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Sample records for enzyme ubiquitination state

  1. Close identity between alternatively folded state N2 of ubiquitin and the conformation of the protein bound to the ubiquitin-activating enzyme.

    PubMed

    Kitazawa, Soichiro; Kameda, Tomoshi; Kumo, Ayumi; Yagi-Utsumi, Maho; Baxter, Nicola J; Kato, Koichi; Williamson, Mike P; Kitahara, Ryo

    2014-01-28

    We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N2 state is 97% populated. This allows us to characterize the structure of the "pure" N2 state of ubiquitin. The N2 state shows a substantial change in the orientation of strand β5 compared to that of the normal folded N1 state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion.

  2. A Strategy for Modulation of Enzymes in the Ubiquitin System

    PubMed Central

    Ernst, Andreas; Avvakumov, George; Tong, Jiefei; Fan, Yihui; Zhao, Yanling; Alberts, Philipp; Persaud, Avinash; Walker, John R; Neculai, Ana-Mirela; Neculai, Dante; Vorobyov, Andrew; Garg, Pankaj; Beatty, Linda; Chan, Pak-Kei; Juang, Yu-Chi; Landry, Marie-Claude; Yeh, Christina; Zeqiraj, Elton; Karamboulas, Konstantina; Allali-Hassani, Abdellah; Vedadi, Masoud; Tyers, Mike; Moffat, Jason; Sicheri, Frank; Pelletier, Laurence; Durocher, Daniel; Raught, Brian; Rotin, Daniela; Yang, Jianhua; Moran, Michael F; Dhe-Paganon, Sirano; Sidhu, Sachdev S

    2013-01-01

    The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system. PMID:23287719

  3. Ubiquitin enzymes in the regulation of immune responses

    PubMed Central

    Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

    2017-01-01

    Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

  4. Steady-state kinetic studies reveal that the anti-cancer target Ubiquitin-Specific Protease 17 (USP17) is a highly efficient deubiquitinating enzyme.

    PubMed

    Hjortland, Nicole M; Mesecar, Andrew D

    2016-12-15

    USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x10(3)) M(-1)sec(-1)) than other human USPs studied to date. Analytical size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering studies suggest that the quaternary structure of USP17 is a monomer. Steady-state kinetic studies show that USP17 efficiently hydrolyzes both ubiquitin-AMC (kcat = 1.5 sec(-1) and Km = 1.0 μM) and ubiquitin-rhodamine110 (kcat = 1.8 sec(-1) and Km = 2.0 μM) substrates. Ubiquitin chain cleavage assays reveal that USP17 efficiently cleaves di-ubiquitin chains with Lys(11), Lys(33), Lys(48) and Lys(63) linkages and tetra-ubiquitin chains with Lys(11), Lys(48) and Lys(63) linkages but is inefficient in cleaving di-ubiquitin chains with Lys(6), Lys(27), or Lys(29) linkages or linear ubiquitin chains. The substrate specificity of USP17 is most similar to that of USP1, where both USPs display higher specificity than other characterized members of the USP family.

  5. UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination*

    PubMed Central

    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.

    2014-01-01

    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 UBE2E3C145S 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-43K263E ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43K263E 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

  6. Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes

    PubMed Central

    Liu, Xianpeng; Zhao, Bo; Sun, Limin; Bhuripanyo, Karan; Wang, Yiyang; Bi, Yingtao; Davuluri, Ramana V.; Duong, Duc M.; Nanavati, Dhaval; Yin, Jun; Kiyokawa, Hiroaki

    2017-01-01

    Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6. PMID:28134249

  7. Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes.

    PubMed

    Liu, Xianpeng; Zhao, Bo; Sun, Limin; Bhuripanyo, Karan; Wang, Yiyang; Bi, Yingtao; Davuluri, Ramana V; Duong, Duc M; Nanavati, Dhaval; Yin, Jun; Kiyokawa, Hiroaki

    2017-01-30

    Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.

  8. Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer.

    PubMed

    Voutsadakis, Ioannis A

    2013-02-01

    Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Sub-types of breast cancer defined by the expression of steroid hormones and Her2/Neu oncogene have distinct prognosis and undergo different therapies. Besides differing in their phenotype, sub-types of breast cancer display various molecular lesions that participate in their pathogenesis. BRCA1 is one of the common hereditary cancer predisposition genes and encodes for an ubiquitin ligase. Ubiquitin ligases or E3 enzymes participate together with ubiquitin activating enzyme and ubiquitin conjugating enzymes in the attachment of ubiquitin (ubiquitination) in target proteins. Ubiquitination is a post-translational modification regulating multiple cell functions. It also plays important roles in carcinogenesis in general and in breast carcinogenesis in particular. Ubiquitin conjugating enzymes are a central component of the ubiquitination machinery and are often perturbed in breast cancer. This paper will discuss ubiquitin and ubiquitin-like proteins conjugating enzymes participating in breast cancer pathogenesis, their relationships with other proteins of the ubiquitination machinery and their role in phenotype of breast cancer sub-types.

  9. Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1*

    PubMed Central

    Sarkari, Feroz; Wheaton, Keith; La Delfa, Anthony; Mohamed, Majda; Shaikh, Faryal; Khatun, Rahima; Arrowsmith, Cheryl H.; Frappier, Lori; Saridakis, Vivian; Sheng, Yi

    2013-01-01

    Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system. PMID:23603909

  10. 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.

  11. Ubiquitin editing enzyme UCH L1 and microtubule dynamics

    PubMed Central

    Bheda, Anjali; Gullapalli, Anuradha; Caplow, Michael; Pagano, Joseph S.; Shackelford, Julia

    2015-01-01

    Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ∼50 kDa band of UCH L1 (not the normal ∼25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. the inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ∼50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins. PMID:20160478

  12. 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.

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

    PubMed Central

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    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

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

    SciTech Connect

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

    2016-04-06

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. 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 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.

  15. RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

    PubMed Central

    Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

    2013-01-01

    RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

  16. High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ. [Triticum vulgare

    SciTech Connect

    Sullivan, M.L.; Callis, J.; Vierstra, R.D. )

    1990-10-01

    The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with {sup 125}I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin {sup 125}I-lysozyme conjugates ({epsilon}-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion ({alpha}-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated.

  17. 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.

  18. OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function

    PubMed Central

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

    2012-01-01

    SUMMARY 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. PMID:22325355

  19. Catalytic proficiency of ubiquitin conjugation enzymes: balancing pK(a) suppression, entropy, and electrostatics.

    PubMed

    Markin, Craig J; Saltibus, Linda F; Kean, Melissa J; McKay, Ryan T; Xiao, Wei; Spyracopoulos, Leo

    2010-12-22

    Biological organisms orchestrate coordinated responses to external stimuli through temporal fluctuations in protein-protein interaction networks using molecular mechanisms such as the synthesis and recognition of polyubiquitin (polyUb) chains on signaling adaptor proteins. One of the pivotal chemical steps in ubiquitination involves reaction of a lysine amino group with a thioester group on an activated E2, or ubiquitin conjugation enzyme, to form an amide bond between Ub and a target protein. In this study, we demonstrate a nominal 14-fold range for the rate of the chemical step, k(cat), catalyzed by different E2 enzymes using non-steady-state, single-turnover assays. However, the observed range for k(cat) is as large as ∼100-fold for steady-state, single-turnover assays. Biochemical assays were used in combination with measurement of the underlying protein-protein interaction kinetics using NMR line-shape and ZZ-exchange analyses to determine the rate of polyUb chain synthesis catalyzed by the heterodimeric E2 enzyme Ubc13-Mms2. Modest variations in substrate affinity and k(cat) can achieve functional diversity in E2 mechanism, thereby influencing the biological outcomes of polyubiquitination. E2 enzymes achieve reaction rate enhancements through electrostatic effects such as suppression of substrate lysine pK(a) and stabilization of transition states by the preorganized, polar enzyme active site as well as the entropic effects of binding. Importantly, modestly proficient enzymes such as E2s maintain the ability to tune reaction rates; this may confer a biological advantage for achieving specificity in the diverse cellular roles for which these enzymes are involved.

  20. Ubiquitin-dependent proteolytic pathway in wheat germ: Isolation of multiple forms of ubiquitin-activating enzyme, E1

    SciTech Connect

    Hatfield, P.M.; Vierstra, R.D. )

    1989-01-24

    Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.

  1. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    PubMed

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

  2. Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

    PubMed Central

    Nangia-Makker, Pratima; Balan, Vitaly; Morelli, Matteo; Kothayer, Hend; Westwell, Andrew D.; Shekhar, Malathy P.V.

    2013-01-01

    Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2–M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDAMB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs. PMID:23339190

  3. Novel inhibitors of Rad6 ubiquitin conjugating enzyme: design, synthesis, identification, and functional characterization.

    PubMed

    Sanders, Matthew A; Brahemi, Ghali; Nangia-Makker, Pratima; Balan, Vitaly; Morelli, Matteo; Kothayer, Hend; Westwell, Andrew D; Shekhar, Malathy P V

    2013-04-01

    Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

  4. 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.

  5. Molecular cloning and characterization of pigeon (Columba liva) ubiquitin and ubiquitin-conjugating enzyme genes from pituitary gland library.

    PubMed

    Gao, Peng-fei; Cao, Guo-qing; Zhao, Hui-ting; Zhang, Gui-xian; Jiang, Yu-suo; Wang, Qin-de

    2009-01-01

    In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54x10(6) pfu/mL and 1.80x10(6) pfu/mL and the titers of amplified libraries were 1.89x10(8) pfu/mL and 2.32x10(9 )pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a(+) expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.

