID:17597759

  15. E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development

    PubMed Central

    Kulkarni, Madhura; Smith, Harold E.

    2008-01-01

    Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model. PMID:18636104

  16. Ubiquitin-conjugating enzyme Cdc34 mediates cadmium resistance in budding yeast through ubiquitination of the transcription factor Met4

    SciTech Connect

    Hwang, Gi-Wook; Furuchi, Takemitsu; Naganuma, Akira

    2007-11-23

    Overexpression of the ubiquitin-conjugating enzyme Cdc34 conferred strong cadmium resistance on budding yeast. Proteasome activity, which is involved in the degradation of ubiquitinated proteins, was not essential for the acquisition of resistance to cadmium. The overexpression of Cdc34 accelerated the ubiquitination of the transcription factor Met4 and reduced expression of MET25 gene, which is a target of Met4. A MET25-disrupted strain of yeast was more resistant to cadmium than was the wild-type strain, but overexpression of Cdc34 in the MET25-disrupted cells did not affect sensitivity to cadmium. Met25 is an enzyme that catalyzes the synthesis of homocysteine from sulfide (S{sup 2-}) and O-acetylhomocysteine and we detected the increased production of S{sup 2-} upon overexpression of Cdc34. Our results suggest that overexpression of Cdc34 inactivates Met4 and interferes with expression of the MET25, with subsequent production of CdS, which has low toxicity, and, thus, a decrease in the cadmium toxicity.

  17. An Allosteric Inhibitor of the Human Cdc34 Ubiquitin-Conjugating Enzyme

    SciTech Connect

    Ceccarelli, Derek F.; Tang, Xiaojing; Pelletier, Benoit; Orlicky, Stephen; Xie, Weilin; Plantevin, Veronique; Neculai, Dante; Chou, Yang-Chieh; Ogunjimi, Abiodun; Al-Hakim, Abdallah; Varelas, Xaralabos; Koszela, Joanna; Wasney, Gregory A.; Vedadi, Masoud; Dhe-Paganon, Sirano; Cox, Sarah; Xu, Shuichan; Lopez-Girona, Antonia; Mercurio, Frank; Wrana, Jeff; Durocher, Daniel; Meloche, Sylvain; Webb, David R.; Tyers, Mike; Sicheri, Frank

    2011-09-06

    In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF{sup Skp2} substrate p27{sup Kip1}. CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

  18. Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34 ▿

    PubMed Central

    Sadowski, Martin; Suryadinata, Randy; Lai, Xianning; Heierhorst, Jörg; Sarcevic, Boris

    2010-01-01

    Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination. PMID:20194622

  19. Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1)

    PubMed Central

    Nasveschuk, Christopher G.; Wang, Wei; Quade, Bettina; Zhang, Gan; Kuchta, Robert D.; Phillips, Andrew J.; Liu, Xuedong

    2012-01-01

    Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells. PMID:22279528

  20. Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalis.

    PubMed

    Yokota, Naoto; Harada, Yoshito; Sawada, Hitoshi

    2010-07-01

    The ubiquitin-proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin-conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole-mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. PMID:20578064

  1. An optimal ubiquitin-proteasome pathway in the nervous system: the role of deubiquitinating enzymes

    PubMed Central

    Ristic, Gorica; Tsou, Wei-Ling; Todi, Sokol V.

    2014-01-01

    The Ubiquitin-Proteasome Pathway (UPP), which is critical for normal function in the nervous system and is implicated in various neurological diseases, requires the small modifier protein ubiquitin to accomplish its duty of selectively degrading short-lived, abnormal or misfolded proteins. Over the past decade, a large class of proteases collectively known as deubiquitinating enzymes (DUBs) has increasingly gained attention in all manners related to ubiquitin. By cleaving ubiquitin from another protein, DUBs ensure that the UPP functions properly. DUBs accomplish this task by processing newly translated ubiquitin so that it can be used for conjugation to substrate proteins, by regulating the “where, when, and why” of UPP substrate ubiquitination and subsequent degradation, and by recycling ubiquitin for re-use by the UPP. Because of the reliance of the UPP on DUB activities, it is not surprising that these proteases play important roles in the normal activities of the nervous system and in neurodegenerative diseases. In this review, we summarize recent advances in understanding the functions of DUBs in the nervous system. We focus on their role in the UPP, and make the argument that understanding the UPP from the perspective of DUBs can yield new insight into diseases that result from anomalous intra-cellular processes or inter-cellular networks. Lastly, we discuss the relevance of DUBs as therapeutic options for disorders of the nervous system. PMID:25191222

  2. Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7

    PubMed Central

    Whitcomb, Elizabeth A; Taylor, Allen

    2009-01-01

    Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increases in gene products dictated by changes in transcription. Of the dozens of ubiquitin conjugating enzymes, or E2s, functions in control of the cell cycle have been defined for only UbcH10 and Ubc3/Cdc34. Each of these E2s works primarily with one ubiquitin ligase or E3. Here we show that another E2, UbcH7 is a regulator of S phase of the cell cycle. Over-expression of UbcH7 delays entry into S phase whereas depletion of UbcH7 increases the length of S phase and decreases cell proliferation. Additionally, the level of the checkpoint kinase Chk1 increases upon UbcH7 depletion while the level of phosphorylated PTEN decreases. Taken together, these data indicate that the length of S phase is controlled in part by UbcH7 through a PTEN/Akt/Chk1 pathway. Potential mechanisms by which UbcH7 controls Chk1 levels both directly and indirectly, as well as the length of S phase are discussed and additional functions for UbcH7 are reviewed. PMID:19664228

  3. Mutation in E1, the Ubiquitin Activating Enzyme, Reduces Drosophila Lifespan and Results in Motor Impairment

    PubMed Central

    Liu, Hsiu-Yu; Pfleger, Cathie M.

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease. PMID:23382794

  4. Conjugation of the Ubiquitin Activating Enzyme UBE1 with the Ubiquitin-Like Modifier FAT10 Targets It for Proteasomal Degradation

    PubMed Central

    Bialas, Johanna; Groettrup, Marcus; Aichem, Annette

    2015-01-01

    The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway. PMID:25768649

  5. The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...

  6. A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

    PubMed Central

    Gao, Yong-Guang; Zhou, Chen-Jie; Zhang, Yu-Hang; Hu, Hong-Yu

    2014-01-01

    The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes. PMID:25207809

  7. Mechanism of Polyubiquitin Chain Recognition by the Human Ubiquitin Conjugating Enzyme Ube2g2*

    PubMed Central

    Bocik, William E.; Sircar, Aroop; Gray, Jeffrey J.; Tolman, Joel R.

    2011-01-01

    Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μm in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ∼3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length. PMID:21098018

  8. Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected Penaeus monodon

    PubMed Central

    Jeena, K.; Prasad, K. Pani; Pathan, Mujahid Khan; Babu, P. Gireesh

    2012-01-01

    White spot syndrome virus (WSSV) is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF) 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc) was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i) was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry. PMID:25049679

  9. Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work

    PubMed Central

    Citterio, Elisabetta

    2015-01-01

    Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ub ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes. Here, we discuss the current knowledge of how ub-mediated signaling at DSBs is controlled by DUBs, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer. PMID:26442100

  10. Ubiquitin modifications

    PubMed Central

    Swatek, Kirby N; Komander, David

    2016-01-01

    Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation. PMID:27012465

  11. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  12. Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP Mechanistic Insights into a Minimalistic E1 Enzyme

    SciTech Connect

    Bacik, John-Paul; Walker, John R.; Ali, Mohsin; Schimmer, Aaron D.; Dhe-Paganon, Sirano

    2010-08-30

    E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys{sup 250}) is part of the adenylation domain in an {alpha}-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.

  13. The Ubiquitination Machinery of the Ubiquitin System

    PubMed Central

    Callis, Judy

    2014-01-01

    The protein ubiquitin is a covalent modifier of proteins, including itself. The ubiquitin system encompasses the enzymes required for catalysing attachment of ubiquitin to substrates as well as proteins that bind to ubiquitinated proteins leading them to their final fate. Also included are activities that remove ubiquitin independent of, or in concert with, proteolysis of the substrate, either by the proteasome or proteases in the vacuole. In addition to ubiquitin encoded by a family of fusion proteins, there are proteins with ubiquitin-like domains, likely forming ubiquitin's β-grasp fold, but incapable of covalent modification. However, they serve as protein-protein interaction platforms within the ubiquitin system. Multi-gene families encode all of these types of activities. Within the ubiquitination machinery “half” of the ubiquitin system are redundant, partially redundant, and unique components affecting diverse developmental and environmental responses in plants. Notably, multiple aspects of biotic and abiotic stress responses require, or are modulated by, ubiquitination. Finally, aspects of the ubiquitin system have broad utility: as components to enhance gene expression or to regulate protein abundance. This review focuses on the ubiquitination machinery: ubiquitin, unique aspects about the synthesis of ubiquitin and organization of its gene family, ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases, or E3s. Given the large number of E3s in Arabidopsis this review covers the U box, HECT and RING type E3s, with the exception of the cullin-based E3s. PMID:25320573

  14. Arabidopsis ubiquitin-conjugating enzyme UBC22 is required for female gametophyte development and likely involved in Lys11-linked ubiquitination.

    PubMed

    Wang, Sheng; Cao, Ling; Wang, Hong

    2016-05-01

    Protein ubiquitination is critical for numerous processes in eukaryotes. The ubiquitin-conjugating enzyme (E2) is required for ubiquitination. The Arabidopsis genome has approximately 37 E2 genes, but in vivo functions for most of them remain unknown. In this study we observed that knockout mutants of Arabidopsis UBC22 had much-reduced silique length and seed number, with nearly 90% of ovules aborted. Analyses revealed that the majority of mutant embryo sacs displayed severe defects and often contained no gamete nuclei. There was no difference between mutant and wild-type Arabidopsis at the megaspore mother cell stage; however, the functional megaspore was either not present or appeared abnormal in a large portion of mutant ovules, suggesting that the defect started with functional megaspore degeneration in the mutants. Degeneration continued during megagametogenesis, such that the percentage of mature embryo sacs without any gamete nuclei was much greater than the percentage of developing ovules without a functional megaspore and, in addition, various abnormalities in megagametogenesis were observed. Additionally, heterozygous plants had only 13.1% of ovules aborted, indicating that the heterozygous sporophytic tissues could affect the development of the mutant female gametophyte. UBC22 is the sole member of an Arabidopsis E2 subfamily, and is more closely related to one type of E2s in animals that catalyzes Lys11-specific ubiquitination. Indeed, our results showed that Arabidopsis UBC22 could catalyze ubiquitin dimer formation in vitro in a Lys11-dependent manner, suggesting that it likely catalyzes Lys11-linked ubiquitination in plants. This study has thus identified one biochemical property of UBC22 and revealed a novel function in female gametophyte development. PMID:27069118

  15. Arabidopsis ubiquitin-conjugating enzyme UBC22 is required for female gametophyte development and likely involved in Lys11-linked ubiquitination

    PubMed Central

    Wang, Sheng; Cao, Ling; Wang, Hong

    2016-01-01

    Protein ubiquitination is critical for numerous processes in eukaryotes. The ubiquitin-conjugating enzyme (E2) is required for ubiquitination. The Arabidopsis genome has approximately 37 E2 genes, but in vivo functions for most of them remain unknown. In this study we observed that knockout mutants of Arabidopsis UBC22 had much-reduced silique length and seed number, with nearly 90% of ovules aborted. Analyses revealed that the majority of mutant embryo sacs displayed severe defects and often contained no gamete nuclei. There was no difference between mutant and wild-type Arabidopsis at the megaspore mother cell stage; however, the functional megaspore was either not present or appeared abnormal in a large portion of mutant ovules, suggesting that the defect started with functional megaspore degeneration in the mutants. Degeneration continued during megagametogenesis, such that the percentage of mature embryo sacs without any gamete nuclei was much greater than the percentage of developing ovules without a functional megaspore and, in addition, various abnormalities in megagametogenesis were observed. Additionally, heterozygous plants had only 13.1% of ovules aborted, indicating that the heterozygous sporophytic tissues could affect the development of the mutant female gametophyte. UBC22 is the sole member of an Arabidopsis E2 subfamily, and is more closely related to one type of E2s in animals that catalyzes Lys11-specific ubiquitination. Indeed, our results showed that Arabidopsis UBC22 could catalyze ubiquitin dimer formation in vitro in a Lys11-dependent manner, suggesting that it likely catalyzes Lys11-linked ubiquitination in plants. This study has thus identified one biochemical property of UBC22 and revealed a novel function in female gametophyte development. PMID:27069118

  16. Importin-11, a nuclear import receptor for the ubiquitin-conjugating enzyme, UbcM2.

    PubMed

    Plafker, S M; Macara, I G

    2000-10-16

    Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes. We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame. Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments. A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11. Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP. UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments. Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta. These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family. PMID:11032817

  17. Vitexin inhibits polyubiquitin synthesis by the ubiquitin-conjugating enzyme E2-25K.

    PubMed

    Helms, Kimberli M; Wilson, Randall C; Ogungbe, Ifedayo V; Setzer, William N; Twigg, Pamela D

    2011-10-01

    An extract of bark from the tropical rainforest plant Byrsonima crassifolia was screened for inhibition of diubiquitin formation by the human ubiquitin-conjugating enzyme E2-25K. Activity assays with both the full-length enzyme and a truncated, active catalytic UBC domain revealed that the extract contained inhibitory properties. Separation of the extract into individual components and additional screens identified vitexin as the active inhibitor. An IC50 for vitexin was calculated to be approximately 0.5 mM. Molecular modeling simulations were used to predict the mode of inhibition and NMR spectra were used to confirm the binding site of vitexin to E2-25K. PMID:22164771

  18. Role of the Deubiquitylating Enzyme DmUsp5 in Coupling Ubiquitin Equilibrium to Development and Apoptosis in Drosophila melanogaster

    PubMed Central

    Kovács, Levente; Nagy, Olga; Pál, Margit; Udvardy, Andor; Popescu, Octavian; Deák, Péter

    2015-01-01

    Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent. PMID:25806519

  19. Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities*

    PubMed Central

    Choi, Yun-Seok; Lee, Yun-Ju; Lee, Seo-Yeon; Shi, Lei; Ha, Jung-Hye; Cheong, Hae-Kap; Cheong, Chaejoon; Cohen, Robert E.; Ryu, Kyoung-Seok

    2015-01-01

    The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184–196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r11–183 (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1CYGY) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1CC93S-[15N]UBK48R oxyester displayed two-state conformational exchange, whereas the Ube2r1CC93S/YGY-[15N]UBK48R oxyester showed predominantly one state. Together with NMR studies that compared UBK48R oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity. PMID:25471371

  20. Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increase...

  1. Electrophilic adduction of ubiquitin activating enzyme E1 by N,N-diethyldithiocarbamate inhibits ubiquitin activation and is accompanied by striatal injury in the rat.

    PubMed

    Viquez, Olga M; Caito, Samuel W; McDonald, W Hayes; Friedman, David B; Valentine, William M

    2012-11-19

    Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens

  2. Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes.

    PubMed

    Geurink, Paul P; van Tol, Bianca D M; van Dalen, Duco; Brundel, Paul J G; Mevissen, Tycho E T; Pruneda, Jonathan N; Elliott, Paul R; van Tilburg, Gabriëlle B A; Komander, David; Ovaa, Huib

    2016-05-01

    Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner. PMID:26996281

  3. Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

    PubMed Central

    Geurink, Paul P.; van Tol, Bianca D.M.; van Dalen, Duco; Brundel, Paul J.G.; Mevissen, Tycho E.T.; Pruneda, Jonathan N.; Elliott, Paul R.; van Tilburg, Gabriëlle B.A.; Komander, David; Ovaa, Huib

    2016-01-01

    Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner. PMID:26996281

  4. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

  5. E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft

    PubMed Central

    Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

    2015-01-01

    Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation. PMID:26463729

  6. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    SciTech Connect

    Imura, Yoshiyuki Molho, Melissa; Chuang, Chingkai; Nagy, Peter D.

    2015-10-15

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

  7. Molecular Basis of Lys-63-linked Polyubiquitination Inhibition by the Interaction between Human Deubiquitinating Enzyme OTUB1 and Ubiquitin-conjugating Enzyme UBC13*

    PubMed Central

    Sato, Yusuke; Yamagata, Atsushi; Goto-Ito, Sakurako; Kubota, Keiko; Miyamoto, Rikako; Nakada, Shinichiro; Fukai, Shuya

    2012-01-01

    UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation. PMID:22679021

  8. An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in th nematode Caenorhabditis elegans.

    PubMed Central

    Zhen, M; Schein, J E; Baillie, D L; Candido, E P

    1996-01-01

    The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5. UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins. Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults. This suggests that the functions of this type of E2 are developmentally regulated in C.elegans. This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages. Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development. One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site. Images PMID:8670823

  9. Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation.

    PubMed Central

    Treier, M; Seufert, W; Jentsch, S

    1992-01-01

    Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes. Images PMID:1310935

  10. Structure of full-length ubiquitin-conjugating enzyme E2-25K (huntingtin-interacting protein 2)

    SciTech Connect

    Wilson, Randall C.; Hughes, Ronny C.; Flatt, Justin W.; Meehan, Edward J.; Ng, Joseph D.; Twigg, Pamela D.

    2009-08-07

    The ubiquitin-conjugating enzyme E2-25K has been identified as a huntingtin (the key protein in Huntington's disease) interacting protein and has been shown to play a role in mediating the toxicity of A{beta}, the principal protein involved in Alzheimer's disease pathogenesis. E2-25K is a dual-domain protein with an ubiquitin-associated (UBA) domain as well as a conserved ubiquitin-conjugating (UBC) domain which catalyzes the formation of a covalent bond between the C-terminal glycine of an ubiquitin molecule and the {var_epsilon}-amine of a lysine residue on the acceptor protein as part of the ubiquitin-proteasome pathway. The crystal structures of E2-25K M172A mutant protein at pH 6.5 and pH 8.5 were determined to 1.9 and 2.2 {angstrom} resolution, respectively. Examination of the structures revealed domain-domain interactions between the UBC and UBA domains which have not previously been reported

  11. A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

    PubMed Central

    Osaka, F; Seino, H; Seno, T; Yamao, F

    1997-01-01

    A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis. PMID:9154838

  12. The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb.

    PubMed Central

    Wyndham, A. M.; Baker, R. T.; Chelvanayagam, G.

    1999-01-01

    A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important. PMID:10386876

  13. Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

    PubMed Central

    Schwartz, A L; Trausch, J S; Ciechanover, A; Slot, J W; Geuze, H

    1992-01-01

    As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell. Images PMID:1376922

  14. Ubiquitin is a Novel Substrate for Human Insulin-Degrading Enzyme

    PubMed Central

    Ralat, Luis A.; Kalas, Vasilios; Zheng, Zhongzhou; Goldman, Robert D.; Sosnick, Tobin R.; Tang, Wei-Jen

    2011-01-01

    Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β (Aβ), peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (kcat = 2 sec-1) followed by a slow cleavage between residues 72-73 (kcat = 0.07 sec-1), thereby producing the inactive Ub1-74 and Ub1-72. IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼90 fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG ‹ 0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE. PMID:21185309

  15. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    PubMed Central

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  16. Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation.

    PubMed

    Stephen, A G; Trausch-Azar, J S; Handley-Gearhart, P M; Ciechanover, A; Schwartz, A L

    1997-04-18

    The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus. PMID:9099746

  17. Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme.

    PubMed

    Hill, Spencer; Harrison, Joseph S; Lewis, Steven M; Kuhlman, Brian; Kleiger, Gary

    2016-06-01

    Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains. PMID:27044868

  18. Effete, an E2 ubiquitin-conjugating enzyme with multiple roles in Drosophila development and chromatin organization

    PubMed Central

    Cipressa, Francesca; Cenci, Giovanni

    2013-01-01

    The Drosophila effete gene encodes an extremely conserved class I E2 ubiquitin-conjugating enzyme. Growing evidence indicates that Eff is involved in many cellular processes including eye development, maintenance of female germline stem cells, and regulation of apoptosis. Eff is also a major component of Drosophila chromatin and it is particularly enriched in chromatin with repressive properties. In addition, Eff is required for telomere protection and to prevent telomere fusion. Consistent with its multiple roles in chromatin maintenance, Eff is also one of the rare factors that modulate both telomere-induced and heterochromatin-induced position effect variegation. PMID:24088712

  19. Novel control of S-phase of the cell cycle by ubiquitin conjugating enzyme H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle dependent changes in l...

  20. Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

    PubMed

    Günther Sillero, María A; de Diego, Anabel; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2008-05-15

    T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis. PMID:18378215

  1. Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties

    PubMed Central

    Qi, Chao; Li, De-Feng; Feng, Lei; Hou, Yanjie; Sun, Hui; Wang, Da-Cheng; Liu, Wei

    2015-01-01

    Ubiquitination is a post-translational modification that is involved in myriad cellar regulation and disease pathways. The ubiquitin-conjugating enzyme (E2) is an important player in the ubiquitin transfer pathway. Although many E2 structures are available, not all E2 families have known structures, and three-dimensional structures from fungal organisms other than yeast are lacking. We report here the crystal structure of UbcA1, which is a novel ubiquitin-conjugating enzyme identified from the edible and medicinal mushroom Agrocybe aegerita and displays potential antitumor properties. The protein belongs to the Ube2w family and shows similar biochemical characteristics to human Ube2w, including monomer-dimer equilibrium in solution, α-NH2 ubiquitin-transfer activity and a mechanism to recognize backbone atoms of intrinsically disordered N-termini in substrates. Its structure displays a unique C-terminal conformation with an orientation of helix α3 that is completely different from the reported E2 structures but similar to a recently reported NMR ensemble of Ube2w. A mutagenesis study on this novel enzyme revealed that an intact C-terminus is significant for protein dimerization and enzymatic activity. As the first crystallized full-length protein of this family, UbcA1 may supersede the truncated X-ray structure of Ube2w (PDB entry 2A7L) as the representative structure of the Ube2w family. PMID:26525192

  2. The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice*

    PubMed Central

    Koenig, Paul-Albert; Nicholls, Peter K.; Schmidt, Florian I.; Hagiwara, Masatoshi; Maruyama, Takeshi; Frydman, Galit H.; Watson, Nicki; Page, David C.; Ploegh, Hidde L.

    2014-01-01

    ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1−/− mice have reduced viability and fail to thrive early after birth. Male Ube2j1−/− mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1−/− elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control. PMID:25320092

  3. The E2 ubiquitin-conjugating enzyme UBE2J1 is required for spermiogenesis in mice.

    PubMed

    Koenig, Paul-Albert; Nicholls, Peter K; Schmidt, Florian I; Hagiwara, Masatoshi; Maruyama, Takeshi; Frydman, Galit H; Watson, Nicki; Page, David C; Ploegh, Hidde L

    2014-12-12

    ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1(-/-) mice have reduced viability and fail to thrive early after birth. Male Ube2j1(-/-) mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1(-/-) elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control. PMID:25320092

  4. Degradation of the deubiquitinating enzyme USP33 is mediated by p97 and the ubiquitin ligase HERC2.

    PubMed

    Chan, Nickie C; den Besten, Willem; Sweredoski, Michael J; Hess, Sonja; Deshaies, Raymond J; Chan, David C

    2014-07-11

    Because the deubiquitinating enzyme USP33 is involved in several important cellular processes (β-adrenergic receptor recycling, centrosome amplification, RalB signaling, and cancer cell migration), its levels must be carefully regulated. Using quantitative mass spectrometry, we found that the intracellular level of USP33 is highly sensitive to the activity of p97. Knockdown or chemical inhibition of p97 causes robust accumulation of USP33 due to inhibition of its degradation. The p97 adaptor complex involved in this function is the Ufd1-Npl4 heterodimer. Furthermore, we identified HERC2, a HECT domain-containing E3 ligase, as being responsible for polyubiquitination of USP33. Inhibition of p97 causes accumulation of polyubiquitinated USP33, suggesting that p97 is required for postubiquitination processing. Thus, our study has identified several key molecules that control USP33 degradation within the ubiquitin-proteasome system. PMID:24855649

  5. Decoding Ubiquitin for Mitosis

    PubMed Central

    Fournane, Sadek; Krupina, Ksenia; Kleiss, Charlotte

    2012-01-01

    Conjugation of ubiquitin (ubiquitination) to substrate proteins is a widespread modification that ensures fidelity of many cellular processes. During mitosis, different dynamic morphological transitions have to be coordinated in a temporal and spatial manner to allow for precise partitioning of the genetic material into two daughter cells, and ubiquitination of key mitotic factors is believed to provide both directionality and fidelity to this process. While directionality can be achieved by a proteolytic type of ubiquitination signal, the fidelity is often determined by various types of ubiquitin conjugation that does not target substrates for proteolysis by the proteasome. An additional level of complexity is provided by various ubiquitin-interacting proteins that act downstream of the ubiquitinated substrate and can serve as “decoders” for the ubiquitin signal. They may, specifically reverse ubiquitin attachment (deubiquitinating enzymes, DUBs) or, act as a receptor for transfer of the ubiquitinated substrate toward downstream signaling components and/or subcellular compartments (ubiquitin-binding proteins, UBPs). In this review, we aim at summarizing the knowledge and emerging concepts about the role of ubiquitin decoders, DUBs, and UBPs that contribute to faithful regulation of mitotic division. PMID:23634257

  6. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

    SciTech Connect

    Butz, Nicole; Ruetz, Stephan; Natt, Francois; Hall, Jonathan; Weiler, Jan; Mestan, Juergen; Ducarre, Monique; Grossenbacher, Rita; Hauser, Patrick; Kempf, Dominique; Hofmann, Francesco . E-mail: francesco.hofmann@pharma.novartis.com

    2005-02-15

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.

  7. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system. PMID:27570077

  8. An Unusual Transmembrane Helix in the Endoplasmic Reticulum Ubiquitin Ligase Doa10 Modulates Degradation of Its Cognate E2 Enzyme*

    PubMed Central

    Kreft, Stefan G.; Hochstrasser, Mark

    2011-01-01

    In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ∼130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ΦPΦXXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ∼5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2. PMID:21467040

  9. Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

    SciTech Connect

    Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin; Kim, Hye Jeong; Shin, Sang Hyun; Lee, Jai Heon; Kim, Doh Hoon; Oh, Ju Sung; Oh, Boung-Jun; Jung, Ho Won; Chung, Young Soo

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

  10. A newly discovered ubiquitin-conjugating enzyme E2 correlated with the cryogenic autolysis of Volvariella volvacea.

    PubMed

    Gong, Ming; Wang, Hong; Chen, Mingjie; Bao, Dapeng; Zhu, Qiuming; Tan, Qi

    2016-05-25

    In Volvariella volvacea, a species of edible mushroom, cryogenic autolysis is a typical part of abnormal metabolism. Previous functional annotation cluster analyses of cold-induced gene expression profiles have shown that the ubiquitin-conjugating enzyme E2 (UBE2), rather than the cyclin-like F-box domain alone, forms the functional cluster. In this study, analysis of gene expression profiling showed that only one type of UBE2 in V. volvacea (UBEV2) was significantly up-regulated. Further quantitative real-time PCR analysis confirmed that the expression of UBEV2 was significantly up-regulated (P<0.05) after cold-treatment lasting 4, 6, and 8h. This provided evidence that UBEV2 was closely correlated with cryogenic autolysis. The specific distribution of UBEV2 in recently diverged herb decay fungi indicated that UBEV2 was not evolutionarily correlated with early diverging fungi. Phylogenetic analysis indicated that UBEV2 was generated by horizontal gene transfer (HGT) from the ancestry of Selaginella moellendorffii UBE2. Further relative time estimation and detection of natural selection showed that there has been recent positive selection after HGT in UBEV2. Molecular modeling and logo analysis showed that the cysteine-cysteine motif is the characteristic of the UBEV2 family. These observations indicate that UBEV2 is a new type of UBE2 correlated with the cryogenic autolysis of V. volvacea. PMID:26927519

  11. Ubiquitin-conjugating enzyme UbcH10 promotes gastric cancer growth and is a potential biomarker for gastric cancer.

    PubMed

    Yang, Mengxuan; Qu, Yingying; Shi, Rongliang; Wu, Xubo; Su, Chang; Hu, Zhiqiu; Chang, Qimeng; Liu, Shaoqun; Pan, Gaofeng; Lei, Ming; Xie, Fubo; Tu, Shiwei; Tao, Weikang; Zhou, He; Hu, Gang; Zhang, Ziping

    2016-08-01

    Gastric cancer is a fatal disease and the availability of early diagnostic methods is limited. There is an urgent need to identify effective targets for early diagnosis and therapeutics. UbcH10 is a ubiquitin-conjugating enzyme with high expression in various types of cancers. In the present study, several gastric tumor cell lines with high or low expression of UbcH10 were exploited to study the role of UbcH10 in gastric cancer. Knockdown of UbcH10 expression using siRNA in gastric cancer cell lines with high expression of UbcH10 resulted in reduced proliferation, increased cisplatin-induced apoptosis and reduced serum-induced ERK, Akt and p38 phosphorylation signaling. In agreement, overexpression of UbcH10 in gastric cancer cell lines with low expression of UbcH10 led to enhanced cell proliferation and resistance to cisplatin-induced apoptosis. Most importantly, IHC analyses showed that the UbcH10 protein was expressed at a high level in most patient gastric cancer tissues, but was absent in adjacent mesenchyme tissues. These data suggest that UbcH10 may promote gastric cancer growth and can serve as a biomarker for diagnosis or as a target for novel therapeutics in gastric cancer. PMID:27349176

  12. Effete, a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

    PubMed Central

    Cipressa, Francesca; Romano, Sabrina; Centonze, Silvia; zur Lage, Petra I.; Vernì, Fiammetta; Dimitri, Patrizio; Gatti, Maurizio; Cenci, Giovanni

    2013-01-01

    Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression. PMID:23821599

  13. Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice.

    PubMed

    Wang, Ya; Xu, Meng-yun; Liu, Jian-ping; Wang, Mu-gui; Yin, Hai-qing; Tu, Ju-min

    2014-07-01

    The ubiquitin (Ub)-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance (or post-replication repair) via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especially in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction (PCR) indicated that OsUbc13 transcripts could be detected in all tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma; the expression of OsUbc13 was induced by low temperature, methylmethane sulfate (MMS), and H(2)O(2), but repressed by mannitol, abscisic acid (ABA), and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC (correlated with apoptosis), OsMADS1 (important for development of floral organs), OsB22EL8 (related to reactive oxygen species (ROS) scavenging and DNA protection), and OsCROC-1 (required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance). The molecular characterization provides a foundation for the functional study of OsUbc13. PMID:25001222

  14. The role of the ubiquitin-editing enzyme A20 in diseases of the central nervous system and other pathological processes

    PubMed Central

    Abbasi, Asghar; Forsberg, Kirsi; Bischof, Felix

    2015-01-01

    In recent years, the ubiquitin-editing enzyme A20 has been shown to control a large set of molecular pathways involved in the regulation of protective as well as self-directed immune responses. Here, we assess the current and putative roles of A20 in inflammatory, vascular and degenerative diseases of the central nervous system and explore future directions of research. PMID:26124703

  15. Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR

    PubMed Central

    Fasshuber, Hannes Klaus; Lakomek, Nils-Alexander; Habenstein, Birgit; Loquet, Antoine; Shi, Chaowei; Giller, Karin; Wolff, Sebastian; Becker, Stefan; Lange, Adam

    2015-01-01

    By applying [1-13C]- and [2-13C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 Å to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region β1-β2 but also for β-strands β1, β2, β3, and β5 as well as for the loop connecting α1 and β3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales. PMID:25644665

  16. Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition

    PubMed Central

    Liu, Zhu; Gong, Zhou; Jiang, Wen-Xue; Yang, Ju; Zhu, Wen-Kai; Guo, Da-Chuan; Zhang, Wei-Ping; Liu, Mai-Li; Tang, Chun

    2015-01-01

    A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition. DOI: http://dx.doi.org/10.7554/eLife.05767.001 PMID:26090905

  17. Ubiquitination of inositol-requiring enzyme 1 (IRE1) by the E3 ligase CHIP mediates the IRE1/TRAF2/JNK pathway.

    PubMed

    Zhu, Xu; Zhang, Ju; Sun, Huiying; Jiang, Cuicui; Dong, Yusheng; Shan, Qiang; Su, Siyuan; Xie, Yingying; Xu, Ningzhi; Lou, Xiaomin; Liu, Siqi

    2014-10-31

    Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys(545) and Lys(828), were targeted for Lys(63)-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys(545) and Lys(828), respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR. PMID:25225294

  18. A sesquiterpene lactone from a medicinal herb inhibits proinflammatory activity of TNF-α by inhibiting ubiquitin-conjugating enzyme UbcH5.

    PubMed

    Liu, Li; Hua, Yaping; Wang, Dan; Shan, Lei; Zhang, Yuan; Zhu, Junsheng; Jin, Huizi; Li, Honglin; Hu, Zhenlin; Zhang, Weidong

    2014-10-23

    UbcH5 is the key ubiquitin-conjugating enzyme catalyzing ubiquitination during TNF-α-triggered NF-κB activation. Here, we identified an herb-derived sesquiterpene lactone compound IJ-5 as a preferential inhibitor of UbcH5 and explored its therapeutic value in inflammatory and autoimmune disease models. IJ-5 suppresses TNF-α-induced NF-κB activation and inflammatory gene transcription by inhibiting the ubiquitination of receptor-interacting protein 1 and NF-κB essential modifier, which is essential to IκB kinase activation. Mechanistic investigations revealed that IJ-5 preferentially binds to and inactivates UbcH5 by forming a covalent adduct with its active site cysteine and thereby preventing ubiquitin conjugation to UbcH5. In preclinical models, pretreatment of IJ-5 exhibited potent anti-inflammatory activity against TNF-α- and D-galactosamine-induced hepatitis and collagen-induced arthritis. These findings highlight the potential of UbcH5 as a therapeutic target for anti-TNF-α interventions and provide an interesting lead compound for the development of new anti-inflammation agents. PMID:25200604

  19. A specific endpoint assay for ubiquitin.

    PubMed Central

    Rose, I A; Warms, J V

    1987-01-01

    Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented. PMID:3031643

  20. The de-ubiquitinating enzyme ataxin-3 does not modulate disease progression in a knock-in mouse model of Huntington disease

    PubMed Central

    Zeng, Li; Tallaksen-Greene, Sara J.; Wang, Bo; Albin, Roger L.; Paulson, Henry L.