  6. 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

  7. 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.

  8. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    PubMed

    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.

  9. Chemical synthesis of phosphorylated ubiquitin and diubiquitin exposes positional sensitivities of e1-e2 enzymes and deubiquitinases.

    PubMed

    Bondalapati, Somasekhar; Mansour, Wissam; Nakasone, Mark A; Maity, Suman Kumar; Glickman, Michael H; Brik, Ashraf

    2015-05-11

    Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63-linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1-E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. The ubiquitin conjugating enzyme, TaU4 regulates wheat defence against the phytopathogen Zymoseptoria tritici

    PubMed Central

    Millyard, Linda; Lee, Jack; Zhang, Cunjin; Yates, Gary; Sadanandom, Ari

    2016-01-01

    Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria. PMID:27759089

  11. E2 ubiquitin conjugating enzymes regulate the deubiquitinating activity of OTUB1

    PubMed Central

    Wiener, Reuven; DiBello, Anthony T.; Lombardi, Patrick; Guzzo, Catherine M.; Zhang, Xiangbin; Matunis, Michael J.; Wolberger, Cynthia

    2014-01-01

    OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin. PMID:23955022

  12. Evolution of the ubiquitin-activating enzyme Uba1 (E1)

    NASA Astrophysics Data System (ADS)

    Allan, Douglas C.; Phillips, J. C.

    2017-10-01

    Ubiquitin tags diseased proteins and initiates an enzyme conjugation cascade, which has three stages. The first-stage enzyme Uba1 (E1) has evolved only modestly from slime mold to humans, and is > 14 times larger than Ub. Here we use critical point thermodynamic scaling theory to connect Uba1 (E1) evolution from yeast and slime mold to fruit flies and humans to subtle changes in its amino acid sequences.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  14. 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

  15. A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity1[OPEN

    PubMed Central

    Connor, Richard A.

    2017-01-01

    Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. PMID:27909045

  16. Ubiquitin editing enzyme UCH L1 and microtubule dynamics: implication in mitosis.

    PubMed

    Bheda, Anjali; Gullapalli, Anuradha; Caplow, Michael; Pagano, Joseph S; Shackelford, Julia

    2010-03-01

    Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, western blot analysis of tubulin fractions after polymerization revealed the presence of a specific approximately 50 kDa band of UCH L1 (not the normal approximately 25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the approximately 50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.

  17. Types of Ubiquitin Ligases.

    PubMed

    Morreale, Francesca Ester; Walden, Helen

    2016-03-24

    Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

  18. Mechanistic Studies of Substrate-assisted Inhibition of Ubiquitin-activating Enzyme by Adenosine Sulfamate Analogues

    PubMed Central

    Chen, Jesse J.; Tsu, Christopher A.; Gavin, James M.; Milhollen, Michael A.; Bruzzese, Frank J.; Mallender, William D.; Sintchak, Michael D.; Bump, Nancy J.; Yang, Xiaofeng; Ma, Jingya; Loke, Huay-Keng; Xu, Qing; Li, Ping; Bence, Neil F.; Brownell, James E.; Dick, Lawrence R.

    2011-01-01

    Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102–111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5′-O-sulfamoyl-N6-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PPi exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine

  19. Expression of ubiquitin-related enzymes in the suprachiasmatic nucleus with special reference to ubiquitin carboxy-terminal hydrolase UchL1.

    PubMed

    Dong, Xin; Yagita, Kazuhiro; Zhang, Jing; Okamura, Hitoshi

    2005-04-01

    There is growing evidence that ubiquitin-proteasome system plays an important role for the generation of circadian rhythms in mice as in Drosophila. Here we examined the expression of ubiquitin-related enzymes (Ubce5, UbcM4, Ube2v, Ube2d2, UchL1, UchL3, Ubp41, UfdlL, beta-TrCP) in the suprachiasmatic nucleus (SCN). At mRNA level, the-expression of these enzymes were faint to moderate except ubiquitin carboxy-terminal hydrolase L1 (UchL1), a dominant deubiquitinating salvaging enzyme. Although strongly expressed in the SCN, UchL1 mRNA did not show the rhythm in the SCN in both light-dark and constant dark conditions.

  20. High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.

    PubMed

    Carvalho, Andreia F; Pinto, Manuel P; Grou, Cláudia P; Vitorino, Rui; Domingues, Pedro; Yamao, Fumiaki; Sá-Miranda, Clara; Azevedo, Jorge E

    2012-07-01

    Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.

  1. E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP.

    PubMed

    Soss, Sarah E; Yue, Yuanyuan; Dhe-Paganon, Sirano; Chazin, Walter J

    2011-06-17

    The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.

  2. Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores

    PubMed Central

    Nir, Iftach; Huttner, Diana; Meller, Amit

    2015-01-01

    Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of

  3. Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores.

    PubMed

    Nir, Iftach; Huttner, Diana; Meller, Amit

    2015-05-05

    Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of

  4. Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase*

    PubMed Central

    Burns, Kristin E.; McAllister, Fiona E.; Schwerdtfeger, Carsten; Mintseris, Julian; Cerda-Maira, Francisca; Noens, Elke E.; Wilmanns, Matthias; Hubbard, Stevan R.; Melandri, Francesco; Ovaa, Huib; Gygi, Steven P.; Darwin, K. Heran

    2012-01-01

    Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development. PMID:22942282

  5. Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein▿

    PubMed Central

    Li, Zhenghe; Barajas, Daniel; Panavas, Tadas; Herbst, David A.; Nagy, Peter D.

    2008-01-01

    To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication. PMID:18463149

  6. 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

  7. Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

    PubMed

    Bédard, Nathalie; Jammoul, Samer; Moore, Tamara; Wykes, Linda; Hallauer, Patricia L; Hastings, Kenneth E M; Stretch, Cynthia; Baracos, Vickie; Chevalier, Stéphanie; Plourde, Marie; Coyne, Erin; Wing, Simon S

    2015-09-01

    The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

  8. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  9. 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

  10. Crystal structure of the human ubiquitin-activating enzyme 5 (UBA5) bound to ATP: mechanistic insights into a minimalistic E1 enzyme.

    PubMed

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

    2010-06-25

    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(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.

  11. 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.

  12. 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

  13. The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis

    PubMed Central

    Onizawa, Michio; Oshima, Shigeru; Schulze-Topphoff, Ulf; Oses-Prieto, Juan A; Lu, Timothy; Tavares, Rita; Prodhomme, Thomas; Duong, Bao; Whang, Michael I.; Advincula, Rommel; Agelidis, Alex; Barrera, Julio; Wu, Hao; Burlingame, Alma; Malynn, Barbara A.; Zamvil, Scott S.; Ma, Averil

    2015-01-01

    A20 is an anti-inflammatory protein linked to multiple human diseases, however the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We now find that A20 deficient T cells and fibroblasts are susceptible to caspase independent and RIPK3 dependent necroptosis. Global RIPK3 deficiency significantly rescues the survival of A20 deficient mice. A20 deficient cells exhibit exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 undergoes physiological ubiquitination at lysine 5 (K5), and this ubiquitination event supports the formation of RIPK1-RIPK3 complexes. The catalytic cysteine of A20’s deubiquitinating motif is required for inhibiting RIPK3 ubiquitination and RIPK1-RIPK3 complex formation. These studies link A20 and RIPK3 ubiquitination to necroptotic cell death, and suggest new mechanisms by which A20 may prevent inflammatory disease. PMID:25939025

  14. Deubiquitinating enzymes Ubp2 and Ubp15 regulate endocytosis by limiting ubiquitination and degradation of ARTs

    PubMed Central

    Ho, Hsuan-Chung; MacGurn, Jason A.; Emr, Scott D.

    2017-01-01

    Endocytic down-regulation of cell-surface proteins is a fundamental cellular process for cell survival and adaptation to environmental stimuli. Ubiquitination of cargo proteins serves as the sorting signal for downstream trafficking and relies on the arrestin-related trafficking adaptor (ART)-Rsp5 ubiquitin ligase adaptor network in yeast. Hence proper regulation of the abundance and activity of these ligase–adaptor complexes is critical for main­tenance of optimal plasma membrane protein composition. Here we report that the stability of ARTs is regulated by the deubiquitinating enzymes (DUBs) Ubp2 and Ubp15. By counteracting the E3 ubiquitin ligase Rsp5, Ubp2 and Ubp15 prevent hyperubiquitination and proteasomal degradation of ARTs. Specifically, we show that loss of both Ubp2 and Ubp15 results in a defect in Hxt6 endocytosis associated with Art4 instability. Our results uncover a novel function for DUBs in the endocytic pathway by which Ubp2 and Ubp15 positively regulate the ART-Rsp5 network. PMID:28298493

  15. Herpes simplex virus 1-infected cell protein 0 contains two E3 ubiquitin ligase sites specific for different E2 ubiquitin-conjugating enzymes

    PubMed Central

    Hagglund, Ryan; Van Sant, Charles; Lopez, Pascal; Roizman, Bernard

    2002-01-01

    Infected cell protein 0 (ICP0) of herpes simplex virus 1, a multifunctional ring finger protein, enhances the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular and viral proteins, and is associated with the degradation of several cellular proteins. Sequences encoded by exon 2 of ICP0 (residues 20–241) bind the UbcH3 (cdc34) ubiquitin-conjugating enzyme, and its carboxy terminus expresses a ubiquitin ligase activity demonstrable by polyubiquitylation of cdc34 in vitro. We report that: (i) The physical interaction of cdc34 and ICP0 leads to its degradation. Thus, substitution of ICP0 aspartate 199 with alanine attenuates the degradation of cdc34 and its binding to the ICP0 ring finger domain. (ii) Substitution of residue 620 reported to abolish the interaction with a ubiquitin-specific protease has no effect on the function of ubiquitin ligase. (iii) ICP0 contains an additional distinct E3 ligase activity specific for the UbcH5a- and UbcH6 E2-conjugating enzymes mapping to the ring finger domain. This is, to our knowledge, the first identification of a viral protein with at least two physically separated E3 ligase activities with different E2 specificities. The results suggest that each activity may target different proteins. PMID:11805320

  16. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Specificity and disease in the ubiquitin system.