    2013-01-01

    Ataxin-3 is a deubiquitinating enzyme (DUB) that participates in ubiquitin-dependent protein quality control pathways and, based on studies in model systems, may be neuroprotective against toxic polyglutamine proteins such as the Huntington's disease (HD) protein, huntingtin (htt). HD is one of at least nine polyglutamine neurodegenerative diseases in which disease-causing proteins accumulate in ubiquitin-positive inclusions within neurons. In studies crossing mice null for ataxin-3 to an established HD knock-in mouse model (HdhQ200), we tested whether loss of ataxin-3 alters disease progression, perhaps by impairing the clearance of mutant htt or the ubiquitination of inclusions. While loss of ataxin-3 mildly exacerbated age-dependent motor deficits, it did not alter inclusion formation, ubiquitination of inclusions or levels of mutant or normal htt. Ataxin-3, itself a polyglutamine-containing protein with multiple ubiquitin binding domains, was not observed to localize to htt inclusions. Changes in neurotransmitter receptor binding known to occur in HD knock-in mice also were not altered by the loss of ataxin-3, although we unexpectedly observed increased GABAA receptor binding in the striatum of HdhQ200 mice, which has not previously been noted. Finally, we confirmed that CNS levels of hsp70 are decreased in HD mice as has been reported in other HD mouse models, regardless of the presence or absence of ataxin-3. We conclude that while ataxin-3 may participate in protein quality control pathways, it does not critically regulate the handling of mutant htt or contribute to major features of disease pathogenesis in HD. PMID:24683430

  1. A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme.

    PubMed Central

    Kok, K; Hofstra, R; Pilz, A; van den Berg, A; Terpstra, P; Buys, C H; Carritt, B

    1993-01-01

    The chromosomal region 3p21 is thought to be the site of a lung tumor suppressor gene. We recently cloned a gene from this region that has greatly reduced expression in almost all lung tumor cell lines examined, in spite of being widely expressed in a variety of other tumor and nontumor cell types. We report here the sequence of this gene and show that it has significant homology to the genes encoding the ubiquitin-activating enzymes of three species, including humans. This suggests it is a second, autosomal member of this gene family in humans and may play a role in the ubiquitin conjugation pathway, which is of central importance in all eukaryotes. PMID:8327486

  2. Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea

    PubMed Central

    Cao, Shiyun; Hepowit, Nathaniel; Maupin-Furlow, Julie A.

    2015-01-01

    Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease. PMID:26010867

  3. Transition state theory for enzyme kinetics.

    PubMed

    Truhlar, Donald G

    2015-09-15

    This article is an essay that discusses the concepts underlying the application of modern transition state theory to reactions in enzymes. Issues covered include the potential of mean force, the quantization of vibrations, the free energy of activation, and transmission coefficients to account for nonequilibrium effect, recrossing, and tunneling. PMID:26008760

  4. Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

    PubMed Central

    Sadiq, Fouzia; Crompton, Leslie A; Scaife, Jes R; Lomax, Michael A

    2008-01-01

    Background Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between

  5. Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments

    PubMed Central

    E, Zhiguo; Zhang, Yuping; Li, Tingting; Wang, Lei; Zhao, Heming

    2015-01-01

    Ubiquitin-conjugating enzyme E2s (UBCs), which catalyze the transfer of ubiquitin to substrate or E3 ligases, are key enzymes in ubiquitination modifications of target proteins. However, little is known about the knowledge of UBC gene family in rice. In this study, a total of 39 UBC encoding genes, which all contained an UBC domain with a cysteine active site, were identified in the rice genome. These were classified into fifteen distinct subfamilies based upon their sequence similarity and phylogenetic relationships. A subset of 19 OsUBC genes exhibited chromosomal duplication; 4 and 15 OsUBC genes were tandemly and segmentally duplicated, respectively. Comprehensive analyses were performed to investigate the expression profiles of OsUBC genes in various stages of vegetative and reproductive development using data from EST, Microarrays, MPSS, and real-time PCR. Many OsUBC genes exhibited abundant and tissue-specific expression patterns. Moreover, 14 OsUBCs were found to be differentially expressed under treatments with drought, or salt stresses. The expression analysis after treatments with IAA, 6-BA, GA and ABA indicated that almost all OsUBC genes were responsive to at least two of the four hormones. Several genes were significantly down-regulated under all of the hormone treatments, and most of the genes reduced by 6-BA were also reduced by GA. This study will facilitate further studies of the OsUBC gene family and provide useful clues for functional validation of OsUBCs in rice. PMID:25902049

  6. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

    PubMed

    Lechtenberg, Bernhard C; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K; Ware, Carl F; Mace, Peter D; Riedl, Stefan J

    2016-01-28

    Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  7. A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

    PubMed Central

    Wing, S S; Bédard, N; Morales, C; Hingamp, P; Trasler, J

    1996-01-01

    The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins. PMID:8754804

  8. Substrate-Assisted Inhibition of Ubiquitin-like Protein-Activating Enzymes: The NEDD8 E1 Inhibitor MLN4924 Forms a NEDD8-AMP Mimetic In Situ

    SciTech Connect

    Brownell, James E.; Sintchak, Michael D.; Gavin, James M.; Liao, Hua; Bruzzese, Frank J.; Bump, Nancy J.; Soucy, Teresa A.; Milhollen, Michael A.; Yang, Xiaofeng; Burkhardt, Anne L.; Ma, Jingya; Loke, Huay-Keng; Lingaraj, Trupti; Wu, Dongyun; Hamman, Kristin B.; Spelman, James J.; Cullis, Courtney A.; Langston, Steven P.; Vyskocil, Stepan; Sells, Todd B.; Mallender, William D.; Visiers, Irache; Li, Ping; Claiborne, Christopher F.; Rolfe, Mark; Bolen, Joseph B.; Dick, Lawrence R.

    2010-11-15

    The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

  9. DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells

    PubMed Central

    Békés, Miklós; Okamoto, Keiji; Crist, Sarah B.; Jones, Mathew J.; Chapman, Jessica R.; Brasher, Bradley; Melandri, Francesco; Ueberheide, Beatrix; Denchi, Eros Lazzerini; Huang, Tony T.

    2013-01-01

    Summary The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof of principle experiment, ectopic expression of the uncleavable ubiquitin stabilized mono-ubiquitinated PCNA in the absence of DNA damage, and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified previously unknown ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool to interrogate cell signaling pathways. PMID:24210823

  10. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  11. Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle.

    PubMed

    Granado, Miriam; Priego, Teresa; Martín, Ana I; Villanúa, Maria Angeles; López-Calderón, Asunción

    2005-12-01

    Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis. PMID:16030067

  12. Downregulation of Ubiquitin-conjugating Enzyme UBE2D3 Promotes Telomere Maintenance and Radioresistance of Eca-109 Human Esophageal Carcinoma Cells

    PubMed Central

    Yang, Hui; Wu, Lin; Ke, Shaobo; Wang, Wenbo; Yang, Lei; Gao, Xiaojia; Fang, Hongyan; Yu, Haijun; Zhong, Yahua; Xie, Conghua; Zhou, Fuxiang; Zhou, Yunfeng

    2016-01-01

    Ubiquitin-conjugating enzyme UBE2D3 is an important member of the ubiquitin-proteasome pathways. Our previous study showed that the expression of UBE2D3 was negatively related to human telomerase reverse transcriptase (hTERT) and radioresistance in human breast cancer cells. However, in esophageal carcinoma, the exact effects and mechanisms of UBE2D3 in radioresistance remain unclear. This study shows that UBE2D3 knockdown was associated with significant increases in radioresistance to X-rays, telomerase activity, telomere length, and telomere shelterins. UBE2D3 knockdown-mediated radioresistance was related to a decrease in the spontaneous and ionizing radiation-induced apoptosis, resulting from a decrease in the Bax/Bcl-2 ratio. Furthermore, UBE2D3 downregulation was associated with increased G1-S phase transition and prolonged IR-induced G2/M arrest through over expression of cyclin D1, decrease of CDC25A expression and promotion of the ATM/ATR-Chk1-CDC25C pathway. Moreover, UBE2D3 downregulation reduced spontaneous DNA double-strand breaks and accelerated the repair of DNA damage induced by IR. The current data thus demonstrate that UBE2D3 downregulation enhances radioresistance by increased telomere homeostasis and prolonged IR-induced G2/M arrest, but decreases the IR-induced apoptosis and the number of DNA damage foci. These results suggest that UBE2D3 might be a potential molecular target to improve radiotherapy effects in esophageal carcinoma. PMID:27326259

  13. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    PubMed Central

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  14. Enzyme stabilization: state of the art.

    PubMed

    Gianfreda, L; Scarfi, M R

    1991-02-01

    'Enzyme stabilization' is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies. PMID:2008180

  15. The ubiquitin-conjugating enzyme UBE2E3 and its import receptor importin-11 regulate the localization and activity of the antioxidant transcription factor NRF2

    PubMed Central

    Plafker, Kendra S.; Plafker, Scott M.

    2015-01-01

    The transcription factor NF-E2 p45–related factor (Nrf2) induces the expression of cytoprotective proteins that maintain and restore redox homeostasis. Nrf2 levels and activity are tightly regulated, and three subcellular populations of the transcription factor have been identified. During homeostasis, the majority of Nrf2 is degraded in the cytoplasm by ubiquitin (Ub)-mediated degradation. A second population is transcriptionally active in the nucleus, and a third population localizes to the outer mitochondrial membrane. Still unresolved are the mechanisms and factors that govern Nrf2 distribution between its subcellular locales. We show here that the Ub-conjugating enzyme UBE2E3 and its nuclear import receptor importin 11 (Imp-11) regulate Nrf2 distribution and activity. Knockdown of UBE2E3 reduces nuclear Nrf2, decreases Nrf2 target gene expression, and relocalizes the transcription factor to a perinuclear cluster of mitochondria. In a complementary manner, Imp-11 functions to restrict KEAP1, the major suppressor of Nrf2, from prematurely extracting the transcription factor off of a subset of target gene promoters. These findings identify a novel pathway of Nrf2 modulation during homeostasis and support a model in which UBE2E3 and Imp-11 promote Nrf2 transcriptional activity by restricting the transcription factor from partitioning to the mitochondria and limiting the repressive activity of nuclear KEAP1. PMID:25378586

  16. The interaction of banana MADS-box protein MuMADS1 and ubiquitin-activating enzyme E-MuUBA in post-harvest banana fruit.

    PubMed

    Liu, Ju-Hua; Zhang, Jing; Jia, Cai-Hong; Zhang, Jian-Bin; Wang, Jia-Shui; Yang, Zi-Xian; Xu, Bi-Yu; Jin, Zhi-Qiang

    2013-01-01

    KEY MESSAGE : The interaction of MuMADS1 and MuUBA in banana was reported, which will help us to understand the mechanism of the MADS-box gene in regulating banana fruit development and ripening. The ubiquitin-activating enzyme E1 gene fragment MuUBA was obtained from banana (Musa acuminata L.AAA) fruit by the yeast two-hybrid method using the banana MADS-box gene MuMADS1 as bait and 2-day post-harvest banana fruit cDNA library as prey. MuMADS1 interacted with MuUBA. The interaction of MuMADS1 and MuUBA in vivo was further proved by bimolecular fluorescence complementation assay. Real-time quantitative PCR evaluation of MuMADS1 and MuUBA expression patterns in banana showed that they are highly expressed in the ovule 4 stage, but present in low levels in the stem, which suggests a simultaneously differential expression action exists for both MuMADS1 and MuUBA in different tissues and developmental fruits. MuMADS1 and MuUBA expression was highly stimulated by exogenous ethylene and suppressed by 1-methylcyclopropene. These results indicated that MuMADS1 and MuUBA were co-regulated by ethylene and might play an important role in post-harvest banana fruit ripening. PMID:23007689

  17. Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.

    PubMed

    Koken, M H; Hoogerbrugge, J W; Jasper-Dekker, I; de Wit, J; Willemsen, R; Roest, H P; Grootegoed, J A; Hoeijmakers, J H

    1996-01-10

    RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a

  18. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    PubMed

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  19. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis.

    PubMed

    Plechanovová, Anna; Jaffray, Ellis G; Tatham, Michael H; Naismith, James H; Hay, Ronald T

    2012-09-01

    Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate. PMID:22842904

  20. Conformational Sub-states and Populations in Enzyme Catalysis.

    PubMed

    Agarwal, P K; Doucet, N; Chennubhotla, C; Ramanathan, A; Narayanan, C

    2016-01-01

    Enzyme function involves substrate and cofactor binding, precise positioning of reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme-substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surrounding environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems. PMID:27497171

  1. Insights into the "free state" enzyme reaction kinetics in nanoconfinement.

    PubMed

    Wang, Chen; Ye, De-Kai; Wang, Yun-Yi; Lu, Tao; Xia, Xing-Hua

    2013-04-21

    The investigation of enzyme reaction kinetics in nanoconfined spaces mimicking the conditions in living systems is of great significance. Here, a nanofluidics chip integrated with an electrochemical detector has been designed for studying "free state" enzyme reaction kinetics in nanoconfinement. The nanofluidics chip is fabricated using the UV-ablation technique developed in our group. The enzyme and substrate solutions are simultaneously supplied from two single streams into a nanochannel through a Y-shaped junction. The laminar flow forms in the front of the nanochannel, then the two liquids fully mix at their downstream where a homogeneous enzyme reaction occurs. The "free state" enzyme reaction kinetics in nanoconfinement can thus be investigated in this laminar flow based nanofluidics device. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of beta-d-glucose. The reaction product hydrogen peroxide (H2O2) can be electrochemically detected by a microelectrode aligning to the end of nanochannel. The steady-state electrochemical current responding to various glucose concentrations is used to evaluate the activity of the "free state" GOx under nanoconfinement conditions. The effect of liquid flow rate, enzyme concentration, and nanoconfinement on reaction kinetics has been studied in detail. Results show that the "free state" GOx activity increases significantly compared to the immobilized enzyme and bath system, and the GOx reaction rate in the nanochannel is two-fold faster than that in bulk solution, demonstrating the importance of "free state" and spatial confinement for the enzyme reaction kinetics. The present approach provides an effective method for exploiting the "free state" enzyme activity in nanospatial confinement. PMID:23429726

  2. An Operational Definition of the Steady State in Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Barnsley, E. A.

    1990-01-01

    The Briggs-Haldane assumption is used as the basis for the development of a kinetic model for enzyme catalysis. An alternative definition of the steady state and examples of realistic mechanisms are provided. (KR)

  3. In Vitro Ubiquitination: Self-Ubiquitination, Chain Formation, and Substrate Ubiquitination Assays.

    PubMed

    Maspero, Elena; Polo, Simona

    2016-01-01

    Ubiquitination of proteins in vitro has evolved as an indispensable tool for the functional analysis of this posttranslational modification. In vitro ubiquitination is particularly helpful to study conjugation mechanisms. The efficiency of the ubiquitination reaction depends in part on the quality of the enzymes utilized. Here we introduce the assay developed in our lab to study HECT E3 ligases. It involves bacterially expressed E1, His-tagged Ube2D3 (also called UbcH5c, the best E2 for Nedd4), untagged Nedd4, and untagged ubiquitin (Ub). As tags may impair specific activity of the enzymes or even interfere with the enzymatic reaction, they should be avoided, removed, or kept to a minimal size whenever possible, unless proven to be without consequence. The protocol described here is suitable for other E3 ligases capable of forming Ub chains as pseudo-product of the enzyme reaction. It is also adapted to include substrates. In this case, substrates should be tagged and purified after the reaction is completed to allow the detection of the ubiquitinated products. PMID:27613033

  4. The role of histone ubiquitination during spermatogenesis.

    PubMed

    Sheng, Kai; Liang, Xiaotong; Huang, Sizhou; Xu, Wenming

    2014-01-01

    Protein ubiquitin-proteasome (ubiquitin-proteasome) system is the major mechanism responsible for protein degradation in eukaryotic cell. During spermatogenesis, the replacement of histone by protamine is vital for normal sperm formation, which is involved in ubiquitination enzymes expressed in testis. Recently, histone ubiquitin ligases have been shown to play critical roles in several aspects of spermatogenesis, such as meiotic sex chromosome inactivation (MSCI), DNA damage response, and spermiogenesis. In this review, we highlight recent progress in the discovery of several histone ubiquitin ligases and elaborate mechanisms of how these enzymes are involved in these processes through knockout mouse model. Using Huwe1, UBR2, and RNF8 as examples, we emphasized the diverse functions for each enzyme and the broad involvement of these enzymes in every stage, from spermatogonia differentiation and meiotic division to spermiogenesis; thus histone ubiquitin ligases represent a class of enzymes, which play important roles in spermatogenesis through targeting histone for ubiquitination and therefore are involved in transcription regulation, epigenetic modification, and other processes essential for normal gametes formation. PMID:24963488

  5. Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

    SciTech Connect

    Misaghi, S.; Galardy, P.J.; Meester, W.J.; Ovaa, H.; Ploegh, H.L.; Gaudet, R.