    PubMed

    Chaugule, Viduth K; Walden, Helen

    2016-02-01

    Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation.

  19. The ubiquitin conjugating enzyme UbcH7, controls cell migration

    USDA-ARS?s Scientific Manuscript database

    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 ...

  20. Investigation of genetic variants in ubiquitin enzyme genes involved in the modulation of neurodevelopmental processes: a role in schizophrenia susceptibility?

    PubMed

    Andrews, Jessica L; Fernandez-Enright, Francesca

    2014-11-24

    Despite extensive research during the last few decades, the etiology of schizophrenia remains unclear. Evidence of both genetic and environmental influences in the developmental profile of schizophrenia has grown, and due to the complexity of this disorder, a polygenic aspect has been associated with this neuropsychiatric pathology. Unfortunately, no diagnostic strategies based on biological measurement or genetic testing is currently available for schizophrenia. Gene-expression profiling and recent protein studies have shown a decrease in the expression of ubiquitin pathway proteins in the prefrontal cortex of schizophrenia patients. We have examined single nucleotide polymorphisms (or SNPs) within three genes from the ubiquitin protein system: the ubiquitin conjugating enzyme E2D1 (UBE2D1) gene, the E3 SUMO-protein ligase protein inhibitor of activated STAT 2 (PIAS2) gene, and the E3 ubiquitin ligase F-box and leucine-rich repeat protein 21 (FBXL21) gene, in a Caucasian case-control population for schizophrenia. After Bonferroni correction for multiple testing was applied, no significant associations were reported for any of the tested SNPs. Additional genetic analyses will be necessary to fully explore the role of these three genes in schizophrenia. Regarding the rising interest in ubiquitin-related proteins as a therapeutic target in other pathologies such as cancer, further research into the role of ubiquitin pathways in schizophrenia seems topical and timely.

  1. Expression patterns of ubiquitin conjugating enzyme UbcM2 during mouse embryonic development.

    PubMed

    Yanjiang, Xing; Hongjuan, He; Tiantian, Gu; Yan, Zhang; Zhijun, Huang; Qiong, Wu

    2012-01-01

    Ubiquitin conjugating enzyme UbcM2 (Ubiquitin-conjugating enzymes from Mice, the number reveals the identification order) has been implicated in many critical processes, such like growth-inhibiting, mediating cell proliferation and regulation of some transcription factor, but the expression profile during mouse embryo development remains unclear. Hereby, during mid-later embryonic stage, the expression patterns of UbcM2 were examined using in situ hybridization and quantitative real-time PCR (qRT-PCR). The signals were significantly intense in central nervous system and skeletal system, weak in tongue, heart, lung, liver, and kidney. In the central nervous system, UbcM2 was principally expressed in thalamus, external germinal layer of cerebellum (EGL), mitral cell layer of olfactory bulb, hippocampus, marginal zone and ventricular zone of cerebral cortex, and spinal cord. In the skeletal system, UbcM2 was primarily expressed in proliferating cartilage. Furthermore, qRT-PCR analysis displayed that the expression of UbcM2 was ubiquitous at E15.5, most prominent in brain, weaker in lung liver and kidney, accompanied by the lowest level in tongue and heart. During brain development, the expression level of UbcM2 first ascended and then decreased from E12.5 to E18.5, the peak of which sustained starting at E14.5 until E16.5. Together, these results suggest that UbcM2 may play potential roles in the development of mouse diverse tissues and organs, particularly in the development of brain and skeleton.

  2. 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.

  3. Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata.

    PubMed

    Miao, Hong-Xia; Qin, Yong-Hua; Ye, Zi-Xing; Hu, Gui-Bing

    2013-01-25

    Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from 'Wuzishatangju' (self-incompatible, SI) and 'Shatangju' (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between 'Wuzishatangju' and 'Shatangju', respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of 'Wuzishatangju' and 'Shatangju'. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of 'Shatangju' was approximately 10-fold higher than in anthers of 'Wuzishatangju'. The highest expression level of CrUBE1 was detected in pistils at 7days after self-pollination of 'Wuzishatangju', which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from 'Wuzishatangju' and 'Shatangju' were successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was significantly inhibited with increasing of CrUBE1 protein concentrations from 'Wuzishatangju'. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya.

    PubMed

    Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

    2017-01-01

    Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya.

  5. The ESCRT-III-interacting deubiquitinating enzyme AMSH3 is essential for degradation of ubiquitinated membrane proteins in Arabidopsis thaliana.

    PubMed

    Katsiarimpa, Anthi; Muñoz, Alfonso; Kalinowska, Kamila; Uemura, Tomohiro; Rojo, Enrique; Isono, Erika

    2014-04-01

    Post-translational modification by ubiquitin plays a key role in the regulation of endocytic degradation in which ubiquitinated plasma membrane cargos are transported to the vacuole for degradation dependent on the ESCRT (endosomal sorting complex required for transport) machinery. Arabidopsis AMSH3 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 3) is a deubiquitinating enzyme that interacts with at least two subunits of the ESCRT-III machinery, VPS2.1 and VPS24.1. amsh3 null mutation causes seedling lethality, and amsh3 null mutants show defects in multiple intracellular trafficking pathways. In this study, we further analyzed the amsh3 mutant phenotype and showed that amsh3 accumulates membrane-associated ubiquitinated proteins, supporting the indication that AMSH3 functions in ubiquitin-mediated endocytic degradation. In accordance with this, an enzymatic inactive variant of AMSH3 inhibits the AvrPtoB-dependent endocytic degradation of CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Furthermore, we showed that the interaction of AMSH3 with ESCRT-III is important for its function in planta. Together, our data indicate the importance of AMSH3 and the AMSH3-ESCRT-III interaction for deubiquitination and degradation of ubiquitinated membrane substrates in plants.

  6. 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

  7. Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins.

    PubMed

    Chen, An-Jing; Wang, Shuai; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

    2011-08-01

    Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.

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

    PubMed Central

    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; Oualid, Farid El; Ovaa, Huib

    2016-01-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin 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 ubiquitin-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 activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

  9. NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20

    PubMed Central

    Verheugen, Eveline; Taghon, Tom; Lambrecht, Bart N.

    2016-01-01

    Natural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage specification and differentiation remain poorly understood. Here, we demonstrate that the ubiquitin-modifying enzyme TNFAIP3/A20, an upstream regulator of T cell receptor (TCR) signaling in T cells, is an essential cell-intrinsic regulator of NKT differentiation. A20 is differentially expressed during NKT cell development, regulates NKT cell maturation, and specifically controls the differentiation and survival of NKT1 and NKT2, but not NKT17, sublineages. Remaining A20-deficient NKT1 and NKT2 thymocytes are hyperactivated in vivo and secrete elevated levels of Th1 and Th2 cytokines after TCR ligation in vitro. Defective NKT development was restored by compound deficiency of MALT1, a key downstream component of TCR signaling in T cells. These findings therefore show that negative regulation of TCR signaling during NKT development controls the differentiation and survival of NKT1 and NKT2 cells. PMID:27551157

  10. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya

    PubMed Central

    Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

    2017-01-01

    Background Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). Methodology/Principal findings In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. Conclusions To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya. PMID:28231288

  11. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

    PubMed

    Yi, Y-J; Zimmerman, S W; Manandhar, G; Odhiambo, J F; Kennedy, C; Jonáková, V; Maňásková-Postlerová, P; Sutovsky, M; Park, C-S; Sutovsky, P

    2012-04-01

    Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation

  12. Synergistic effect of two E2 ubiquitin conjugating enzymes in SCFhFBH1 catalyzed polyubiquitination

    PubMed Central

    Choi, Jin Sun; Kim, Sunhong; Kim, Kidae; Myung, Pyung Keun; Park, Sung Goo; Seo, Yeon-Soo; Park, Byoung Chul

    2015-01-01

    Ubiquitination is a post translational modification which mostly links with proteasome dependent protein degradation. This process has been known to play pivotal roles in the number of biological events including apoptosis, cell signaling, transcription and translation. Although the process of ubiquitination has been studied extensively, the mechanism of polyubiquitination by multi protein E3 ubiquitin ligase, SCF complex remains elusive. In the present study, we identified UbcH5a as a novel stimulating factor for poly-ubiquitination catalyzed by SCFhFBH1 using biochemical fractionations and MALDI-TOF. Moreover, we showed that recombinant UbcH5a and Cdc34 synergistically stimulate SCFhFBH1 catalyzed polyubiquitination in vitro. These data may provide an important cue to understand the mechanism how the SCF complex efficiently polyubiquitinates target substrates. [BMB Reports 2015; 48(1): 25-29] PMID:24667174

  13. The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells

    PubMed Central

    Plafker, Kendra S.; Farjo, Krysten M.; Wiechmann, Allan F.; Plafker, Scott M.