    2009-03-24

    Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

  6. Identification, Analysis and Prediction of Protein Ubiquitination Sites

    PubMed Central

    Radivojac, Predrag; Vacic, Vladimir; Haynes, Chad; Cocklin, Ross R.; Mohan, Amrita; Heyen, Joshua W.; Goebl, Mark G.; Iakoucheva, Lilia M.

    2009-01-01

    Summary Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associated mutations. UbPred is available at http://www.ubpred.org PMID:19722269

  7. RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen.

    PubMed

    Strzalka, Wojciech; Bartnicki, Filip; Pels, Katarzyna; Jakubowska, Agata; Tsurimoto, Toshiki; Tanaka, Katsunori

    2013-02-01

    The proliferating cell nuclear antigen (PCNA) is post-translationally modified by ubiquitin in yeast and mammalian cells. It is widely accepted that in yeast mono- and polyubiquitinated PCNA is involved in distinct pathways of DNA postreplication repair. This study showed an interaction between plant ubiquitin and PCNA in the plant cell. Using different approaches, it was demonstrated that Arabidopsis RAD5a ubiquitin ligase is involved in the post-translational modification of plant PCNA. A detailed analysis of the properties of selected Arabidopsis ubiquitin-conjugating enzymes (AtUBC) has shown that a plant homologue of yeast RAD6 (AtUBC2) is sufficient to monoubiquitinate AtPCNA in the absence of ubiquitin ligase. Using different combinations of selected AtUBC proteins together with AtRAD5a, it was demonstrated that plants have potential to use different pathways to ubiquitinate PCNA. The analysis of Arabidopsis PCNA1 and PCNA2 did not demonstrate substantial differences in the ubiquitination pattern between these two proteins. The major ubiquitination target of Arabidopsis PCNA, conserved in eukaryotes, is lysine 164. Taken together, the presented results clearly demonstrate the involvement of Arabidopsis UBC and RAD5a proteins in the ubiquitination of plant PCNA at lysine 164. The data show the complexity of the plant ubiquitination system and open new questions about its regulation in the plant cell. PMID:23314815

  8. The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange

    PubMed Central

    Clerici, Marcello; Luna-Vargas, Mark P. A.; Faesen, Alex C.; Sixma, Titia K.

    2014-01-01

    Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP–Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP–Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP–Ubl domain promotes a change of a switching loop near the active site. This ‘allosteric regulation of product discharge’ provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes. PMID:25404403

  9. Equilibrium Binding and Steady-State Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Dunford, H. Brian

    1984-01-01

    Points out that equilibrium binding and steady-state enzyme kinetics have a great deal in common and that related equations and error analysis can be cast in identical forms. Emphasizes that if one type of problem solution is taught, the other is also taught. Various methods of data analysis are evaluated. (JM)

  10. Nucleotide Docking: Prediction of Reactant State Complexes for Ribonuclease Enzymes

    SciTech Connect

    Elsasser, Brigitta M.; Fels, Gregor

    2010-12-01

    Ribonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools - MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0 - for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies.

  11. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  12. The role of allostery in the ubiquitin-proteasome system

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2012-01-01

    The Ubiquitin-Proteasome System is involved in many cellular processes including protein degradation. Degradation of a protein via this system involves two successive steps: ubiquitination and degradation. Ubiquitination tags the target protein with ubiquitin-like proteins, such as ubiquitin, SUMO and NEDD8, via a cascade involving three enzymes: activating enzyme E1, conjugating enzyme E2, and E3 ubiquitin ligases. The proteasomes recognize the ubiquitin-like protein tagged substrate proteins and degrade them. Accumulating evidence indicates that allostery is a central player in the regulation of ubiquitination, as well as deubiquitination and degradation. Here, we provide an overview of the key mechanistic roles played by allostery in all steps of these processes, and highlight allosteric drugs targeting them. Throughout the review, we emphasize the crucial mechanistic role played by linkers in allosterically controlling the Ubiquitin-Proteasome System action by biasing the sampling of the conformational space, which facilitate the catalytic reactions of the ubiquitination and degradation. Finally, we propose that allostery may similarly play key roles in the regulation of molecular machines in the cell, and as such allosteric drugs can be expected to be increasingly exploited in therapeutic regimes. PMID:23234564

  13. Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry

    PubMed Central

    Scott, Daniel; Layfield, Robert; Oldham, Neil J

    2015-01-01

    Nanoelectrospray ionization-mass spectrometry and ion mobility-mass spectrometry have been used to study the interactions of the large, multidomain, and conformationally flexible deubiquitinating enzyme ubiquitin specific protease 5 (USP5) with mono- and poly-ubiquitin (Ub) substrates. Employing a C335A active site mutant, mass spectrometry was able to detect the stable and cooperative binding of two mono-Ub molecules at the Zinc-finger ubiquitin binding protein (ZnF-UBP) and catalytic site domains of USP5. Tetra-ubiquitin, in contrast, bound to USP5 with a stoichiometry of 1 : 1, and formed additional interactions with USP5's two ubiquitin associated domains (UBAs). Charge-state distribution and ion mobility analysis revealed that both mono- and tetra-ubiquitin bound to the compact conformation of USP5 only, and that tetra-ubiquitin binding was able to shift the conformational distribution of USP5 from a mixture of extended and compact forms to a completely compact conformation. PMID:25970461

  14. [Progress in ubiquitin, ubiquitin chain and protein ubiquitination].

    PubMed

    Lan, Qiuyan; Gao, Yuan; Li, Yanchang; Hong, Xuechuan; Xu, Ping

    2016-01-01

    Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes. PMID:27363196

  15. The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation.

    PubMed

    von Delbrück, Maximilian; Kniss, Andreas; Rogov, Vladimir V; Pluska, Lukas; Bagola, Katrin; Löhr, Frank; Güntert, Peter; Sommer, Thomas; Dötsch, Volker

    2016-06-16

    Ubiquitin conjugation is an essential process modulating protein function in eukaryotic cells. Surprisingly, little is known about how the progressive assembly of ubiquitin chains is managed by the responsible enzymes. Only recently has ubiquitin binding activity emerged as an important factor in chain formation. The Ubc7 activator Cue1 carries a ubiquitin binding CUE domain that substantially stimulates K48-linked polyubiquitination mediated by Ubc7. Our results from NMR-based analysis and in vitro ubiquitination reactions point out that two parameters accelerate ubiquitin chain assembly: the increasing number of CUE binding sites and the position of CUE binding within a growing chain. In particular, interactions with a ubiquitin moiety adjacent to the acceptor ubiquitin facilitate chain elongation. These data indicate a mechanism for ubiquitin binding in which Cue1 positions Ubc7 and the distal acceptor ubiquitin for rapid polyubiquitination. Disrupting this mechanism results in dysfunction of the ERAD pathway by a delayed turnover of substrates. PMID:27264873

  16. Structural Basis for Ubiquitin Recognition and Autoubiquitination by Rabex-5

    SciTech Connect

    Lee,S.; Tsai, Y.; Mattera, R.; Smith, W.; Kostelansky, M.; Weissman, A.; Bonifacino, J.; Hurley, J.

    2006-01-01

    Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-Angstroms resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with {approx}29-{mu}M affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with {approx}22-{mu}M affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.

  17. OptZyme: Computational Enzyme Redesign Using Transition State Analogues

    PubMed Central

    Grisewood, Matthew J.; Gifford, Nathanael P.; Pantazes, Robert J.; Li, Ye; Cirino, Patrick C.; Janik, Michael J.; Maranas, Costas D.

    2013-01-01

    OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., kcat or kcat/KM) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that KM correlates (R2 = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, kcat/KM correlates (R2 = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and kcat correlates (R2 = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved KM, kcat, and kcat/KM for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving KM, kcat, or kcat/KM. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S. PMID:24116038

  18. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    PubMed Central

    Taylor, Allen

    2011-01-01

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in non-canonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. While many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin conjugation enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin conjugation enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells appear to be an indicator of mild oxidative stress. PMID:21530648

  19. E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Yount, Jacob S.

    2015-01-01

    Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection. PMID:26263374

  20. UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation

    PubMed Central

    Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

    2013-01-01

    In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)–E2 (ubiquitin-conjugating enzyme)–E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl. PMID:23172288

  1. Dynamic ubiquitination drives herpesvirus neuroinvasion

    PubMed Central

    Huffmaster, Nicholas J.; Sollars, Patricia J.; Richards, Alexsia L.; Pickard, Gary E.; Smith, Gregory A.

    2015-01-01

    Neuroinvasive herpesviruses display a remarkable propensity to enter the nervous system of healthy individuals in the absence of obvious trauma at the site of inoculation. We document a repurposing of cellular ubiquitin during infection to switch the virus between two invasive states. The states act sequentially to defeat consecutive host barriers of the peripheral nervous system and together promote the potent neuroinvasive phenotype. The first state directs virus access to nerve endings in peripheral tissue, whereas the second delivers virus particles within nerve fibers to the neural ganglia. Mutant viruses locked in either state remain competent to overcome the corresponding barrier but fail to invade the nervous system. The herpesvirus “ubiquitin switch” may explain the unusual ability of these viruses to routinely enter the nervous system and, as a consequence, their prevalence in human and veterinary hosts. PMID:26407585

  2. Synthesis and in vitro anticancer evaluation of some 4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors.

    PubMed

    Kothayer, Hend; Spencer, Sebastian M; Tripathi, Kaushlendra; Westwell, Andrew D; Palle, Komaraiah

    2016-04-15

    Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our compound names as 'new triazines', have been synthesized, based on the previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate anticancer agents TZ9 and 4-amino-N'-phenyl-6-(arylamino)-1,3,5-triazine-2-carbohydrazides. Synthesis of the target compounds was readily accomplished in two steps from either bis-aryl/aryl biguanides via reaction of phenylhydrazine or hydrazinehydrate with key 4-amino-6-bis(arylamino)/(arylamino)-1,3,5-triazine-2-carboxylate intermediates. These new triazine derivatives were evaluated for their abilities to inhibit Rad6B ubiquitin conjugation and in vitro anticancer activity against several human cancer cell lines: ovarian (OV90 and A2780), lung (H1299 and A549), breast (MCF-7 and MDA-MB231) and colon (HT29) cancer cells by MTS assays. All the 10 new triazines exhibited superior Rad6B inhibitory activities in comparison to selective Rad6 inhibitor TZ9 that was reported previously. Similarly, new triazines also showed better IC50 values in survival assays of various tumor cell lines. Particularly, new triazines 6a-c, exhibited lower IC50 (3.3-22μM) values compared to TZ9. PMID:26965855

  3. Ubiquitin--the kiss of death goes Nobel. Will you be quitting?

    PubMed

    Behuliak, M; Celec, P; Gardlik, R; Palffy, R

    2005-01-01

    The Nobel prize in chemistry 2004 was given to Aaron Ciechanover, Avram Hershko and Irwin Rose for their discovery of the ubiquitin mediated proteolysis. Years of research have shown that the ubiquitin pathway plays a crucial role in the cellular metabolism and its regulation. These scientists together with Alexander Varshavsky have identified the most important elements of this pathway as well as their interactions. The ubiquitin pathway degrades intracellular proteins with an ubiquitin chain being the tag that marks proteins assigned for degradation. This process is mediated by ubiquitin-activating enzyme (El), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3). Mono-ubiquitination and deubiquitination play a classic regulatory role in numerous processes including cell-cycle, transcription, translation, DNA repair, stress response etc. This article tries to summarize current knowledge on the molecular basis of the ubiqutin pathway. (Fig. 1, Ref. 52.) PMID:16026142

  4. Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5

    PubMed Central

    Lee, Sangho; Tsai, Yien Che; Mattera, Rafael; Smith, William J.; Kostelansky, Michael S.; Weissman, Allan M.; Bonifacino, Juan S.; Hurley, James H.

    2006-01-01

    Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination, and contains an intrinsic ubiquitin E3 ligase activity, all of which require an N-terminal A20 zinc finger and an immediately C-terminal helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5 Å resolution shows that Rabex-5:ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin binding domain, an inverted ubiquitin interaction motif (IUIM), that binds with ~29 μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ~22 μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc finger diaromatic patch mediates E3 ligase activity by directly recruiting a ubiquitin-loaded ubiquitin conjugating enzyme. PMID:16462746

  5. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    NASA Technical Reports Server (NTRS)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  6. Targeting the Ubiquitin Pathway for Cancer Treatment

    PubMed Central

    Liu, Jia; Shaik, Shavali; Dai, Xiangpeng; Wu, Qiong; Zhou, Xiuxia; Wang, Zhiwei; Wei, Wenyi

    2015-01-01

    Proteasome-mediated degradation is a common mechanism by which cells renew their intracellular proteins and maintain protein homeostasis. In this process, the E3 ubiquitin ligases are responsible for targeting specific substrates (proteins) for ubiquitin-mediated degradation. However, in cancer cells, the stability and the balance between oncoproteins and tumor suppressor proteins are disturbed in part due to deregulated proteasome-mediated degradation. This ultimately leads to either stabilization of oncoprotein(s) or increased degradation of tumor suppressor(s), contributing to tumorigenesis and cancer progression. Therefore, E3 ubiquitin ligases including the SCF types of ubiquitin ligases have recently evolved as promising therapeutic targets for the development of novel anti-cancer drugs. In this review, we highlighted the critical components along the ubiquitin pathway including E1, E2, various E3 enzymes and DUBs that could serve as potential drug targets and also described the available bioactive compounds that target the ubiquitin pathway to control various cancers. PMID:25481052

  7. Stabilization of an unusual salt bridge in ubiquitin by the extra C-terminal domain of the proteasome-associated deubiquitinase UCH37 as a mechanism of its exo specificity

    PubMed Central

    Morrow, Marie E.; Kim, Myung-Il; Ronau, Judith A.; Sheedlo, Michael J.; White, Rhiannon R.; Chaney, Joseph; Paul, Lake N.; Lill, Markus A.; Artavanis-Tsakonas, Katerina; Das, Chittaranjan

    2013-01-01

    Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs) - about 100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from Trichinella spiralis in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intra-molecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB-ubiquitin structures in the protein data bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo

  8. Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.

    PubMed

    Wang, Bo; Merillat, Sean A; Vincent, Michael; Huber, Amanda K; Basrur, Venkatesha; Mangelberger, Doris; Zeng, Li; Elenitoba-Johnson, Kojo; Miller, Richard A; Irani, David N; Dlugosz, Andrzej A; Schnell, Santiago; Scaglione, Kenneth Matthew; Paulson, Henry L

    2016-02-01

    UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems. PMID:26601958

  9. The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases.

    PubMed

    Pruneda, Jonathan N; Durkin, Charlotte H; Geurink, Paul P; Ovaa, Huib; Santhanam, Balaji; Holden, David W; Komander, David

    2016-07-21

    Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins. PMID:27425412

  10. THE ROLE OF E3 LIGASES IN THE UBIQUITIN-DEPENDENT REGULATION OF SPERMATOGENESIS*

    PubMed Central

    Richburg, John H.; Myers, Jessica L.; Bratton, Shawn B.

    2014-01-01

    The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis. PMID:24632385

  11. Neuronal ubiquitin homeostasis

    PubMed Central

    Hallengren, Jada; Chen, Ping-Chung; Wilson, Scott M.

    2013-01-01

    Neurons have highly specialized intracellular compartments that facilitate the development and activity of the nervous system. Ubiquitination is a post-translational modification that controls many aspects of neuronal function by regulating protein abundance. Disruption of this signaling pathway has been demonstrated in neurological disorders such as Parkinson’s disease, Amyotrophic Lateral Sclerosis and Angleman Syndrome. Since many neurological disorders exhibit ubiquitinated protein aggregates, the loss of neuronal ubiquitin homeostasis may be an important contributor of disease. This review discusses the mechanisms utilized by neurons to control the free pool of ubiquitin necessary for normal nervous system development and function as well as new roles of protein ubiquitination in regulating synaptic activity. PMID:23686613

  12. The location of ubiquitin in Lethocerus arthrin.

    PubMed

    Galkin, Vitold E; Orlova, Albina; Lukoyanova, Natalya; VanLoock, Margaret S; Hååg, Petra; Bullard, Belinda; Egelman, Edward H

    2003-01-24

    Arthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation. The stoichiometry of arthrin to actin in Lethocerus indirect flight muscle, 1:6, suggests that there would be one arthrin molecule for each Tm-Tn (tropomyosin-troponin) complex. The appearance of arthrin after tropomyosin and troponin in Drosophila development is consistent with the Tm-Tn complex determining which actin subunit is targeted for conjugation with ubiquitin. We have used a new approach of three-dimensional reconstruction of helical filaments, the iterative helical real space reconstruction method, to extract segments of homogeneous arthrin out of long filaments where the conformation of the ubiquitin is more heterogeneous. Surprisingly, the location of the ubiquitin is on the face of actin subdomain 1, opposite to where tropomyosin binds in the "off" state, suggesting that there could not be a direct interaction between the ubiquitin and the tropomyosin. It is possible that the troponin complex in the "on" state that is bound to one actin strand makes an unfavorable contact with a ubiquitin molecule attached to the opposite actin strand. This might be the basis for a destabilization of the on state at rest length. Lys118 is the most likely residue to which the ubiquitin is conjugated, based upon fitting atomic structures of actin and ubiquitin into the reconstruction. PMID:12507467

  13. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  14. Decoding the ubiquitin-mediated pathway of arthropod disease vectors.