    2008-01-01

    Purpose Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. Methods UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27Kip1 were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for β-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. Results UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27Kip1, and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. Conclusions UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation. PMID:18614808

  14. Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair

    PubMed Central

    Gilon, Tamar; Chomsky, Orna; Kulka, Richard G.

    2000-01-01

    Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of β-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Δ and sec61-2) did not inhibit the degradation of the β-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p. PMID:10982838

  15. 14-kDa ubiquitin-conjugating enzyme: structure of the rat gene and regulation upon fasting and by insulin.

    PubMed

    Wing, S S; Banville, D

    1994-07-01

    Upon fasting, an increase in proteolysis occurs in rat skeletal muscle and is associated with increased levels of ubiquitin-protein conjugates. As this suggests that formation of conjugates may be activated upon fasting, we studied the expression of the gene encoding the 14-kDa ubiquitin-conjugating enzyme (E2(14k)). A cDNA encoding rat E2(14k) was isolated and used to probe Northern blots of RNA from extensor digitorum longus muscles of fed, fasted, and refed rats. Two mRNA transcripts of 1.2 and 1.8 kb were observed. Isolation and sequencing of a genomic clone determined that these transcripts arise from differential sites of polyadenylation. The 1.2-kb transcript increased threefold upon fasting at 2 days and returned to normal with refeeding. Northern analysis of RNA from various tissues of fed and fasted rats showed that E2(14k) mRNA was expressed at high levels in testes, moderate levels in muscle, heart, and brain, but low levels in liver and kidney. Upon fasting, increases in mRNA levels were seen in muscle, heart, liver, and kidney. In vitro, in rat L6 myotubes, insulin lowered levels of E2(14k) mRNA. Because E2s catalyze the first irreversible reaction in the pathway and E2(14k) gene expression appears to change in parallel with the changes in levels of ubiquitinated proteins and rates of proteolysis, conjugation mediated by this E2 may be a rate-limiting step in the pathway. This is the first demonstration of direct hormonal regulation of a gene in the ubiquitin system and argues strongly for a role of the ubiquitin system in the metabolic response to fasting in skeletal muscle.

  16. 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

  17. An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

    PubMed

    Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

    2014-01-01

    Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc

  18. DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer.

    PubMed

    Hernández-Pérez, S; Cabrera, E; Salido, E; Lim, M; Reid, L; Lakhani, S R; Khanna, K K; Saunus, J M; Freire, R

    2017-08-17

    Correct control of DNA replication is crucial to maintain genomic stability in dividing cells. Inappropriate re-licensing of replicated origins is associated with chromosomal instability (CIN), a hallmark of cancer progression that at the same time provides potential opportunities for therapeutic intervention. Geminin is a critical inhibitor of the DNA replication licensing factor Cdt1. To properly achieve its functions, Geminin levels are tightly regulated through the cell cycle by ubiquitin-dependent proteasomal degradation, but the de-ubiquitinating enzymes (DUBs) involved had not been identified. Here we report that DUB3 and USP7 control human Geminin. Overexpression of either DUB3 or USP7 increases Geminin levels through reduced ubiquitination. Conversely, depletion of DUB3 or USP7 reduces Geminin levels, and DUB3 knockdown increases re-replication events, analogous to the effect of Geminin depletion. In exploring potential clinical implications, we found that USP7 and Geminin are strongly correlated in a cohort of invasive breast cancers (P<1.01E-08). As expected, Geminin expression is highly prognostic. Interestingly, we found a non-monotonic relationship between USP7 and breast cancer-specific survival, with both very low or high levels of USP7 associated with poor outcome, independent of estrogen receptor status. Altogether, our data identify DUB3 and USP7 as factors that regulate DNA replication by controlling Geminin protein stability, and suggest that USP7 may be involved in Geminin dysregulation during breast cancer progression.

  19. Activity and cellular functions of the deubiquitinating enzyme and polyglutamine disease protein ataxin-3 are regulated by ubiquitination at lysine 117.

    PubMed

    Todi, Sokol V; Scaglione, K Matthew; Blount, Jessica R; Basrur, Venkatesha; Conlon, Kevin P; Pastore, Annalisa; Elenitoba-Johnson, Kojo; Paulson, Henry L

    2010-12-10

    Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs themselves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic ataxin-3. Further studies indicate that ubiquitin-dependent activation of ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances ataxin-3 activity independent of the known ubiquitin-binding sites in ataxin-3, most likely through a direct conformational change in or near the catalytic domain.

  20. Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening.

    PubMed

    Wang, Yuying; Wang, Weihao; Cai, Jianghua; Zhang, Yanrui; Qin, Guozheng; Tian, Shiping

    2014-01-01

    Fruits are unique to flowering plants and play a central role in seed maturation and dispersal. Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit. To better understand the regulatory mechanisms underlying fruit ripening, we report here the first comprehensive analysis of the nuclear proteome in tomato fruits. Nuclear proteins were isolated from tomatoes in different stages of ripening, and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. We show that the proteins whose abundances change during ripening stages are involved in various cellular processes. We additionally evaluate changes in the nuclear proteome in the ripening-deficient mutant, ripening-inhibitor (rin), carrying a mutation in the transcription factor RIN. A set of proteins were identified and particular attention was paid to SlUBC32 and PSMD2, the components of ubiquitin-proteasome pathway. Through chromatin immunoprecipitation and gel mobility shift assays, we provide evidence that RIN directly binds to the promoters of SlUBC32 and PSMD2. Moreover, loss of RIN function affects protein ubiquitination in nuclei. SlUBC32 encodes an E2 ubiquitin-conjugating enzyme and a genome-wide survey of the E2 gene family in tomatoes identified five more E2s as direct targets of RIN. Virus-induced gene silencing assays show that two E2s are involved in the regulation of fruit ripening. Our results uncover a novel function of protein ubiquitination, identifying specific E2s as regulators of fruit ripening. These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.

  1. Evidence for Two New Solution States of Ubiquitin by IMS–MS Analysis

    PubMed Central

    2015-01-01

    Ion mobility spectrometry coupled with mass spectrometry (IMS–MS) is used to investigate the populations of different states for ubiquitin in water:methanol solutions. In these experiments, ubiquitin is electrosprayed from 20 water:methanol (100:0 to 5:95, pH = 2) solutions, ranging from native to denaturing conditions. With an increased percentage of methanol in solution, ubiquitin ions ([M + 7H]7+ to [M + 12H]12+) show substantial variations in both charge state distributions and ion mobility distributions. Analysis of these data provides evidence for the existence of five ubiquitin states in solution: the native N state, favored in solutions of 100:0 to 70:30 water:methanol for the +7 and +8 charge states; the more helical A state and a new closely related A′ state, favored in solutions of 70:30 to 5:95 water:methanol for the +9 to +12 charge states; the unfolded U state, populated in 40:60 to 5:95 water:methanol solutions for the +8 to +10 and +12 charge states; and a new low-abundance state termed the B state, observed for 100:0 to 70:30 water:methanol solutions in the +8 to +10 and +12 charge states. The relative abundances for different states in different solutions are determined. The analysis presented here provides insight into how solution structures evolve into anhydrous conformations and demonstrates the utility of IMS–MS methods as a means of characterizing populations of conformers for proteins in solution. PMID:24625065

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. The ubiquitin-conjugating enzyme, Ubc1, indirectly regulates SNF1 kinase activity via Forkhead-dependent transcription

    PubMed Central

    Jiao, Rubin; Lobanova, Liubov; Waldner, Amanda; Fu, Anthony; Xiao, Linda; Harkness, Troy A.; Arnason, Terra G.

    2016-01-01

    The SNF1 kinase in Saccharomyces cerevisiae is an excellent model to study the regulation and function of the AMP-dependent protein kinase (AMPK) family of serine-threonine protein kinases. Yeast discoveries regarding the regulation of this non-hormonal sensor of metabolic/environmental stress are conserved in higher eukaryotes, including poly-ubiquitination of the α-subunit of yeast (Snf1) and human (AMPKα) that ultimately effects subunit stability and enzyme activity. The ubiquitin-cascade enzymes responsible for targeting Snf1 remain unknown, leading us to screen for those that impact SNF1 kinase function. We identified the E2, Ubc1, as a regulator of SNF1 kinase function. The decreased Snf1 abundance found upon deletion of Ubc1 is not due to increased degradation, but instead is partly due to impaired SNF1 gene expression, arising from diminished abundance of the Forkhead 1/2 proteins, previously shown to contribute to SNF1 transcription. Ultimately, we report that the Fkh1/2 cognate transcription factor, Hcm1, fails to enter the nucleus in the absence of Ubc1. This implies that Ubc1 acts indirectly through transcriptional effects to modulate SNF1 kinase activity. PMID:28357323

  4. 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.