    PubMed

    Choy, Anthony; Severo, Maiara S; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J; Pedra, Joao H F

    2013-01-01

    Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

  15. Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

    PubMed Central

    Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

    2013-01-01

    Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

  16. Substrate specificity of the ubiquitin and Ubl proteases

    PubMed Central

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  17. The mechanism of OTUB1-mediated inhibition of ubiquitination

    SciTech Connect

    Wiener, Reuven; Zhang, Xiangbin; Wang, Tao; Wolberger, Cynthia

    2013-04-08

    Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13-Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13-Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13-Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13-Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13-Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13-Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1

  18. Development of inhibitors in the ubiquitination cascade.

    PubMed

    Zhang, Wei; Sidhu, Sachdev S

    2014-01-21

    The ubiquitin proteasome system (UPS) is essential in regulating myriad aspects of protein functions. It is therefore a fundamentally important regulatory mechanism that impacts most if not all aspects of cellular processes. Indeed, malfunction of UPS components is implicated in human diseases such as neurodegenerative and immunological disorders and many cancers. The success of proteasome inhibitors in cancer therapy suggests that modulating enzymes in the ubiquitination cascade would be clinically important for therapeutic benefits. In this review, we summarize advances in developing inhibitors of a variety of UPS components. In particular, we highlight recent work done on the protein engineering of ubiquitin as modulators of the UPS, a novel approach that may shed light on innovative drug discovery in the future. PMID:24239534

  19. The Importance of Ubiquitination and Deubiquitination in Cellular Reprogramming

    PubMed Central

    Suresh, Bharathi; Lee, Junwon; Kim, Kye-Seong; Ramakrishna, Suresh

    2016-01-01

    Ubiquitination of core stem cell transcription factors can directly affect stem cell maintenance and differentiation. Ubiquitination and deubiquitination must occur in a timely and well-coordinated manner to regulate the protein turnover of several stemness related proteins, resulting in optimal embryonic stem cell maintenance and differentiation. There are two switches: an E3 ubiquitin ligase enzyme that tags ubiquitin molecules to the target proteins for proteolysis and a second enzyme, the deubiquitinating enzyme (DUBs), that performs the opposite action, thereby preventing proteolysis. In order to maintain stemness and to allow for efficient differentiation, both ubiquitination and deubiquitination molecular switches must operate properly in a balanced manner. In this review, we have summarized the importance of the ubiquitination of core stem cell transcription factors, such as Oct3/4, c-Myc, Sox2, Klf4, Nanog, and LIN28, during cellular reprogramming. Furthermore, we emphasize the role of DUBs in regulating core stem cell transcriptional factors and their function in stem cell maintenance and differentiation. We also discuss the possibility of using DUBs, along with core transcription factors, to efficiently generate induced pluripotent stem cells. Our review provides a relatively new understanding regarding the importance of ubiquitination/deubiquitination of stem cell transcription factors for efficient cellular reprogramming. PMID:26880980

  20. Global approaches to understanding ubiquitination

    PubMed Central

    Kaiser, Peter; Huang, Lan

    2005-01-01

    Ubiquitination - the linkage of one or more molecules of the protein ubiquitin to another protein - regulates a wide range of biological processes in all eukaryotes. We review the proteome-wide strategies that are being used to study aspects of ubiquitin biology, including substrates, components of the proteasome and ubiquitin ligases, and deubiquitination. PMID:16207362

  1. The early history of the ubiquitin field

    PubMed Central

    Varshavsky, Alexander

    2006-01-01

    This is a personal account of the early history of ubiquitin research, by one of its protagonists. The field of ubiquitin and regulated protein degradation was created in the 1980s, largely through the complementary discoveries by the laboratory of A. Hershko (Technion, Haifa, Israel) and by my laboratory, then at MIT (Cambridge, MA). I describe the elegant insights by Hershko and his colleagues that yielded the initial understanding of ubiquitin conjugation and ubiquitin-mediated proteolysis in cell extracts, including the identification of E1, E2, and E3 enzymes. These advances were followed by a set of interconnected discoveries in my laboratory that revealed the biology of the ubiquitin system, i.e., its necessity for the protein degradation in vivo, its specific physiological functions (in the cell cycle, DNA repair, protein synthesis, transcriptional regulation, and stress responses), the source of its selectivity (specific degradation signals in short-lived proteins), and its key mechanistic attributes, such as the polyubiquitin chain and the subunit selectivity of protein degradation. The above biological (function-based) insights produced the main discovery of the physiological regulation by intracellular protein degradation. These advances caused the enormous expansion of the ubiquitin field in the 1990s. Together with the initial discovery of ubiquitin-mediated proteolysis by Hershko and coworkers, our biological discoveries in the 1980s led to a radically changed understanding of the logic of intracellular circuits, as it became clear that the control through regulated protein degradation rivals, and often surpasses in significance, the classical regulation through transcription and translation. PMID:16501229

  2. Pioglitazone Attenuates Drug-Eluting Stent-Induced Proinflammatory State in Patients by Blocking Ubiquitination of PPAR

    PubMed Central

    Wang, Zhongxia; Zhang, Tao; Sun, Lizhe; Li, Ruifeng; Wei, Yuanyuan; Yuan, Zuyi; Liu, Junhui; Chen, Tao

    2016-01-01

    The inflammatory response after polymer-based drug-eluting stent (DES) placement has recently emerged as a major concern. The biologic roles of peroxisome proliferator-activated receptor-γ (PPAR-γ) activators thiazolidinedione (TZD) remain controversial in cardiovascular disease. Herein, we investigated the antiinflammatory effects of pioglitazone (PIO) on circulating peripheral blood mononuclear cells (MNCs) in patients after coronary DES implantation. Methods and Results. Twenty-eight patients with coronary artery disease and who underwent DES implantations were randomly assigned to pioglitazone (30 mg/d; PIO) or placebo (control; Con) treatment in addition to optimal standard therapy. After 12 weeks of treatment, plasma concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9) were significantly decreased in PIO group compared to the Con group (P = 0.035, 0.011, 0.008, and 0.012, resp.). DES-induced mRNA expressions of IL-6, TNF-α, and MMP-9 in circulating MNC were significantly blocked by PIO (P = 0.031, 0.012, and 0.007, resp.). In addition, PIO markedly inhibited DES-enhanced NF-κB function and DES-blocked PPAR-γ activity. Mechanically, DES induced PPAR-γ ubiquitination and degradation in protein level, which can be totally reversed by PIO. Conclusion. PIO treatment attenuated DES-induced PPAR loss, NF-κB activation, and proinflammation, indicating that PIO may have a novel direct protective role in modulating proinflammation in DES era. PMID:27403152

  3. Dengue Virus Genome Uncoating Requires Ubiquitination

    PubMed Central

    Byk, Laura A.; Iglesias, Néstor G.; De Maio, Federico A.; Gebhard, Leopoldo G.; Rossi, Mario

    2016-01-01

    ABSTRACT The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. PMID:27353759

  4. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

    SUMMARY Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated. PMID:20409276

  5. A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins.

    PubMed

    Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

    2016-10-01

    Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity. PMID:27325501

  6. Visualizing K48 Ubiquitination during Presynaptic Formation By Ubiquitination-Induced Fluorescence Complementation (UiFC)

    PubMed Central

    Pinto, Maria J.; Pedro, Joana R.; Costa, Rui O.; Almeida, Ramiro D.

    2016-01-01

    In recent years, signaling through ubiquitin has been shown to be of great importance for normal brain development. Indeed, fluctuations in ubiquitin levels and spontaneous mutations in (de)ubiquitination enzymes greatly perturb synapse formation and neuronal transmission. In the brain, expression of lysine (K) 48-linked ubiquitin chains is higher at a developmental stage coincident with synaptogenesis. Nevertheless, no studies have so far delved into the involvement of this type of polyubiquitin chains in synapse formation. We have recently proposed a role for polyubiquitinated conjugates as triggering signals for presynaptic assembly. Herein, we aimed at characterizing the axonal distribution of K48 polyubiquitin and its dynamics throughout the course of presynaptic formation. To accomplish so, we used an ubiquitination-induced fluorescence complementation (UiFC) strategy for the visualization of K48 polyubiquitin in live hippocampal neurons. We first validated its use in neurons by analyzing changing levels of polyubiquitin. UiFC signal is diffusely distributed with distinct aggregates in somas, dendrites and axons, which perfectly colocalize with staining for a K48-specific antibody. Axonal UiFC aggregates are relatively stable and new aggregates are formed as an axon grows. Approximately 65% of UiFC aggregates colocalize with synaptic vesicle clusters and they preferentially appear in the axonal domains of axo-somatodendritic synapses when compared to isolated axons. We then evaluated axonal accumulation of K48 ubiquitinated signals in bead-induced synapses. We observed rapid accumulation of UiFC signal and endogenous K48 ubiquitin at the sites of newly formed presynapses. Lastly, we show by means of a microfluidic platform, for the isolation of axons, that presynaptic clustering on beads is dependent on E1-mediated ubiquitination at the axonal level. Altogether, these results indicate that enrichment of K48 polyubiquitin at the site of nascent presynaptic

  7. The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression.

    PubMed

    Böhm, Stefanie; Szakal, Barnabas; Herken, Benjamin W; Sullivan, Meghan R; Mihalevic, Michael J; Kabbinavar, Faiz F; Branzei, Dana; Clark, Nathan L; Bernstein, Kara A

    2016-02-26

    DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7. PMID:26740628

  8. Ubiquitin Activates Patatin-Like Phospholipases from Multiple Bacterial Species

    PubMed Central

    Anderson, David M.; Sato, Hiromi; Dirck, Aaron T.; Feix, Jimmy B.

    2014-01-01

    Phospholipase A2 enzymes are ubiquitously distributed throughout the prokaryotic and eukaryotic kingdoms and are utilized in a wide array of cellular processes and physiological and immunological responses. Several patatin-like phospholipase homologs of ExoU from Pseudomonas aeruginosa were selected on the premise that ubiquitin activation of this class of bacterial enzymes was a conserved process. We found that ubiquitin activated all phospholipases tested in both in vitro and in vivo assays via a conserved serine-aspartate catalytic dyad. Ubiquitin chains versus monomeric ubiquitin were superior in inducing catalysis, and ubiquitin-like proteins failed to activate phospholipase activity. Toxicity studies in a prokaryotic dual-expression system grouped the enzymes into high- and low-toxicity classes. Toxicity measured in eukaryotic cells also suggested a two-tiered classification but was not predictive of the severity of cellular damage, suggesting that each enzyme may correspond to unique properties perhaps based on its specific biological function. Additional studies on lipid binding preference suggest that some enzymes in this family may be differentially sensitive to phosphatidyl-4,5-bisphosphate in terms of catalytic activation enhancement and binding affinity. Further analysis of the function and amino acid sequences of this enzyme family may lead to a useful approach to formulating a unifying model of how these phospholipases behave after delivery into the cytoplasmic compartment. PMID:25404699

  9. Downstream activation of NF-κB in the EDA-A1/EDAR signalling in Sjögren's syndrome and its regulation by the ubiquitin-editing enzyme A20.

    PubMed

    Sisto, M; Barca, A; Lofrumento, D D; Lisi, S

    2016-05-01

    Sjögren's syndrome (SS) is an autoimmune disease and the second most common chronic systemic rheumatic disorder. Prevalence of primary SS in the general population has been estimated to be approximately 1-3%, whereas secondary SS has been observed in 10-20% of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma. Despite this, its exact aetiology and pathogenesis are largely unexplored. Nuclear factor-kappa B (NF-κB) signalling mechanisms provide central controls in SS, but how these pathways intersect the pathological features of this disease is unclear. The ubiquitin-editing enzyme A20 (tumour necrosis factor-α-induced protein 3, TNFAIP3) serves as a critical inhibitor on NF-κB signalling. In humans, polymorphisms in the A20 gene or a deregulated expression of A20 are often associated with several inflammatory disorders, including SS. Because A20 controls the ectodysplasin-A1 (EDA-A1)/ectodysplasin receptor (EDAR) signalling negatively, and the deletion of A20 results in excessive EDA1-induced NF-κB signalling, this work investigates the expression levels of EDA-A1 and EDAR in SS human salivary glands epithelial cells (SGEC) and evaluates the hypothesis that SS SGEC-specific deregulation of A20 results in excessive EDA1-induced NF-κB signalling in SS. Our approach, which combines the use of siRNA-mediated gene silencing and quantitative pathway analysis, was used to elucidate the role of the A20 target gene in intracellular EDA-A1/EDAR/NF-κB pathway in SS SGEC, holding significant promise for compound selection in drug discovery. PMID:26724675

  10. Crystal Structure of a Ube2S-Ubiquitin Conjugate

    PubMed Central

    Lorenz, Sonja; Bhattacharyya, Moitrayee; Feiler, Christian; Rape, Michael; Kuriyan, John

    2016-01-01

    Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. PMID:26828794

  11. Crystal Structure of a Ube2S-Ubiquitin Conjugate.

    PubMed

    Lorenz, Sonja; Bhattacharyya, Moitrayee; Feiler, Christian; Rape, Michael; Kuriyan, John

    2016-01-01

    Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a "donor" ubiquitin and a primary amino group of an "acceptor" ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. PMID:26828794

  12. Ubiquitination and filamentous structure of cytidine triphosphate synthase.

    PubMed

    Pai, Li-Mei; Wang, Pei-Yu; Lin, Wei-Cheng; Chakraborty, Archan; Yeh, Chau-Ting; Lin, Yu-Hung

    2016-07-01

    Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5'-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure. PMID:27116391

  13. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

    SciTech Connect

    Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

    2001-09-11

    Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.

  14. Ubiquitin signaling in immune responses

    PubMed Central

    Hu, Hongbo; Sun, Shao-Cong

    2016-01-01

    Ubiquitination has emerged as a crucial mechanism that regulates signal transduction in diverse biological processes, including different aspects of immune functions. Ubiquitination regulates pattern-recognition receptor signaling that mediates both innate immune responses and dendritic cell maturation required for initiation of adaptive immune responses. Ubiquitination also regulates the development, activation, and differentiation of T cells, thereby maintaining efficient adaptive immune responses to pathogens and immunological tolerance to self-tissues. Like phosphorylation, ubiquitination is a reversible reaction tightly controlled by the opposing actions of ubiquitin ligases and deubiquitinases. Deregulated ubiquitination events are associated with immunological disorders, including autoimmune and inflammatory diseases. PMID:27012466

  15. Deubiquitinase–based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

    PubMed Central

    Komander, David

    2016-01-01

    Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins. PMID:25633630

  16. Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

    NASA Astrophysics Data System (ADS)

    Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

    2013-08-01

    Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

  17. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  18. Comparison of mHTT Antibodies in Huntington's Disease Mouse Models Reveal Specific Binding Profiles and Steady-State Ubiquitin Levels with Disease Development.

    PubMed

    Bayram-Weston, Zubeyde; Jones, Lesley; Dunnett, Stephen B; Brooks, Simon P

    2016-01-01

    Huntington's disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. PMID:27196694

  19. Comparison of mHTT Antibodies in Huntington’s Disease Mouse Models Reveal Specific Binding Profiles and Steady-State Ubiquitin Levels with Disease Development

    PubMed Central

    Bayram-Weston, Zubeyde; Jones, Lesley; Dunnett, Stephen B.; Brooks, Simon P.

    2016-01-01

    Huntington’s disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. PMID:27196694

  20. Ubiquitination in Signaling to and Activation of IKK

    PubMed Central

    Chen, Zhijian J.

    2013-01-01

    A role of polyubiquitination in the activation of IκB kinase (IKK) through a proteasome-independent mechanism was first reported in 1996, but the physiological significance of this finding was not clear until 2000 when TRAF6 was found to be a ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination. Since then, several proteins known to regulate IKK have been linked to the ubiquitin pathway. These include the deubiquitination enzymes CYLD and A20 that inhibit IKK, and the ubiquitin binding proteins NEMO and TAB2 which are the regulatory subunits of IKK and TAK1 kinase complexes, respectively. Now accumulating evidence strongly supports a central role of K63 polyubiquitination in IKK activation by multiple immune and inflammatory pathways. Interestingly, recent research suggests that some alternative ubiquitin chains such as linear or K11 ubiquitin chains may also play a role in certain pathways such as the TNF pathway. Here I present a historical narrative of the discovery of the role of ubiquitin in IKK activation, review recent advances in understanding the role and mechanism of ubiquitin-mediated IKK activation, and raise some questions to be resolved in future research. PMID:22435549

  1. Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.

    PubMed

    Crowe, Sean O; Pham, Grace H; Ziegler, Jacob C; Deol, Kirandeep K; Guenette, Robert G; Ge, Ying; Strieter, Eric R

    2016-08-17

    Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains. PMID:27256865

  2. Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

    PubMed Central

    Kamadurai, Hari B; Qiu, Yu; Deng, Alan; Harrison, Joseph S; MacDonald, Chris; Actis, Marcelo; Rodrigues, Patrick; Miller, Darcie J; Souphron, Judith; Lewis, Steven M; Kurinov, Igor; Fujii, Naoaki; Hammel, Michal; Piper, Robert; Kuhlman, Brian; Schulman, Brenda A

    2013-01-01

    Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI: http://dx.doi.org/10.7554/eLife.00828.001 PMID:23936628

  3. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

    PubMed Central

    Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

    2015-01-01

    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

  4. Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4).

    PubMed

    Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V; Vertegaal, Alfred C O

    2015-06-19

    Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

  5. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state.

    PubMed

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Sauer-Eriksson, A Elisabeth; Sauer, Uwe H; Wolf-Watz, Magnus

    2015-01-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or 'invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions. PMID:26138143

  6. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    NASA Astrophysics Data System (ADS)

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-07-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or `invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  7. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    PubMed Central

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-01-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions. PMID:26138143

  8. Channel Catfish, Ictalurus punctatus, ubiquitin carboxy-terminal hydrolase L5 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One of them is ubiquitin carboxy-terminal hydrolase (UCT). In this report, we cloned, sequenced and characterized the channel catfish UCT L5 cDNA....

  9. Detection of a single enzyme molecule based on a solid-state nanopore sensor

    NASA Astrophysics Data System (ADS)

    Tan, ShengWei; Gu, DeJian; Liu, Hang; Liu, QuanJun

    2016-04-01

    The nanopore sensor as a high-throughput and low-cost technology can detect a single molecule in a solution. In the present study, relatively large silicon nitride (Si3N4) nanopores with diameters of ∼28 and ∼88 nm were fabricated successfully using a focused Ga ion beam. We have used solid-state nanopores with various sizes to detect the single horseradish peroxidase (HRP) molecule and for the first time analyzed single HRP molecular translocation events. In addition, a real-time monitored single enzyme molecular biochemical reaction and a translocation of the product of enzyme catalysis substrates were investigated by using a Si3N4 nanopore. Our nanopore system showed a high sensitivity in detecting single enzyme molecules and a real-time monitored single enzyme molecular biochemical reaction. This method could also be significant for studying gene expression or enzyme dynamics at the single-molecule level.

  10. Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs.

    PubMed

    Xolalpa, Wendy; Mata-Cantero, Lydia; Aillet, Fabienne; Rodriguez, Manuel S

    2016-01-01

    Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities. PMID:27613034

  11. Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

    PubMed

    Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

    2015-01-01

    Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. PMID:25043555

  12. Characterization and tissue expression of channel catfish (Ictalurus punctatus, Rafinesque 1818) ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One of them is ubiquitin carboxy-terminal hydrolase L5 (UCT L5), which deubiquitylates the polyubiquitin chain into ubiquitin. In this report, ...

  13. Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

    PubMed Central

    Mortensen, Franziska; Schneider, Daniel; Barbic, Tanja; Sladewska-Marquardt, Anna; Kühnle, Simone; Marx, Andreas; Scheffner, Martin

    2015-01-01

    Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein. PMID:26216987

  14. SUMO-targeted ubiquitin ligases.

    PubMed

    Sriramachandran, Annie M; Dohmen, R Jürgen

    2014-01-01

    Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. PMID:24018209

  15. A Generic Platform for Cellular Screening Against Ubiquitin Ligases

    PubMed Central

    Maculins, Timurs; Carter, Nikki; Dorval, Thierry; Hudson, Kevin; Nissink, J. Willem M.; Hay, Ronald T.; Alwan, Husam

    2016-01-01

    Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class. PMID:26743172

  16. Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

    ERIC Educational Resources Information Center

    Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

    2006-01-01

    This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

  17. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini

    PubMed Central

    Tatham, Michael H.; Plechanovová, Anna; Jaffray, Ellis G.; Salmen, Helena; Hay, Ronald T.

    2013-01-01

    The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation. PMID:23560854

  18. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini.

    PubMed

    Tatham, Michael H; Plechanovová, Anna; Jaffray, Ellis G; Salmen, Helena; Hay, Ronald T

    2013-07-01

    The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13-UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation. PMID:23560854

  19. Ubiquitination of inducible nitric oxide synthase is required for its degradation

    PubMed Central

    Kolodziejski, Pawel J.; Musial, Aleksandra; Koo, Ja-Seok; Eissa, N. Tony

    2002-01-01

    Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. We have previously shown that iNOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether iNOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (E1) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40°C) resulted in inhibition of iNOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation. PMID:12221289

  20. Production of cellulase enzymes during the solid-state fermentation of empty palm fruit bunch fiber.

    PubMed

    Kim, Seonghun; Kim, Chul Ho

    2012-01-01

    Penicillium verruculosum COKE4E is a fungal strain isolated from bituminous coal. The microorganism cultivated in a minimal medium supplemented with Avicel, carboxymethylcellulose, and oat spelt xylan produced cellulase enzymes as exhibiting carboxymethylcellulase (CMCase), Avicelase, xylanase, and cellobiosidase activities. In this study, the productivity of the extracellular enzymes in the strain was evaluated by using empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass, as a substrate for solid-state bioconversion. The highest cellulase activities were observed after 6 days of fermentation at pH 6.0 and 30 °C. The enzymes were secreted as cellulosomes for the degradation of EPFBF as a sole carbon source. Focused ion beam analysis showed that P. verruculosum COKE4E produced cellulolytic enzymes that were able to effectively biodegrade EPFBF during solid-state fermentation. In this process, 6.5 U of CMCase, 6.8 U of Avicelase, and 8.8 U of xylanase per gram of dry solid EPFBF were produced. These results demonstrate that EPFBF may be a potential raw material in solid-state fermentation for the production of cellulase enzymes to be used for biofuel production. PMID:22052232

  1. Ubiquitin in health and disease.

    PubMed

    Mayer, R J; Arnold, J; László, L; Landon, M; Lowe, J

    1991-06-13

    Studies in recent years have shown that ubiquitin has increasingly important functions in eukaryotic cells; roles which were previously not suspected in healthy and diseased cells. The interplay between molecular pathological and molecular cell biological findings has indicated that ubiquitin may be pivotal in the cell stress response in chronic degenerative and viral diseases. Furthermore, the studies have led to the notion that ubiquitination may not only serve as a signal for nonlysosomal protein degradation but may be a unifying covalent protein modification for the major intracellular protein catabolic systems; these can act to identify proteins for cytosolic proteinases or direct intact and fragmented proteins into the lysosome system for breakdown to amino acids. This unifying role could explain why ubiquitin is restricted to eukaryotic cells, which possess extensive endomembrane systems in addition to a nuclear envelope. Protein ubiquitination is a feature of most filamentous inclusions and certain other intracellular conglomerates that are found in some degenerative and viral diseases. The detection of ubiquitin-protein conjugates is not of great diagnostic importance in these diseases. Protein ubiquitination is not only essential for the normal physiological turnover of proteins but appears to have been adapted as part of an intracellular surveillance system that can be activated by altered, damaged, or foreign proteins and organelles. The purpose of this system is to isolate and eliminate these noxious structures from the cell: as a cytoprotective mechanism this appears to have evolved in the cell akin perhaps to an 'intracellular immune system'. Other heat shock proteins such as hsp 70 may be involved in this process. It is apparent that ubiquitin has a role in embryonic development. Protein ubiquitination is presumably involved in the reorganisation of cytoplasm that accompanies cell differentiation. Ubiquitin is also necessary for the gross

  2. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    SciTech Connect

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  3. Structure and ubiquitin binding of the ubiquitin-interacting motif

    SciTech Connect

    Fisher,R.; Wang, B.; Alam, S.; Higginson, D.; Robinson, H.; Sundquist, C.; Hill, C.

    2003-01-01

    Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (K{sub d} = 0.1-1 mM), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 {angstrom} resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

  4. Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

    PubMed Central

    Schwartzkopff, Benjamin; Platta, Harald W.; Hasan, Sohel; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-01-01

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. PMID:26182377

  5. Ni(II) affects ubiquitination of core histones H2B and H2A

    SciTech Connect

    Karaczyn, Aldona A.; Golebiowski, Filip; Kasprzak, Kazimierz S. . E-mail: kasprkaz@mail.ncifcrf.gov

    2006-10-15

    The molecular mechanisms of nickel-induced malignant cell transformation include effects altering the structure and covalent modifications of core histones. Previously, we found that exposure of cells to Ni(II) resulted in truncation of histones H2A and H2B and thus elimination of some modification sites. Here, we investigated the effect of Ni(II) on one such modification, ubiquitination, of histones H2B and H2A in nuclei of cultured 1HAEo- and HPL1D human lung cells. After 1-5 days of exposure, Ni(II) up to 0.25 mM stimulated mono-ubiquitination of both histones, while at higher concentrations a suppression was found. Di-ubiquitination of H2A was not affected except for a drop after 5 days at 0.5 mM Ni(II). The decrease in mono-ubiquitination coincided with the appearance of truncated H2B that lacks the K120 ubiquitination site. However, prevention of truncation did not avert the decrease of H2B ubiquitination, indicating mechanistic independence of these effects. The changes in H2B ubiquitination did not fully coincide with concurrent changes in the nuclear levels of the ubiquitin-conjugating enzymes Rad6 and UbcH6. Overall, our results suggest that dysregulation of H2B ubiquitination is a part of Ni(II) adverse effects on gene expression and DNA repair which may assist in cell transformation.

  6. Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor

    PubMed Central

    Ungermannova, Dana; Parker, Seth J.; Nasveschuk, Christopher G.; Chapnick, Douglas A.; Phillips, Andrew J.; Kuchta, Robert D.; Liu, Xuedong

    2012-01-01

    Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo. PMID:22274912

  7. Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter.

    PubMed Central

    Gitan, R S; Eide, D J

    2000-01-01

    The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels. At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis. The zinc transporter is subsequently degraded in the vacuole. This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal. In this report, we examine the role of ubiquitin conjugation in this process. First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis. Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis. Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis. This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached. These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc. PMID:10677350

  8. Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome.

    PubMed

    Reichard, Eden L; Chirico, Giavanna G; Dewey, William J; Nassif, Nicholas D; Bard, Katelyn E; Millas, Nickolas E; Kraut, Daniel A

    2016-08-26

    In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process. PMID:27405762

  9. Solid-state enzyme deactivation in air and in organic solvents

    SciTech Connect

    Toscano, G.; Pirozzi, D.; Maremonti, M.; Greco, G. Jr. . Dipartimento di Ingegneria Chimica)

    1994-09-05

    Thermal deactivation of solid-state acid phosphatase is analyzed, both in the presence and in the absence of organic solvents. The thermal deactivation profile departs from first order kinetics and shows an unusual, temperature-dependent, asymptotic value of residual activity. The process is described by a phenomenological equation, whose theoretical implications are also discussed. The total amount of buffer salts in the enzyme powder dramatically affects enzyme stability in the range 70 to 105 C. The higher salt/protein ratio increases the rate of thermal deactivation. The deactivation rate is virtually unaffected by the presence of organic solvents, independent of their hydrophilicity.

  10. Stabilization of Different Types of Transition States in a Single Enzyme Active Site: QM/MM Analysis of Enzymes in the Alkaline Phosphatase Superfamily

    PubMed Central

    Hou, Guanhua; Cui, Qiang

    2013-01-01

    The first step for the hydrolysis of a phosphate monoester (pNPP2−) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bi-metallic site plays a minor role in accommodating multiple types of transition states, and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states. PMID:23786365

  11. Ubiquitin: molecular modeling and simulations.

    PubMed

    Ganoth, Assaf; Tsfadia, Yossi; Wiener, Reuven

    2013-11-01

    The synthesis and destruction of proteins are imperative for maintaining their cellular homeostasis. In the 1970s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered that certain proteins are tagged by ubiquitin before degradation, a discovery that awarded them the 2004 Nobel Prize in Chemistry. Compelling data gathered during the last several decades show that ubiquitin plays a vital role not only in protein degradation but also in many cellular functions including DNA repair processes, cell cycle regulation, cell growth, immune system functionality, hormone-mediated signaling in plants, vesicular trafficking pathways, regulation of histone modification and viral budding. Due to the involvement of ubiquitin in such a large number of diverse cellular processes, flaws and impairments in the ubiquitin system were found to be linked to cancer, neurodegenerative diseases, genetic disorders, and immunological disorders. Hence, deciphering the dynamics and complexity of the ubiquitin system is of significant importance. In addition to experimental techniques, computational methodologies have been gaining increasing influence in protein research and are used to uncover the structure, stability, folding, mechanism of action and interactions of proteins. Notably, molecular modeling and molecular dynamics simulations have become powerful tools that bridge the gap between structure and function while providing dynamic insights and illustrating essential mechanistic characteristics. In this study, we present an overview of molecular modeling and simulations of ubiquitin and the ubiquitin system, evaluate the status of the field, and offer our perspective on future progress in this area of research. PMID:24113788

  12. Endosomal Transportation via Ubiquitination

    PubMed Central

    Piper, Robert C.; Lehner, Paul J.

    2011-01-01

    Cell survival, growth, differentiation, and homeostasis all rely on exquisite control over the abundance of particular cell surface membrane proteins. Cell surface proteins must respond appropriately to environmental as well as intracellular cues, often undergoing regulated internalization and lysosomal degradation. In addition, cell surface proteins can sustain damage and must be recognized and removed. A unifying mechanism has now emerged for the trafficking of damaged and downregulated proteins to the lysosome by their attachment to ubiquitin, which serves as a sorting signal for clathrin-mediated internalization and sorting into the lumen of late endosomes. Major questions remain as to how this broad system is governed, how it is adapted to meet the needs of particular cell surface proteins, and whether Ub serves as more than a one-way ticket to the lysosome for degradation. Here we highlight recent insights into these questions and the challenges that remain. PMID:21955996

  13. Quantifying Ubiquitin Signaling

    PubMed Central

    Ordureau, Alban; Münch, Christian; Harper, J. Wade

    2015-01-01

    Ubiquitin (UB)-driven signaling systems permeate biology, and are often integrated with other types of post-translational modifications (PTMs), most notably phosphorylation. Flux through such pathways is typically dictated by the fractional stoichiometry of distinct regulatory modifications and protein assemblies as well as the spatial organization of pathway components. Yet, we rarely understand the dynamics and stoichiometry of rate-limiting intermediates along a reaction trajectory. Here, we review how quantitative proteomic tools and enrichment strategies are being used to quantify UB-dependent signaling systems, and to integrate UB signaling with regulatory phosphorylation events. A key regulatory feature of ubiquitylation is that the identity of UB chain linkage types can control downstream processes. We also describe how proteomic and enzymological tools can be used to identify and quantify UB chain synthesis and linkage preferences. The emergence of sophisticated quantitative proteomic approaches will set a new standard for elucidating biochemical mechanisms of UB-driven signaling systems. PMID:26000850

  14. Ubiquitin pathways in neurodegenerative disease

    PubMed Central

    Atkin, Graham; Paulson, Henry

    2014-01-01

    Control of proper protein synthesis, function, and turnover is essential for the health of all cells. In neurons these demands take on the additional importance of supporting and regulating the highly dynamic connections between neurons that are necessary for cognitive function, learning, and memory. Regulating multiple unique synaptic protein environments within a single neuron while maintaining cell health requires the highly regulated processes of ubiquitination and degradation of ubiquitinated proteins through the proteasome. In this review, we examine the effects of dysregulated ubiquitination and protein clearance on the handling of disease-associated proteins and neuronal health in the most common neurodegenerative diseases. PMID:25071440

  15. Chemical Synthesis of Ubiquitin Chains.

    PubMed

    Hemantha, Hosahalli P; Bondalapati, Somasekhar; Singh, Sumeet K; Brik, Ashraf

    2015-01-01

    Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di-ubiquitin chains. PMID:26629614

  16. Ubiquitin and Ubiquitin-Modified Proteins Activate the Pseudomonas aeruginosa T3SS Cytotoxin, ExoU

    PubMed Central

    Anderson, David M.; Schmalzer, Katherine M.; Sato, Hiromi; Casey, Monika; Terhune, Scott S.; Haas, Arthur L.; Feix, Jimmy B.; Frank, Dara W.

    2011-01-01

    Summary Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU posseses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination. PMID:22040088

  17. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  18. TRIM32 is an E3 ubiquitin ligase for dysbindin.