  5. TRIM32 ubiquitin E3 ligase, one enzyme for several pathologies: From muscular dystrophy to tumours.

    PubMed

    Lazzari, Elisa; Meroni, Germana

    2016-10-01

    TRIM32 is a member of the TRIpartite Motif family characterised by the presence of an N-terminal three-domain-module that includes a RING domain, which confers E3 ubiquitin ligase activity, one or two B-box domains and a Coiled-Coil region that mediates oligomerisation. Several TRIM32 substrates were identified including muscular proteins and proteins involved in cell cycle regulation and cell motility. As ubiquitination is a versatile post-translational modification that can affect target turnover, sub-cellular localisation or activity, it is likely that diverse substrates may be differentially affected by TRIM32-mediated ubiquitination, reflecting its multi-faceted roles in muscle physiology, cancer and immunity. With particular relevance for muscle physiology, mutations in TRIM32 are associated with autosomal recessive Limb-Girdle Muscular Dystrophy 2H, a muscle-wasting disease with variable clinical spectrum ranging from almost asymptomatic to wheelchair-bound patients. In this review, we will focus on the ability of TRIM32 to mark specific substrates for proteasomal degradation discussing how the TRIM32-proteasome axis may (i) be important for muscle homeostasis and for the pathogenesis of muscular dystrophy; and (ii) define either an oncogenic or tumour suppressive role for TRIM32 in the context of different types of cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

    PubMed

    Pitluk, Z W; McDonough, M; Sangan, P; Gonda, D K

    1995-03-01

    CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

  7. Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

    PubMed Central

    Pitluk, Z W; McDonough, M; Sangan, P; Gonda, D K

    1995-01-01

    CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination. PMID:7862115

  8. Involvement of Steroid Receptor Coactivators/Ubiquitin Pathway Enzymes in Mammary Gland Tumorigenesis

    DTIC Science & Technology

    2005-06-01

    PRINCIPAL INVESTIGATOR: Xiuhua Gao, M.D., Ph.D. CONTRACTING ORGANIZATION: Baylor College of Medicine Houston, TX 77030 REPORT DATE: June 2005 TYPE OF REPORT...Summary 13 May 2002 - 12 May 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Involvement of Steroid Receptor Coactivators/Ubiquitin Pathway 5b...Usell tc localization of MUcle ~s. Eý a, Eý Ca exaressiorn Crofile; E-p groffle; W1~’, API staining for nUcleUs. E6-AP DAMP -HI +E Figure 2: Effect of

  9. Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product.

    PubMed

    Yin, Gang; Ji, Chaoneng; Wu, Tong; Shen, Zhouliang; Xu, Xin; Xie, Yi; Mao, Yumin

    2006-05-01

    Ubiquitin charging and activation of class III E2 enzymes has been directly linked to their nuclear import. It has not been published whether other classes E2s also abide by this mechanism. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone that is 2252 base pair in length, encoding a putative 162 amino acid protein, which shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16 (Accession number NP_565110, 51% identity and 71% similarity) at protein level. Bioinformatics analysis revealed that the gene is composed of 7 exons, located on human chromosome 8q13-8q21.1, and that the predicted protein of the gene is a class I E2, for only composed of a conserved approximately 150-amino acid catalytic core, ubiquitin-conjugating enzyme E2 domain (UBC domain). In the C-terminal of the UBC domain sequence, there are two nuclear localization signals (NLSs). RT-PCR showed that this gene is ubiquitously expressed in 16 kinds of normal human tissues, but expression level is very low, unless in human heart, brain, liver, and pancreas. The subcellular localizations of the new human Ubiquitin conjugating enzyme E2 and its mutation were also examined, which showed that the nuclear localization of hUBC16 depended on two conditions: It has NLS, and at the same time, has enzyme active site, too, at least in HEK293 cells.

  10. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells*

    PubMed Central

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-01-01

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells. PMID:26160177

  11. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells.

    PubMed

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-08-21

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

  12. 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

  13. Specificity and disease in the ubiquitin system

    PubMed Central

    Chaugule, Viduth K.; Walden, Helen

    2016-01-01

    Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

  14. Assessment of Nanosecond Time Scale Motions in Native and Non-Native States of Ubiquitin.

    PubMed

    Morozova, Olga B; Yurkovskaya, Alexandra V

    2015-10-01

    The paramagnetic relaxation times of the aromatic and β protons of Tyr59 and His68 residues of the native ubiquitin and of Tyr59 residue of the non-native ubiquitin were determined from an analysis of chemically induced dynamic nuclear polarization (CIDNP) kinetics obtained during the photoreaction of the protein and 2,2'-dipyridyl excited in the triplet state. Using the paramagnetic relaxation times determined earlier for the radicals of free amino acids as an internal standard and assuming that the hyperfine interaction (HFI) anisotropy is very similar for the radicals of free amino acids and the corresponding radicals of amino acid residues in the proteins, we determined parameters that characterize the intramolecular mobility of different protons in native and two non-native states of ubiquitin. The latter are denatured at pH 2 and 57 °C, and the A-state at pH 2 in a 60%/40% methanol/water mixture. The determination of the two parameters of intramolecular mobility (i.e., the correlation time of internal motion, τ(e), and the order parameter, S(2)) was only possible by analyzing paramagnetic relaxation data obtained at two magnetic fields (4.7 and 9.4 T) using nuclear magnetic resonance (NMR) spectrometry. Intramolecular correlation times fall into the submicrosecond-microsecond time scale. Longer correlation times and higher order parameters were found for the less accessible Tyr59 residue than for the His68 residue, as well as for the more buried β protons than for the aromatic protons for both of the protein residues in the native state. For Tyr59, intramolecular mobility increases following the loss of the tertiary structure of ubiquitin. These findings strongly support the reliability of the obtained data.

  15. Lack of the ubiquitin-editing enzyme A20 results in loss of hematopoietic stem cell quiescence

    PubMed Central

    Nakagawa, Masahiro Marshall; Thummar, Keyur; Mandelbaum, Jonathan; Pasqualucci, Laura

    2015-01-01

    A balance between quiescence and proliferation is critical for proper maintenance of the hematopoietic stem cell (HSC) pool. Although a lot is known about hematopoiesis, molecular mechanisms that control HSC quiescence remain largely unknown. The ubiquitin-editing enzyme A20 functions as a central regulator of inflammation and adaptive immunity. Here, we show that a deficiency of A20 in the hematopoietic system causes anemia, lymphopenia, and postnatal lethality. Lack of A20 in HSCs results in diminished pool size, impaired radioprotection, defective repopulation, and loss of quiescence. A20-deficient HSCs display increased IFN-γ signaling, caused by augmented NF-κB activation. Strikingly, deletion of both IFN-γ and A20 in hematopoietic cells results in partial rescue of the HSC phenotype. We anticipate that our experiments will facilitate the understanding of mechanisms through which A20-mediated inflammatory signals control HSC quiescence and functions. PMID:25624445

  16. Expression Profile of Penaeus monodon Ubiquitin Conjugating Enzyme (PmUbc) at Protein Level in White spot syndrome virus Challenged Shrimp.

    PubMed

    Keezhedath, Jeena; Kurcheti, Pani Prasad; Pathan, Mujahid Khan; Babu, Gireesh P; Tripathi, Gayatri; Sudhagar, Arun; Rao, Srinivas P

    2013-06-01

    White spot syndrome virus (WSSV) is one of the major pathogens in shrimp aquaculture. Four proteins of WSSV are predicted to encode a RING H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimps 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, expression profile of Penaeus monodon Ubiquitin conjugating enzyme (PmUbc) was studied at protein level in WSSV challenged shrimp. A time point analysis of the expression of PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge in P. monodon. Recombinant PmUbc (rPmUbc) was produced in prokaryotic expression vector, BL21 (DE3) pLys S. The PmUbc expression pattern was studied by ELISA with rPmUbc antibodies raised in rabbit. A significant increase in PmUbc expression at 24 h post infection (hpi) was observed followed by a decline till 72 hpi. Since the up-regulation and a tremendous decline of PmUbc protein expression was observed at 24 and in 72 hpi respectively in ELISA, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. Many findings have shown that viral infection can up-regulate expression of ubiquitin and that the ubiquitin system plays a key role in the course of viral infection. The present study reveals the expression patterns of PmUbc at protein level in WSSV infected P. monodon. However, further studies are to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this major culprit in shrimp culture industry.

  17. The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

    PubMed

    Rojas, Federico; Koszela, Joanna; Búa, Jacqueline; Llorente, Briardo; Burchmore, Richard; Auer, Manfred; Mottram, Jeremy C; Téllez-Iñón, María Teresa

    2017-06-01

    The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

  18. 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.

  19. 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.

  20. Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB) highly expressed in rat testis.

    PubMed Central

    Wing, S S; Jain, P

    1995-01-01

    Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4

  1. Identification of a gene product induced by hard-surface contact of Colletotrichum gloeosporioides conidia as a ubiquitin-conjugating enzyme by yeast complementation.

    PubMed

    Liu, Z M; Kolattukudy, P E

    1998-07-01

    The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host's surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (ubc1Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1Cg can complement the proteolysis deficiency of the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.

  2. Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development.

    PubMed

    Woodward, Andrew W; Ratzel, Sarah E; Woodward, Erin E; Shamoo, Yousif; Bartel, Bonnie

    2007-06-01

    The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.

  3. Ubiquitin-Based Probes Prepared by Total Synthesis To Profile the Activity of Deubiquitinating Enzymes

    PubMed Central

    de Jong, Annemieke; Merkx, Remco; Berlin, Ilana; Rodenko, Boris; Wijdeven, Ruud H M; El Atmioui, Dris; Yalçin, Zeliha; Robson, Craig N; Neefjes, Jacques J; Ovaa, Huib

    2012-01-01

    Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells. PMID:23011887

  4. Practical steady-state enzyme kinetics.

    PubMed

    Lorsch, Jon R

    2014-01-01

    Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2012-01-01

    SUMMARY 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 of 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 C-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 stabilise the consequent tetrahedral transition state intermediate. PMID:22842904

  6. Decreased H2B monoubiquitination and overexpression of ubiquitin-specific protease enzyme 22 in malignant colon carcinoma.

    PubMed

    Wang, Zijing; Zhu, Linlin; Guo, Tianjiao; Wang, Yiping; Yang, Jinlin

    2015-07-01

    This study aimed to evaluate the expression of H2B monoubiquitination enzyme (uH2B) and ubiquitin-specific protease enzyme 22 (USP22) in colon carcinoma and establish a correlation between the expression of these enzymes and clinicopathological parameters. The modification levels of uH2B and USP22 in 20 noncancerous and 129 cancerous colon samples were studied by immunohistochemistry. We used a dual-rated semiquantitative method to classify the expression according to 3 levels and analyzed these results. uH2B was abundant in the normal colon epithelium, but its expression was decreased in colon cancers (P < .001); the uH2B modification level correlated with tumor differentiation (P < .001), lymph node metastasis (P = .017), distant metastasis (P = .036), and tumor stage (P = .039). The USP22 expression in colon carcinoma was higher than that in normal tissues (P = .007) and negatively correlated with the degree of differentiation (P = .006), invasion (P = .025), lymph node metastasis (P = .026), and tumor stage (P = .044). uH2B and USP22 expression negatively correlated (r = -0.401, P < .001). Patients with uH2B-negative and USP22-positive staining were found to have lower survival rates (30.737 ± 2.866 versus 51.667 ± 2.286 months, P < .001). Positive uH2B and negative USP22 expression remained a statistically significant prognostic indicator in a multivariate Cox regression analysis (hazard ratio, 2.557; 95% confidence interval, 1.043-6.269; P = .04). We conclude that uH2B displays differential staining patterns according to progressive stages of colon cancer, indicating that uH2B may play an important inhibitory role in carcinogenesis. Increased USP22 expression in colon cancer correlated with reduced uH2B expression, and this expression pattern may contribute to tumor progression.

  7. 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.

  8. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

    PubMed

    Ramphan, Suwipa; Khongwichit, Sarawut; Saisawang, Chonticha; Kovanich, Duangnapa; Ketterman, Albert J; Ubol, Sukathida; Auewarakul, Prasert; Roytrakul, Sittiruk; Smith, Duncan R; Kuadkitkan, Atichat

    2017-10-04

    Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV nonstructural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) was transfected into Hela cells, and differential protein expression was detected by 2-dimension (2D) polyacrylamide gel electrophoresis. A total of 21 differentially regulated (6 upregulated, 15 downregulated) spots were observed, of which 5 were identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) was confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 was additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduced levels of infection and E protein expression but did not alter RNA genome levels. These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. De-ubiquitinating enzyme, USP11, promotes transforming growth factor β-1 signaling through stabilization of transforming growth factor β receptor II

    PubMed Central

    Jacko, A M; Nan, L; Li, S; Tan, J; Zhao, J; Kass, D J; Zhao, Y

    2016-01-01

    The transforming growth factor β-1 (TGFβ-1) signaling pathway plays a central role in the pathogenesis of pulmonary fibrosis. Two TGFβ-1 receptors, TβRI and TβRII, mediate this pathway. TβRI protein stability, as mediated by the ubiquitin/de-ubiquitination system, has been well studied; however, the molecular regulation of TβRII still remains unclear. Here we reveal that a de-ubiquitinating enzyme, USP11, promotes TGFβ-1 signaling through de-ubiquitination and stabilization of TβRII. We elucidate the role that mitoxantrone (MTX), an USP11 inhibitor, has in the attenuation of TGFβ-1 signaling. Inhibition or downregulation of USP11 results in increases in TβRII ubiquitination and reduction of TβRII stability. Subsequently, TGFβ-1 signaling is greatly attenuated, as shown by the decreases in phosphorylation of SMAD2/3 levels as well as that of fibronectin (FN) and smooth muscle actin (SMA). Overexpression of USP11 reduces TβRII ubiquitination and increases TβRII stabilization, thereby elevating phosphorylation of SMAD2/3 and the ultimate expression of FN and SMA. Further, elevated expression of USP11 and TβRII were detected in lung tissues from bleomycin-challenged mice and IPF patients. Therefore, USP11 may contribute to the pathogenesis of pulmonary fibrosis by stabilization of TβRII and promotion of TGFβ-1 signaling. This study provides mechanistic evidence for development of USP11 inhibitors as potential antifibrotic drugs for pulmonary fibrosis. PMID:27853171

  11. Transition States and transition state analogue interactions with enzymes.

    PubMed

    Schramm, Vern L

    2015-04-21

    Enzymatic transition states have lifetimes of a few femtoseconds (fs). Computational analysis of enzyme motions leading to transition state formation suggests that local catalytic site motions on the fs time scale provide the mechanism to locate transition states. An experimental test of protein fs motion and its relation to transition state formation can be provided by isotopically heavy proteins. Heavy enzymes have predictable mass-altered bond vibration states without altered electrostatic properties, according to the Born-Oppenheimer approximation. On-enzyme chemistry is slowed in most heavy proteins, consistent with altered protein bond frequencies slowing the search for the transition state. In other heavy enzymes, structural changes involved in reactant binding and release are also influenced. Slow protein motions associated with substrate binding and catalytic site preorganization are essential to allow the subsequent fs motions to locate the transition state and to facilitate the efficient release of products. In the catalytically competent geometry, local groups move in stochastic atomic motion on the fs time scale, within transition state-accessible conformations created by slower protein motions. The fs time scale for the transition state motions does not permit thermodynamic equilibrium between the transition state and stable enzyme states. Isotopically heavy enzymes provide a diagnostic tool for fast coupled protein motions to transition state formation and mass-dependent conformational changes. The binding of transition state analogue inhibitors is the opposite in catalytic time scale to formation of the transition state but is related by similar geometries of the enzyme-transition state and enzyme-inhibitor interactions. While enzymatic transition states have lifetimes as short as 10(-15) s, transition state analogues can bind tightly to enzymes with release rates greater than 10(3) s. Tight-binding transition state analogues stabilize the rare but

  12. New insight into the role of the Cdc34 ubiquitin-conjugating enzyme in cell cycle regulation via Ace2 and Sic1.

    PubMed

    Cocklin, Ross; Heyen, Joshua; Larry, Tolonda; Tyers, Mike; Goebl, Mark

    2011-03-01

    The Cdc34 ubiquitin-conjugating enzyme plays a central role in progression of the cell cycle. Through analysis of the phenotype of a mutant missing a highly conserved sequence motif within the catalytic domain of Cdc34, we discovered previously unrecognized levels of regulation of the Ace2 transcription factor and the cyclin-dependent protein kinase inhibitor Sic1. In cells carrying the Cdc34(tm) mutation, which alters the conserved sequence, the cyclin-dependent protein kinase inhibitor Sic1, an SCF(Cdc4) substrate, has a shorter half-life, while the cyclin Cln1, an SCF(Grr1) substrate, has a longer half-life than in wild-type cells. Expression of the SIC1 gene cluster, which is regulated by Swi5 and Ace2 transcription factors, is induced in CDC34(tm) cells. Levels of Swi5, Ace2, and the SCF(Grr1) targets Cln1 and Cln2 are elevated in Cdc34(tm) cells, and loss of Grr1 causes an increase in Ace2 levels. Sic1 levels are similar in CDC34(tm) ace2Δ and wild-type cells, explaining a paradoxical increase in the steady-state level of Sic1 protein despite its reduced half-life. A screen for mutations that interact with CDC34(tm) uncovered novel regulators of Sic1, including genes encoding the polyubiquitin chain receptors Rad23 and Rpn10. © 2011 by the Genetics Society of America

  13. Aged monkey brains reveal the role of ubiquitin-conjugating enzyme UBE2N in the synaptosomal accumulation of mutant huntingtin

    PubMed Central

    Yin, Peng; Tu, Zhuchi; Yin, An; Zhao, Ting; Yan, Sen; Guo, Xiangyu; Chang, Renbao; Zhang, Lianhe; Hong, Yan; Huang, Xiahe; Zhou, Junxia; Wang, Yingchun; Li, Shihua; Li, Xiao-Jiang

    2015-01-01

    Although misfolded proteins are ubiquitinated and cleared by the proteasome, they can accumulate in synapses in aged neurons to promote synaptic dysfunction in a variety of neurodegenerative diseases, including Huntington's disease (HD), which is caused by polyglutamine expansion in huntingtin. The mechanism behind this aging-related phenomenon is unknown and has been difficult to investigate using animals with short life spans. With brain tissues from longer-lived rhesus monkeys of different ages, we found that aging reduces ubiquitin-proteasomal activity and also increases the level of ubiquitin-conjugating enzyme UBE2N (Ubc13) in synaptosomes. Synaptosomal fractions from aged monkey brain increase in vitro ubiquitinated huntingtin, whereas depletion of UBE2N markedly reduces this increase. Overexpressing UBE2N increases the aggregation of mutant huntingtin, and reducing UBE2N attenuates huntingtin aggregation in cellular and mouse models of HD. Our studies suggest that increased UBE2N plays a critical role in the synaptosomal accumulation of mutant huntingtin with age. PMID:25343992

  14. 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.