    PubMed

    Locke, Matthew; Tinsley, Caroline L; Benson, Matthew A; Blake, Derek J

    2009-07-01

    Mutations in the gene encoding tripartite motif protein 32 (TRIM32) cause two seemingly diverse diseases: limb-girdle muscular dystrophy type 2H (LGMD2H) or sarcotubular myopathy (STM) and Bardet-Biedl syndrome type 11(BBS11). Although TRIM32 is involved in protein ubiquitination, its substrates and the molecular consequences of disease-causing mutations are poorly understood. In this paper, we show that TRIM32 is a widely expressed ubiquitin ligase that is localized to the Z-line in skeletal muscle. Using the yeast two-hybrid system, we found that TRIM32 binds and ubiquitinates dysbindin, a protein implicated in the genetic aetiology of schizophrenia, augmenting its degradation. Small-interfering RNA-mediated knock-down of TRIM32 in myoblasts resulted in elevated levels of dysbindin. Importantly, the LGMD2H/STM-associated TRIM32 mutations, D487N and R394H impair ubiquitin ligase activity towards dysbindin and were mislocalized in heterologous cells. These mutants were able to self-associate and also co-immunoprecipitated with wild-type TRIM32 in transfected cells. Furthermore, the D487N mutant could bind to both dysbindin and its E2 enzyme but was defective in monoubiquitination. In contrast, the BBS11 mutant P130S did not show any biochemical differences compared with the wild-type protein. Our data identify TRIM32 as a regulator of dysbindin and demonstrate that the LGMD2H/STM mutations may impair substrate ubiquitination. PMID:19349376

  19. Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

    PubMed Central

    Landré, Vivien; Rotblat, Barak; Melino, Sonia; Bernassola, Francesca; Melino, Gerry

    2014-01-01

    The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides. PMID:25237759

  20. SUMO, Ubiquitin, UBL Proteins: Implications For Human Diseases - Fifth International Conference.

    PubMed

    Skaug, Brian; Chen, Zhijian J

    2010-04-01

    The fifth international conference on SUMO, Ubiquitin, UBL Proteins: Implications for Human Diseases, held in Houston, included topics covering the latest advances and new targets in the field of protein modification. This conference report highlights selected presentations on the structural characterization of ubiquitination and SUMOylation machinery; the regulation of ubiquitination enzymes, including E3 ligases; the functions and mechanism of action of SUMO-targeted ubiquitin ligases (STUbLs); the regulation of gene expression by SUMO; non-degradative functions of ubiquitin and SUMO in signal transduction; mechanisms and functions of ISG15 conjugation; the interaction of pathogens with host cell SUMOylation machinery; and stabilization of the Axin protein. Investigational drugs discussed include MLN-4924 (Millennium Pharmaceuticals Inc). PMID:20373248

  1. Inhibitors of apoptosis catch ubiquitin.

    PubMed

    Rajalingam, Krishnaraj; Dikic, Ivan

    2009-01-01

    IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation. PMID:19061481

  2. Novel strategies to target the ubiquitin proteasome system in multiple myeloma

    PubMed Central

    Lub, Susanne; Maes, Ken; Menu, Eline; De Bruyne, Elke; Vanderkerken, Karin; Van Valckenborgh, Els

    2016-01-01

    Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of plasma cells in the bone marrow (BM). The success of the proteasome inhibitor bortezomib in the treatment of MM highlights the importance of the ubiquitin proteasome system (UPS) in this particular cancer. Despite the prolonged survival of MM patients, a significant amount of patients relapse or become resistant to therapy. This underlines the importance of the development and investigation of novel targets to improve MM therapy. The UPS plays an important role in different cellular processes by targeted destruction of proteins. The ubiquitination process consists of enzymes that transfer ubiquitin to proteins targeting them for proteasomal degradation. An emerging and promising approach is to target more disease specific components of the UPS to reduce side effects and overcome resistance. In this review, we will focus on different components of the UPS such as the ubiquitin activating enzyme E1, the ubiquitin conjugating enzyme E2, the E3 ubiquitin ligases, the deubiquitinating enzymes (DUBs) and the proteasome. We will discuss their role in MM and the implications in drug discovery for the treatment of MM. PMID:26695547

  3. Orthogonal Ubiquitin Transfer through Engineered E1-E2 Cascades for Protein Ubiquitination

    PubMed Central

    Zhao, Bo; Bhuripanyo, Karan; Zhang, Keya; Kiyokawa, Hiroaki; Schindelin, Hermann; Yin, Jun

    2014-01-01

    SUMMARY Protein modification by ubiquitin (UB) controls diverse cellular processes. UB is conjugated to cellular proteins by sequential transfer through an E1-E2-E3 enzymatic cascade. The cross-activities of 2 E1s, 50 E2s and thousands of E3s encoded by the human genome make it difficult to identify the substrate proteins of a specific E3 enzyme in the cell. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination. PMID:23102221

  4. Unidirectional steady state rates of central metabolism enzymes measured simultaneously in a living plant tissue.

    PubMed

    Roscher, A; Emsley, L; Raymond, P; Roby, C

    1998-09-25

    The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme. PMID:9737962

  5. Structure And Function of the Yeast U-Box-Containing Ubiquitin Ligase Ufd2p

    SciTech Connect

    Tu, D.; Li, W.; Ye, Y.; Brunger, A.T.

    2009-06-04

    Proteins conjugated by Lys-48-linked polyubiquitin chains are preferred substrates of the eukaryotic proteasome. Polyubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). Occasionally, these enzymes only assemble short ubiquitin oligomers, and their extension to full length involves a ubiquitin elongating factor termed E4. Ufd2p, as the first E4 identified to date, is involved in the degradation of misfolded proteins of the endoplasmic reticulum and of a ubiquitin-{beta}-GAL fusion substrate in Saccharomyces cerevisiae. The mechanism of action of Ufd2p is unknown. Here we describe the crystal structure of the full-length yeast Ufd2p protein. Ufd2p has an elongated shape consisting of several irregular Armadillo-like repeats with two helical hairpins protruding from it and a U-box domain flexibly attached to its C terminus. The U-box of Ufd2p has a fold similar to that of the RING (Really Interesting New Gene) domain that is present in certain ubiquitin ligases. Accordingly, Ufd2p has all of the hallmarks of a RING finger-containing ubiquitin ligase: it associates with its cognate E2 Ubc4p via its U-box domain and catalyzes the transfer of ubiquitin from the E2 active site to Ufd2p itself or to an acceptor ubiquitin molecule to form unanchored diubiquitin oligomers. Thus, Ufd2p can function as a bona fide E3 ubiquitin ligase to promote ubiquitin chain elongation on a substrate.

  6. Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

    SciTech Connect

    Maiti, Tushar K.; Permaul, Michelle; Boudreaux, David A.; Mahanic, Christina; Mauney, Sarah; Das, Chittaranjan

    2012-10-25

    Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.

  7. Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

    PubMed

    Morey, Trevor M; Albers, Shawn; Shilton, Brian H; Rylett, R Jane

    2016-05-01

    Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by

  8. Ubiquitin chain diversity at a glance.

    PubMed

    Akutsu, Masato; Dikic, Ivan; Bremm, Anja

    2016-03-01

    Ubiquitin plays an essential role in modulating protein functions, and deregulation of the ubiquitin system leads to the development of multiple human diseases. Owing to its molecular features, ubiquitin can form various homo- and heterotypic polymers on substrate proteins, thereby provoking distinct cellular responses. The concept of multifaceted ubiquitin chains encoding different functions has been substantiated in recent years. It has been established that all possible ubiquitin linkage types are utilized for chain assembly and propagation of specific signals in vivo. In addition, branched ubiquitin chains and phosphorylated ubiquitin molecules have been put under the spotlight recently. The development of novel technologies has provided detailed insights into the structure and function of previously poorly understood ubiquitin signals. In this Cell Science at a Glance article and accompanying poster, we provide an update on the complexity of ubiquitin chains and their physiological relevance. PMID:26906419

  9. ATLs and BTLs, plant-specific and general eukaryotic structurally-related E3 ubiquitin ligases.

    PubMed

    Guzmán, Plinio

    2014-02-01

    Major components of the ubiquitin proteasome system are the enzymes that operate on the transfer of ubiquitin to selected target substrate, known as ubiquitin ligases. The RING finger is a domain that is present in key classes of ubiquitin ligases. This domain coordinates the interaction with a suitable E2 conjugase and the transfer of ubiquitin from the E2 to protein targets. Additional domains coupled to the same polypeptide are important for modulating the function of these ubiquitin ligases. Plants contain several types of E3 ubiquitin ligases that in many cases have expanded as multigene families. Some families are specific to the plant lineage, whereas others may have a common ancestor among plants and other eukaryotic lineages. Arabidopsis Tóxicos en Levadura (ATLs) and BCA2 zinc finger ATLs (BTLs) are two families of ubiquitin ligases that share some common structural features. These are intronless genes that encode a highly related RING finger domain, and yet during evolutionary history, their mode of gene expansion and function is rather different. In each of these two families, the co-occurrence of transmembrane helices or C2/C2 (BZF finger) domains with a selected variation on the RING finger has been subjected to strong selection pressure in order to preserve their unique domain architectures during evolution. PMID:24388516

  10. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes.

    PubMed

    Bekker-Jensen, Simon; Rendtlew Danielsen, Jannie; Fugger, Kasper; Gromova, Irina; Nerstedt, Annika; Lukas, Claudia; Bartek, Jiri; Lukas, Jiri; Mailand, Niels

    2010-01-01

    Regulatory ubiquitylation is emerging as an important mechanism to protect genome integrity in cells exposed to DNA damage. However, the spectrum of known ubiquitin regulators of the DNA damage response (DDR) is limited and their functional interplay is poorly understood. Here, we identify HERC2 as a factor that regulates ubiquitin-dependent retention of repair proteins on damaged chromosomes. In response to ionising radiation (IR), HERC2 forms a complex with RNF8, a ubiquitin ligase involved in the DDR. The HERC2-RNF8 interaction requires IR-inducible phosphorylation of HERC2 at Thr 4827, which in turn binds to the forkhead-associated (FHA) domain of RNF8. Mechanistically, we provide evidence that HERC2 facilitates assembly of the ubiquitin-conjugating enzyme Ubc13 with RNF8, thereby promoting DNA damage-induced formation of Lys 63-linked ubiquitin chains. We also show that HERC2 interacts with, and maintains the levels of, RNF168, another ubiquitin ligase operating downstream of RNF8 (Refs 7, 8). Consequently, knockdown of HERC2 abrogates ubiquitin-dependent retention of repair factors such as 53BP1, RAP80 and BRCA1. Together with the increased radiosensitivity of HERC2-depleted cells, these results uncover a regulatory layer in the orchestration of protein interactions on damaged chromosomes and they underscore the role of ubiquitin-mediated signalling in genome maintenance. PMID:20023648

  11. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d. PMID:26766115

  12. Modulation of Heme/Substrate Binding Cleft of Neuronal Nitric-oxide Synthase (nNOS) Regulates Binding of Hsp90 and Hsp70 Proteins and nNOS Ubiquitination*

    PubMed Central

    Peng, Hwei-Ming; Morishima, Yoshihiro; Pratt, William B.; Osawa, Yoichi

    2012-01-01

    Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885–22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with NG-nitro-l-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca2+-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination. PMID:22128174

  13. Development of microbial-enzyme-mediated decomposition model parameters through steady-state and dynamic analyses

    SciTech Connect

    Wang, Gangsheng; Post, Wilfred M; Mayes, Melanie

    2013-01-01

    We developed a Microbial-ENzyme-mediated Decomposition (MEND) model, based on the Michaelis-Menten kinetics, that describes the dynamics of physically defined pools of soil organic matter (SOC). These include particulate, mineral-associated, dissolved organic matter (POC, MOC, and DOC, respectively), microbial biomass, and associated exoenzymes. The ranges and/or distributions of parameters were determined by both analytical steady-state and dynamic analyses with SOC data from the literature. We used an improved multi-objective parameter sensitivity analysis (MOPSA) to identify the most important parameters for the full model: maintenance of microbial biomass, turnover and synthesis of enzymes, and carbon use efficiency (CUE). The model predicted an increase of 2 C (baseline temperature =12 C) caused the pools of POC-Cellulose, MOC, and total SOC to increase with dynamic CUE and decrease with constant CUE, as indicated by the 50% confidence intervals. Regardless of dynamic or constant CUE, the pool sizes of POC, MOC, and total SOC varied from 8% to 8% under +2 C. The scenario analysis using a single parameter set indicates that higher temperature with dynamic CUE might result in greater net increases in both POC-Cellulose and MOC pools. Different dynamics of various SOC pools reflected the catalytic functions of specific enzymes targeting specific substrates and the interactions between microbes, enzymes, and SOC. With the feasible parameter values estimated in this study, models incorporating fundamental principles of microbial-enzyme dynamics can lead to simulation results qualitatively different from traditional models with fast/slow/passive pools.

  14. Solid state bioconversion of oil palm biomass for ligninase enzyme production.

    PubMed

    Alam, Md Zahangir; Mahmat, Mohd Erman; Muhammad, Nurdina

    2005-01-01

    A laboratory-scale study of bioconversion of local lignocellulosic material, oil palm biomass (OPB) was conducted by evaluating the enzyme production through microbial treatment in solid state bioconversion (SSB). OPB in the form of empty fruit bunches (EFB) was used as a solid substrate and treated with the white-rot fungus, Phanerochaete chrysosporium, to produce ligninase. The results showed that the highest ligninase activity of 400.27 U/liter was obtained at day 12 of fermentation. While the optimum study indicated the enzyme production of 1472.8 U/liter with moisture content of 50%, 578.7 U/liter with 10% v/w of inoculum size, and 721.8 U/liter with co-substrate concentration of 1% (w/w) at days 9, 9 and 12 of fungal treatment, respectively. The parameters glucosamine and reducing sugar were observed to evaluate the growth and substrate utilization in the experiment. PMID:16317964

  15. Compound I in horseradish peroxidase enzyme: Magnetic state assessment by quadratric configuration interaction calculations

    NASA Astrophysics Data System (ADS)

    Zazza, Costantino; Sanna, Nico; Tatoli, Simone; Aschi, Massimiliano; Palma, Amedeo

    Quadratic configuration interaction procedure with single and double electronic excitations (QCISD) has been used, for the first time, to calculate the electronic structure of the Compound I (CpdI), which represents a key intermediate in the catalytic cycle of Horseradish Peroxidase (HRP) enzyme. The QCISD method is applied to lowest quasi-isoenergetic doublet and quartet spin multiplicity and results compared with density functional theory (DFT/B3LYP) data. This investigation shows that, at present, QCISD is more accurate than DFT-based approach in discriminating between the two lowest magnetic states of CpdI complex in HRP enzyme. Such a result opens the possibility of theoretically addressing the reaction mechanism leading to CpdI complex in HRP using a correlated wavefunction based approach.

  16. Production of Cellulolytic and Hemicellulolytic Enzymes From Aureobasidium pulluans on Solid State Fermentation

    NASA Astrophysics Data System (ADS)

    Leite, Rodrigo Simões Ribeiro; Bocchini, Daniela Alonso; da Silva Martins, Eduardo; Silva, Dênis; Gomes, Eleni; da Silva, Roberto

    This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme remaining 100% active when incubated at 75°C for 1 h.

  17. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    PubMed Central

    Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

    2014-01-01

    . We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease. PMID:24660806

  18. A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development*

    PubMed Central

    Franco, Maribel; Seyfried, Nicholas T.; Brand, Andrea H.; Peng, Junmin; Mayor, Ugo

    2011-01-01

    Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system. PMID:20861518

  19. Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

    SciTech Connect

    Artavanis-Tsakonas, Katerina; Weihofen, Wilhelm A.; Antos, John M.; Coleman, Bradley I.; Comeaux, Christy A.; Duraisingh, Manoj T.; Gaudet, Rachelle; Ploegh, Hidde L.

    2010-03-29

    Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.

  20. Ubiquitin-specific protease 24 negatively regulates abscisic acid signalling in Arabidopsis thaliana.

    PubMed

    Zhao, Jinfeng; Zhou, Huapeng; Zhang, Ming; Gao, Yanan; Li, Long; Gao, Ying; Li, Ming; Yang, Yuhong; Guo, Yan; Li, Xueyong

    2016-02-01

    Abscisic acid (ABA) is an important plant hormone integrating environmental stress and plant growth. Protein ubiquitination and deubiquitination are reversible processes catalysed by E3 ubiquitin ligase and deubiquitinating enzyme, respectively. Lots of E3 ubiquitin ligase and transcriptional factors modified by ubiquitination were reported to modulate ABA signalling. However, no deubiquitinating enzyme has been identified that functions in ABA signalling until now. Here, we isolated an ABA overly sensitive mutant, ubp24, in which the gene encoding ubiquitin-specific protease 24 (UBP24, At4g30890) was disrupted by a T-DNA insertion. The ubp24 mutant was hypersensitive to ABA and salt stress in both post-germinative growth and seedling growth. However, stomata closure in the ubp24 mutant was less sensitive to ABA, and the ubp24 mutant showed drought sensitivity. UBP24 possessed deubiquitinating enzyme activity, and the activity was essential for UBP24 function. Additionally, UBP24 formed homodimer in vivo. UBP24 was genetically upstream of ABI2, and the phosphatase activity of protein phosphatase 2C was decreased in the ubp24 mutant compared with the wild type in the presence of ABA. These results uncover an important regulatory role for the ubiquitin-specific protease in response to ABA and salt stress in plant. PMID:26290265

  1. Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

    PubMed Central

    Bulatov, Emil; Ciulli, Alessio

    2015-01-01

    In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

  2. Intracellular Dynamics of the Ubiquitin-Proteasome-System.

    PubMed

    Chowdhury, Maisha; Enenkel, Cordula

    2015-01-01

    The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body's cells. PMID:26339477

  3. The integral membrane enzyme PagP alternates between two dynamically distinct states.

    PubMed

    Hwang, Peter M; Bishop, Russell E; Kay, Lewis E

    2004-06-29

    PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria. A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP beta barrel. The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located. To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity. Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T). The kinetics and thermodynamics of the interchange have been investigated by (1)H-(15)N NMR spectroscopy, with Delta H = -10.7 kcal/mol and Delta S = -37.5 cal/mol.K for the R--> T transition. A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel. In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible. Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis. The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP. PMID:15210985

  4. Salivary enzymes and exhaled air affect Streptococcus salivarius growth and physiological state in complemented artificial saliva.