  15. Issues in high-throughput comparative modelling: a case study using the ubiquitin E2 conjugating enzymes.

    PubMed

    Winn, P J; Battey, J N D; Schleinkofer, K; Banerjee, A; Wade, R C

    2005-02-01

    Sequences of the ubiquitin-conjugating enzyme (UBC or E2) family were used as a test set to investigate issues associated with the high-throughput comparative modelling of protein structures. A semi-automatic method was initially developed with particular emphasis on producing models of a quality suitable for structural comparison. Structural and sequence features of the E2 family were used to improve the sequence alignment and the quality of the structural templates. Initially, failure to correct for subtle structural inconsistencies between templates lead to problems in the comparative analysis of the UBC electrostatic potentials. Modelling of known UBC structures using Modeller 4.0 showed that multiple templates produced, on average, no better models than the use of just one template, as judged by the root-mean-squared deviation between the comparative model and crystal structure backbones. Using four different quality-checking methods, for a given target sequence, it was not possible to distinguish the model most similar to the experimental structure. The UBC models were thus finally modelled using only the crystal structure template with the highest sequence identity to the target to be modelled, and producing only one model solution. Quality checking was used to reject models with obvious structural anomalies (e.g., bad side-chain packing). The resulting models have been used for a comparison of UBC structural features and of their electrostatic potentials. The work was extended through the development of a fully automated pipeline that identifies E2 sequences in the sequence databases, aligns and models them, and calculates the associated electrostatic potential.

  16. 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

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

    PubMed

    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.

  18. Direct observation of an ensemble of stable collapsed states in the mechanical folding of ubiquitin

    PubMed Central

    Garcia-Manyes, Sergi; Dougan, Lorna; Badilla, Carmen L.; Brujić, Jasna; Fernández, Julio M.

    2009-01-01

    Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested by bulk techniques, because the conformational diversity of folding molecules has been experimentally unapproachable. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length and mechanical stability during each stage of folding. Here, we discover that upon hydrophobic collapse, the protein rapidly selects a subset of minimum energy structures that are mechanically weak and essential precursors of the native fold. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. Our results support the validity of statistical mechanics models in describing the folding of a small protein on biological timescales. PMID:19541635

  19. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Transition state theory for enzyme kinetics

    PubMed Central

    Truhlar, Donald G.

    2015-01-01

    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

  1. The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region

    PubMed Central

    Sig Nielsen, Inga; Nielsen, Olaf; Murray, Johanne M.; Thon, Geneviève

    2002-01-01

    Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6. PMID:12456009

  2. The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region.

    PubMed

    Nielsen, Inga Sig; Nielsen, Olaf; Murray, Johanne M; Thon, Geneviève

    2002-08-01

    Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.

  3. Constitutive expression of a peanut ubiquitin-conjugating enzyme gene in Arabidopsis confers improved water-stress tolerance through regulation of stress-responsive gene expression.

    PubMed

    Wan, Xiaorong; Mo, Aiqiong; Liu, Shuai; Yang, Lixia; Li, Ling

    2011-04-01

    Ubiquitin (Ub)-conjugating enzymes (UBCs) are key enzymes involved in ubiquitination. Although UBCs have been shown to play important roles in regulating various aspects of plant growth and development, the role of plant UBCs in abiotic stress response needs to be examined further. Here we report the characterization of a ubiquitin-conjugating enzyme gene AhUBC2 from dehydrated peanut plants. The expression of AhUBC2 gene in peanut plants is responsive to physiological water-stress induced by polyethylene glycol (PEG6000), high salinity, abscisic acid (ABA) or low temperature. The constitutive expression of AhUBC2 gene in wild-type Arabidopsis confers improved tolerance to water-stress induced by sorbitol or soil drought in 35S::AhUBC2 transgenic plants. Constitutive expression of AhUBC2 results in significantly increased expressions of three stress-responsive genes P5CS1, RD29A and KIN1 in 35S::AhUBC2 Arabidopsis grown under normal conditions, whereas the expressions of other four stress-responsive genes NCED3, ABF3, RD29B and RD22 are not affected. The proline level in 35S::AhUBC2 Arabidopsis is significantly higher than that in wild-type Arabidopsis under both soil-drought stressed and control conditions. In contrast, there is no significant difference in the levels of NCED3 transcript and endogenous ABA between wild-type and 35S::AhUBC2 Arabidopsis. These results suggest that constitutive expression of AhUBC2 in Arabidopsis confers improved water-stress tolerance likely through activating an ABA-independent signaling pathway, including regulating the expression of ABA-independent stress-responsive genes and promoting the synthesis of osmolyte proline to protect plants from water deficit.

  4. 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

  5. Autoregulation of the 26S proteasome by in situ ubiquitination

    PubMed Central

    Jacobson, Andrew D.; MacFadden, Andrea; Wu, Zhiping; Peng, Junmin; Liu, Chang-Wei

    2014-01-01

    The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome's ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels. PMID:24743594

  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.

  7. The ubiquitin-conjugating enzyme UbcH6 regulates the transcriptional repression activity of the SCA1 gene product ataxin-1.

    PubMed

    Lee, Soyeon; Hong, Sunghoi; Kang, Seongman

    2008-08-08

    Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. We previously reported that the E2 ubiquitin-conjugating enzyme UbcH6 interacts with and ubiquitinates the ataxin-1 proteins as an E2-substrate cognate pair in the ubiquitin-proteasome system. In the present study, we further investigated whether the function of ataxin-1 is associated with UbcH6 and found that UbcH6 regulates the transcriptional repression activity of ataxin-1. The overexpression of UbcH6 reduced the transcriptional repression activity of ataxin-1. Interestingly, ataxin-1(30Q) was more affected by the presence of UbcH6 than ataxin-1(82Q), implying that the length of the polyglutamine tract in ataxin-1 might be involved in determining the stability of ataxin-1. The half-life of ataxin-1(82Q) was longer than that of ataxin-1(30Q) in the presence of UbcH6. shRNAs targeting UbcH6 enhanced the transcriptional repression activity of ataxin-1. In addition, the overexpression of UbcH6 reduced the formation of ataxin-1 aggregates. Our studies demonstrate that UbcH6 modulates the transcriptional repression activity of ataxin-1 by modulating the degradation of ataxin-1, suggesting that UbcH6 may have some therapeutic potential in the treatment of SCA1.

  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. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ.

    PubMed

    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

    2010-01-15

    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.

  10. Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1.

    PubMed

    Sudha, T; Tsuji, H; Sameshima, M; Matsuda, Y; Kaneda, S; Nagai, Y; Yamao, F; Seno, T

    1995-03-01

    A mouse cell mutant, ts85, containing the temperature-sensitive ubiquitin-activating enzyme was arrested in G2 phase at the non-permissive temperature. In the arrested cells, azure C, a nucleolus-specific stain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes with an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron microscopic examination revealed a cloud of unidentified electron-dense particles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality. FISH with rDNA and NOR silver staining demonstrated that the pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaphase of the stressed cells after release indicated a delayed dissociation of the nucleolus that brought about the abnormal chromosomes in M-phase. ts85 cells transfected with the mouse E1 cDNA recovered growth at the non-permissive temperature and no longer showed abnormal nucleolar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chromosomes.

  11. A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins1

    PubMed Central

    Berndsen, Christopher E.; Wolberger, Cynthia

    2011-01-01

    Ubiquitination is a widely-studied regulatory modification involved in protein degradation, DNA damage repair and the immune response. Conjugation of ubiquitin to a substrate lysine occurs in an enzymatic cascade involving an E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, or E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes. PMID:21771579

  12. Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis

    PubMed Central

    So, Hyun-Ah; Kang, Jee-Sook; Chung, Young Soo; Lee, Jai-Heon

    2013-01-01

    The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase. PMID:23824688

  13. Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis.

    PubMed

    Chung, Eunsook; Cho, Chang-Woo; So, Hyun-Ah; Kang, Jee-Sook; Chung, Young Soo; Lee, Jai-Heon

    2013-01-01

    The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thalianaVrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

  14. lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

    PubMed Central

    2012-01-01

    Background Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg) mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. Results The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among

  15. 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

  16. 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.

  17. Enzyme stabilization: state of the art.

    PubMed

    Gianfreda, L; Scarfi, M R

    1991-02-02

    '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.

  18. Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides.

    PubMed

    Bacchi, Marine; Fould, Benjamin; Jullian, Magali; Kreiter, Aude; Maurras, Amélie; Nosjean, Olivier; Coursindel, Thibault; Puget, Karine; Ferry, Gilles; Boutin, Jean A

    2017-02-15

    Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein "murine double minute 2" (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.

  19. Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme.

    PubMed Central

    Wing, S S; Bedard, N

    1996-01-01

    Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E2(14K)), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am.J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1-3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E2(14K) mRNA levels in L6 myotubes. Insulin suppressed levels of E2(14K) mRNA with an IC50 of 4 x 10(-9) M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E2(14K), also increase in skeletal muscle upon fasting. Reduction of E2(14K) mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5 x 10(-10) M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E2(14K) mRNA levels with an IC50 of 3 x 10(-11) M. DES-IGF-I did not alter rates of transcription of the E2(14K) gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3' non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E2(14K) expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates. PMID

  20. Ubiquitin Ligases: Structure, Function, and Regulation.

    PubMed

    Zheng, Ning; Shabek, Nitzan

    2017-06-20

    Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

  1. 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

  2. Assessing the Native State Conformational Distribution of Ubiquitin by Peptide Acidity

    PubMed Central

    Hernández, Griselda; Anderson, Janet S.; LeMaster, David M.