    PubMed

    Roger, P; Harn-Arsa, S; Delettre, J; Béal, C

    2011-12-01

    To better understand the phenomena governing the establishment of the oral bacterium Streptococcus salivarius in the mouth, the effect of some environmental factors has been studied in complemented artificial saliva, under oral pH and temperature conditions. Three salivary enzymes at physiological concentrations were tested: peroxidase, lysozyme and amylase, as well as injection of exhaled air. Injection of air containing 5% CO2 and 16% O2 induced a deleterious effect on S. salivarius K12, mainly by increasing redox potential. Addition of lysozyme slightly affected the physiological state of S. salivarius by altering membrane integrity. In contrast, peroxidase was not detrimental as it made it possible to decrease the redox potential. The addition of amylase reduced the specific growth rate of S. salivarius by formation of a complex with amylase and mucins, but led to high final biomass, as a result of enzymatic degradation of some nutrients. Finally, this work demonstrated that salivary enzymes had a slight impact on S. salivarius behaviour. It can thus be concluded that this bacterium was well adapted to in-mouth conditions, as it was able to resist certain salivary enzymes, even if tolerance to expired air was affected, as a result of an increased redox potential. PMID:21892611

  5. Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site.

    PubMed

    Roston, Daniel; Demapan, Darren; Cui, Qiang

    2016-06-15

    A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care. PMID:27186960

  6. Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states.

    PubMed

    Hubin, Ellen; Cioffi, Federica; Rozenski, Jef; van Nuland, Nico A J; Broersen, Kerensa

    2016-06-01

    To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met35-Val36 as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity. PMID:26968463

  7. SCF ubiquitin ligase targeted therapies

    PubMed Central

    Skaar, Jeffrey R.; Pagan, Julia K.; Pagano, Michele

    2015-01-01

    Summary The recent clinical successes of inhibitors of the proteasome for the treatment of cancer have highlighted the therapeutic potential of this protein degradation system. Proteasome inhibitors prevent the degradation of numerous proteins, so increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a large number of processes at the cellular and organismal levels, and their misregulation is implicated in many pathologies. SCF ligases are characterized by a high specificity for their substrates, so they represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This review will explore and discuss potential strategies to target SCF-mediated biology to treat human diseases. PMID:25394868

  8. Isotopic mapping of transition-state structural features associated with enzymic catalysis of methyl transfer

    SciTech Connect

    Rodgers, J.; Femec, D.A.; Schowen, R.L.

    1982-06-16

    For comparison of the molecular structures of nonenzymic and enzymic sulfur-to-oxygen transmethylation transition states by the use of kinetic isotope effects, a series of isotopic maps is produced. In these, contours of constant isotope effect are displayed vs. the Pauling bond orders B/sub CS/ and B/sub CO/, for the carbon-sulfur and carbon-oxygen bonds, respectively, taken as independent variables to describe the transition states. Maps are calculated by the BEBOVIB approach for k(CH/sub 3/)/k(CD/sub 3/), k(/sup 12/CH/sub 3/)/k(/sup 13/CH/sub 3/), k(/sup 16/O)/k(/sup 18/O), and k(/sup 32/S)/k(/sup 34/S), with two models for the reaction coordinate, two force-field assumptions, and four temperatures. Nonenzymic isotope effects and isotope effects for catechol-O-methyltransferase action are then used to construct figures on the CH/sub 3//CD/sub 3/ and /sup 12/CH/sub 3///sup 13/CH/sub 3/ maps which correspond to allowed spaces of transition-states structures. Superposition of the figures yields the spaces of transition-state structures simultaneously consistent with both hydrogen and carbon isotope effects. It is concluded that the enzyme compresses the S/sub N/2 transition state and that the compression of the C-O and C-S bonds may well be of the order of 0.15 A per bond and could conceivably be more than twice as large.

  9. A Ubl/ubiquitin switch in the activation of Parkin

    PubMed Central

    Sauvé, Véronique; Lilov, Asparouh; Seirafi, Marjan; Vranas, Marta; Rasool, Shafqat; Kozlov, Guennadi; Sprules, Tara; Wang, Jimin; Trempe, Jean-François; Gehring, Kalle

    2015-01-01

    Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding. PMID:26254305

  10. A Ubl/ubiquitin switch in the activation of Parkin.

    PubMed

    Sauvé, Véronique; Lilov, Asparouh; Seirafi, Marjan; Vranas, Marta; Rasool, Shafqat; Kozlov, Guennadi; Sprules, Tara; Wang, Jimin; Trempe, Jean-François; Gehring, Kalle

    2015-10-14

    Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding. PMID:26254305

  11. Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

    PubMed Central

    Andrews, Logan D.; Fenn, Tim D.; Herschlag, Daniel

    2013-01-01

    Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 1022-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound Pi was determined from pH dependencies of the binding of Pi and tungstate, a Pi analog lacking titratable protons over the pH range of 5–11, and from the 31P chemical shift of bound Pi. The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥108-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and Pi binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition

  12. Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

    PubMed

    Li, Minghui; Kales, Stephen C; Ma, Ke; Shoemaker, Benjamin A; Crespo-Barreto, Juan; Cangelosi, Andrew L; Lipkowitz, Stanley; Panchenko, Anna R

    2016-02-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  13. Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.

    PubMed

    Soss, Sarah E; Rose, Kristie L; Hill, Salisha; Jouan, Sophie; Chazin, Walter J

    2015-01-01

    The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate. PMID:26010904

  14. Sperm ubiquitination in epididymal feline semen.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Varesi, Sara; Nonnis, Simona; Maffioli, Elisa; Negri, Armando; Tedeschi, Gabriella; Luvoni, Gaia Cecilia

    2014-09-01

    Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in

  15. Demonstration of ATP-dependent, ubiquitin-conjugating activities in higher plants

    SciTech Connect

    Vierstra, R.D.

    1986-05-01

    Ubiquitin is a 76 amino acid eucaryotic polypeptide with several important functions that arise from its ability to become covalently ligated to other cytoplasmic and nuclear proteins. Ubiquitin has recently been purified from higher plants and found to be very homologous, both structurally and functionally, to the highly conserved animal form. Here, the authors present evidence that crude extracts from several plants have the capacity to conjugate ubiquitin to other plant proteins using either labelled human or oat ubiquitin as a substrate. The reaction requires ATP and can be detected in soluble extracts from dry seeds, etiolated shoots and green leaves, with etiolated shoot extracts having the highest activity. Mixing experiments indicate that the low activity found with green tissue in vitro is the result of an endogenous inhibitor. The conjugating activities are extremely labile with a half-life of 20 min at 30/sup 0/C. The addition of polyphenol inhibitors fails to protect the system from this inactivation. In addition to conjugating activities, crude plant extracts also have ATP-independent activities that degrade ubiquitin conjugates. These results provide the first evidence that higher plants contain the necessary enzymes for ubiquitin conjugate formation. Further analysis of these activities should help clarify the functions of ubiquitination in plants.

  16. Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

    PubMed Central

    Sepulveda, P; Lopez-Ribot, J L; Gozalbo, D; Cervera, A; Martinez, J P; Chaffin, W L

    1996-01-01

    We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures. PMID:8926122

  17. A Novel Ubiquitin-Specific Protease, UBP43, Cloned from Leukemia Fusion Protein AML1-ETO-Expressing Mice, Functions in Hematopoietic Cell Differentiation

    PubMed Central

    Liu, Li-Qin; Ilaria, Robert; Kingsley, Paul D.; Iwama, Atsushi; van Etten, Richard A.; Palis, James; Zhang, Dong-Er

    1999-01-01

    Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation. PMID:10082570

  18. A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

    PubMed

    Liu, L Q; Ilaria, R; Kingsley, P D; Iwama, A; van Etten, R A; Palis, J; Zhang, D E

    1999-04-01

    Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation. PMID:10082570

  19. Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP.

    PubMed

    Scaglione, K Matthew; Zavodszky, Eszter; Todi, Sokol V; Patury, Srikanth; Xu, Ping; Rodríguez-Lebrón, Edgardo; Fischer, Svetlana; Konen, John; Djarmati, Ana; Peng, Junmin; Gestwicki, Jason E; Paulson, Henry L

    2011-08-19

    The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating, and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination, ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3. PMID:21855799

  20. Smurf1 represses TNF-α production through ubiquitination and destabilization of USP5.

    PubMed

    Qian, Guanghui; Ren, Ying; Zuo, Yibo; Yuan, Yukang; Zhao, Peng; Wang, Xiaofang; Cheng, Qiao; Liu, Jin; Zhang, Liting; Guo, Tingting; Liu, Chang; Zheng, Hui

    2016-06-01

    Ubiquitin-specific peptidase 5 (USP5) has been demonstrated to be critical for the production of Tumor Necrosis Factor-alpha (TNF-α), a pivotal mediator for inflammatory responses. Besides, USP5 regulates p53 activation and DNA repair. However, the mechanism underlying the regulation of USP5, especially its responsible E3 ligase is still unclear. Here we found that Smad ubiquitination regulatory factor 1 (Smurf1) down regulated protein expression of USP5, and the E3 enzyme activity of Smurf1 was required for this function. We also revealed that Smurf1 interacted with USP5 and mediated its degradation via the ubiquitin proteasome pathway. Consequently, Smurf1 inhibited the production of TNF-α through down-regulation of USP5. Taken together, our study for the first time clarified that the E3 ligase Smurf1 regulates USP5 protein stability and USP5-mediated TNF-α production through the ubiquitin proteasome pathway. PMID:27133717

  1. Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control

    PubMed Central

    Mocciaro, Annamaria; Rape, Michael

    2012-01-01

    The covalent modification of proteins with ubiquitin is required for accurate cell division in all eukaryotes. Ubiquitylation depends on an enzymatic cascade, in which E3 enzymes recruit specific substrates for modification. Among ~600 human E3s, the SCF (Skp1–cullin1–F-box) and the APC/C (anaphase-promoting complex/cyclosome) are known for driving the degradation of cell cycle regulators to accomplish irreversible cell cycle transitions. The cell cycle machinery reciprocally regulates the SCF and APC/C through various mechanisms, including the modification of these E3s or the binding of specific inhibitors. Recent studies have provided new insight into the intricate relationship between ubiquitylation and the cell division apparatus as they revealed roles for atypical ubiquitin chains, new mechanisms of substrate and E3 regulation, as well as extensive crosstalk between ubiquitylation enzymes. Here, we review these emerging regulatory mechanisms of ubiquitin-dependent cell cycle control and discuss how their manipulation might provide therapeutic benefits in the future. PMID:22357967

  2. Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

    SciTech Connect

    Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Dani; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.

    2013-08-07

    Ubiquitination is an abundant post-translational modification that consists of covalent attachment of a 76 amino acid residue polypeptide, ubiquitin, to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions. Affinity enrichment of ubiquitinated proteins has enabled the global analysis of this key modification. In this context, the use of ubiquitin-binding domains (UBDs) is a promising, but poorly explored alternative to more broadly used immune-affinity or tagged affinity enrichment methods. Herein we evaluate the application of eight selected UBDs with differing and contrasting affinities for ubiquitination states. We performed a micro-scale proteomic comparison, leading to the identification of ~200 ubiquitinated protein candidates per UBD to facilitate comparisons. Western blot analysis using anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggests that UBDs from Dsk2 and ubiquilin-1 have the broadest specificity capturing most types of ubiquitination, whereas the one from NBR1 seems to be more selective to polyubiquitination. Our data demonstrate that with optimized purification conditions UBDs can be a useful tool for proteomic applications.

  3. Functional assessment of ubiquitin-depended processes under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Zhabereva, Anastasia; Shenkman, Boris S.; Gainullin, Murat; Gurev, Eugeny; Kondratieva, Ekaterina; Kopylov, Arthur

    Ubiquitylation, a widespread and important posttranslational modification of eukaryotic proteins, controls a multitude of critical cellular processes, both in normal and pathological conditions. The present work aims to study involvement of ubiquitin-dependent regulation in adaptive response to the external stimuli. Experiments were carried out on C57BL/6 mice. The microgravity state under conditions of real spaceflight on the biosatellite “BION-M1” was used as a model of stress impact. Additionally, number of control series including the vivarium control and experiments in Ground-based analog were also studied. The aggregate of endogenously ubiquitylated proteins was selected as specific feature of ubiquitin-dependent processes. Dynamic changes of modification pattern were characterized in liver tissue by combination of some methods, particularly by specific isolation of explicit protein pool, followed by immunodetection and/or mass spectrometry-based identification. The main approach includes specific extraction of proteins, modified by multiubiquitin chains of different length and topology. For this purpose two techniques were applied: 1) immunoprecipitation with antibodies against ubiquitin and/or multiubiquitin chains; 2) pull-down using synthetic protein construct termed Tandem Ubiquitin Binding Entities (TUBE, LifeSensors). TUBE represents fusion protein, composed of well characterized ubiquitin-binding domains, and thereby allows specific high-affinity binding and extraction of ubiquitylated proteins. Resulting protein fractions were analyzed by immunoblotting with antibodies against different types of multiubiquitin chains. Using this method we mapped endogenously modified proteins involved in two different types of ubiquitin-dependent processes, namely catabolic and non-catabolic ubiquitylation, in liver tissues, obtained from both control as well as experimental groups of animals, mentioned above. Then, isolated fractions of ubiquitylated proteins

  4. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    PubMed Central

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea), quotient energy (Q10), Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

  5. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation.

    PubMed

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m , and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

  6. Intrinsic Enzyme Dynamics in the Unbound State and Relation to Allosteric Regulation

    PubMed Central

    Bahar, Ivet; Chennubhotla, Chakra; Tobi, Dror

    2007-01-01

    In recent years, there has been a surge in the number of studies exploring the relationship between proteins’ equilibrium dynamics and structural changes involved in function. An emerging concept, supported by both theory and experiments, is that under native state conditions proteins have an intrinsic ability to sample conformations that meet functional requirements. A typical example is the ability of enzymes to sample open and closed forms, irrespective of substrate, succeeded by the stabilization of one form (usually closed) upon substrate binding. This ability is structure-encoded, and plays a key role in facilitating allosteric regulation, which suggests complementing the sequence-encodes-structure paradigm of protein science by structure-encodes-dynamics-encodes-function. The emerging connection implies an evolutionary role in selecting/conserving structures based on their ability to achieve functional dynamics, and in turn, selecting sequences that fold into such ‘apt’ structures. PMID:18024008

  7. Gaseous emissions during the solid state fermentation of different wastes for enzyme production at pilot scale.

    PubMed

    Maulini-Duran, Caterina; Abraham, Juliana; Rodríguez-Pérez, Sheila; Cerda, Alejandra; Jiménez-Peñalver, Pedro; Gea, Teresa; Barrena, Raquel; Artola, Adriana; Font, Xavier; Sánchez, Antoni

    2015-03-01

    The emissions of volatile organic compounds (VOC), CH4, N2O and NH3 during the solid state fermentation process of some selected wastes to obtain different enzymes have been determined at pilot scale. Orange peel+compost (OP), hair wastes+raw sludge (HW) and winterization residue+raw sludge (WR) have been processed in duplicate in 50 L reactors to provide emission factors and to identify the different VOC families present in exhaust gaseous emissions. Ammonia emission from HW fermentation (3.2±0.5 kg Mg(-1) dry matter) and VOC emission during OP processes (18±6 kg Mg(-1) dry matter) should be considered in an industrial application of these processes. Terpenes have been the most emitted VOC family during all the processes although the emission of sulphide molecules during HW SSF is notable. The most emitted compound was dimethyl disulfide in HW and WR processes, and limonene in the SSF of OP. PMID:25545090

  8. Multivariable fluctuation theorems in the steady-state cycle kinetics of single enzyme with competing substrates

    NASA Astrophysics Data System (ADS)

    Ge, Hao

    2012-06-01

    We prove the detailed and integral multivariable fluctuation theorems in the steady-state cycle kinetics of single enzyme with competing substrates for any arbitrary time interval [0, t]. It is shown that the moment generating function for the stochastic number of each enzymatic cycle follows the multivariable fluctuation theorems in the form of Kurchan-Lebowitz-Spohn-type symmetry. These symmetry relations associated with different variables are independent of each other, which may help experimentally determine the thermodynamic affinities from the sample trajectories separately. Furthermore, we also obtain the Kawasaki equalities for the fluctuating chemical work done within each enzymatic cycle. The derivation here is directly based on the generalized Haldane equalities, which say that the forward and backward conditional dwell times for each enzymatic cycle have identical distributions. These symmetries are independent of each other, which may help experimentally determine the thermodynamic affinities from the sample trajectories separately.

  9. Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery

    PubMed Central

    Love, Kerry Routenberg; Pandya, Renuka K.; Spooner, Eric; Ploegh, Hidde L.

    2009-01-01

    Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubls and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling (ABPP) uses chemical probes that are active-site directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein. PMID:19256548

  10. Characterization of a ubiquitinated protein which is externally located in African swine fever virions.

    PubMed Central

    Hingamp, P M; Leyland, M L; Webb, J; Twigger, S; Mayer, R J; Dixon, L K

    1995-01-01

    An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein. PMID:7853518