    2011-01-01

    At equilibrium, every energetically feasible conformation of a protein occurs with a non-zero probability. Quantitative analysis of protein flexibility is thus synonymous with determining the proper Boltzmann-weighting of this conformational distribution. The exchange reactivity of solvent-exposed amide hydrogens greatly varies with conformation, while the short-lived peptide anion intermediate implies an insensitivity to the dynamics of conformational motion. Amides that are well-exposed in model conformational ensembles of ubiquitin vary a million-fold in exchange rates which continuum dielectric methods can predict with an rmsd of 3. However, the exchange rates for many of the more rarely exposed amides are markedly overestimated in the PDB-deposited 2K39 and 2KN5 ubiquitin ensembles, while the 2NR2 ensemble predictions are largely consistent with those of the Boltzmann-weighted conformational distribution sampled at the level of 1%. The correlation between the fraction of solvent-accessible conformations for a given amide hydrogen and the exchange rate constant for that residue provides a useful monitor of the degree of completeness with which a given ensemble has sampled the energetically accessible conformational space. These exchange predictions correlate with the degree to which each ensemble deviates from a set of 46 ubiquitin X-ray structures. Kolmogorov-Smirnov analysis for the distribution of intra- and inter-ensemble pairwise structural rmsd values assisted the identification of a subensemble of 2K39 that eliminates the overestimations of hydrogen exchange rates observed for the full ensemble. The relative merits of incorporating experimental restraints into the conformational sampling process is compared to using these restraints as filters to select subpopulations consistent with the experimental data. PMID:21055867

  3. 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

  4. Observing Single Enzyme Molecules Interconvert between Activity States upon Heating

    PubMed Central

    Rojek, Marcin J.; Walt, David R.

    2014-01-01

    In this paper, we demonstrate that single enzyme molecules of β-galactosidase interconvert between different activity states upon exposure to short pulses of heat. We show that these changes in activity are the result of different enzyme conformations. Hundreds of single β-galactosidase molecules are trapped in femtoliter reaction chambers and the individual enzymes are subjected to short heating pulses. When heating pulses are introduced into the system, the enzyme molecules switch between different activity states. Furthermore, we observe that the changes in activity are random and do not correlate with the enzyme's original activity. This study demonstrates that different stable conformations play an important role in the static heterogeneity reported previously, resulting in distinct long-lived activity states of enzyme molecules in a population. PMID:24465972

  5. 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.

  6. 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.

  7. Conformational Sub-states and Populations in Enzyme Catalysis

    SciTech Connect

    Agarwal, Pratul K; Doucet, Nicholas; Chennubholta, C; Ramanathan, Arvind

    2016-01-01

    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.

  8. 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)

  9. 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)

  10. Conformational Sub-states and Populations in Enzyme Catalysis

    PubMed Central

    Agarwal, Pratul K.; Doucet, Nicolas; Chennubhotla, Chakra; Ramanathan, Arvind; Narayanan, Chitra

    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 allows 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 substates, 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 is 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 (NMR) have been developed for quantitative characterization of these substates and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems. PMID:27497171

  11. In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

    PubMed Central

    Gardner, Richard G.; Shearer, Alexander G.; Hampton, Randolph Y.

    2001-01-01

    Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate. PMID:11390656

  12. Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924).

    PubMed

    Wong, Kit Man; Micel, Lindsey N; Selby, Heather M; Tan, Aik Choon; Pitts, Todd M; Bagby, Stacey M; Spreafico, Anna; Klauck, Peter J; Blakemore, Stephen J; Smith, Peter F; McDonald, Alice; Berger, Allison; Tentler, John J; Eckhardt, S Gail

    2017-02-01

    Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC50 < 0.3 μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

  13. Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase.

    PubMed

    Cadwell, Ken; Coscoy, Laurent

    2005-07-01

    Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.

  14. Identification of enzyme inhibitory mechanisms from steady-state kinetics.

    PubMed

    Fange, David; Lovmar, Martin; Pavlov, Michael Y; Ehrenberg, Måns

    2011-09-01

    Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.

  15. Dissecting the ubiquitin pathway by mass spectrometry

    PubMed Central

    Xu, Ping; Peng, Junmin

    2007-01-01

    Summary Protein modification by ubiquitin is a central regulatory mechanism in eukaryotic cells. Recent proteomics developments in mass spectrometry enable systematic analysis of cellular components in the ubiquitin pathway. Here, we review the advances in analyzing ubiquitinated substrates, determining modified lysine residues, quantifying polyubiquitin chain topologies, as well as profiling deubiquitinating enzymes based on the activity. Moreover, proteomic approaches have been developed for probing the interactome of proteasome and for identifying proteins with ubiquitin-binding domains. Similar strategies have been applied on the studies of the modification by ubiquitin-like proteins as well. These strategies are discussed with respect to their advantages, limitations and potential improvements. While the utilization of current methodologies has rapidly expanded the scope of protein modification by the ubiquitin family, a more active role is anticipated in the functional studies with the emerging of quantitative mass spectrometry. PMID:17055348

  16. Proteasome subunit Rpn13 is a novel ubiquitin receptor

    PubMed Central

    Husnjak, Koraljka; Elsasser, Suzanne; Zhang, Naixia; Chen, Xiang; Randles, Leah; Shi, Yuan; Hofmann, Kay; Walters, Kylie; Finley, Daniel; Dikic, Ivan

    2010-01-01

    Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin via a conserved N-terminal region termed the Pru domain (Pleckstrin-like receptor for ubiquitin), which binds K48-linked diubiquitin with an affinity of ∼90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like domains of the UBL/UBA family of ubiquitin receptors. A synthetic phenotype results in yeast when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Since Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome. PMID:18497817

  17. 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.

  18. Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System*

    PubMed Central

    Singh, Rajesh K.; Zerath, Sylvia; Kleifeld, Oded; Scheffner, Martin; Glickman, Michael H.; Fushman, David

    2012-01-01

    Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin. PMID:23105008

  19. 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.

  20. Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.

    PubMed

    Misaghi, Shahram; Galardy, Paul J; Meester, Wim J N; Ovaa, Huib; Ploegh, Hidde L; Gaudet, Rachelle

    2005-01-14

    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 A 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.

  1. 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

  2. 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)

  3. 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)

  4. Targeting ubiquitination for cancer therapies

    PubMed Central

    Morrow, John Kenneth; Lin, Hui-Kuan; Sun, Shao-Cong; Zhang, Shuxing

    2015-01-01

    Ubiquitination, the structured degradation and turnover of cellular proteins, is regulated by the ubiquitin–proteasome system (UPS). Most proteins that are critical for cellular regulations and functions are targets of the process. Ubiquitination is comprised of a sequence of three enzymatic steps, and aberrations in the pathway can lead to tumor development and progression as observed in many cancer types. Recent evidence indicates that targeting the UPS is effective for certain cancer treatment, but many more potential targets might have been previously overlooked. In this review, we will discuss the current state of small molecules that target various elements of ubiquitination. Special attention will be given to novel inhibitors of E3 ubiquitin ligases, especially those in the SCF family. PMID:26630263

  5. Substrate ground state binding energy concentration is realized as transition state stabilization in physiological enzyme catalysis.

    PubMed

    Britt, Billy Mark

    2004-09-30

    Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.

  6. Ubiquitination in Periodontal Disease: A Review

    PubMed Central

    Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

    2017-01-01

    Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue’s response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin–protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases. PMID:28698506

  7. Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

    PubMed

    Nagel, Marie-Kristin; Kalinowska, Kamila; Vogel, Karin; Reynolds, Gregory D; Wu, Zhixiang; Anzenberger, Franziska; Ichikawa, Mie; Tsutsumi, Chie; Sato, Masa H; Kuster, Bernhard; Bednarek, Sebastian Y; Isono, Erika

    2017-08-22

    Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

  8. 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.

  9. Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (Nicotiana tabacum).

    PubMed

    Bahmani, Ramin; Kim, DongGwan; Lee, Byoung Doo; Hwang, Seongbin

    2017-07-01

    Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca(2+)/H(+) exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.

  10. RSK1 protects P-glycoprotein/ABCB1 against ubiquitin–proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1

    PubMed Central

    Katayama, Kazuhiro; Fujiwara, Chiaki; Noguchi, Kohji; Sugimoto, Yoshikazu

    2016-01-01

    P-glycoprotein (P-gp) is a critical determinant of multidrug resistance in cancer. We previously reported that MAPK inhibition downregulates P-gp expression and that P-gp undergoes ubiquitin–proteasomal degradation regulated by UBE2R1 and SCFFbx15. Here, we investigated the crosstalk between MAPK inhibition and the ubiquitin–proteasomal degradation of P-gp. Proteasome inhibitors or knockdown of FBXO15 and/or UBE2R1 cancelled MEK inhibitor-induced P-gp downregulation. RSK1 phosphorylated Thr162 on UBE2R1 but did not phosphorylate FBXO15. MEK and RSK inhibitors increased UBE2R1-WT but not UBE2R1-T162D and -T162A expression. UBE2R1-T162D showed higher self-ubiquitination and destabilisation than UBE2R1-WT and -T162A. Unlike UBE2R1-WT and -T162A, UBE2R1-T162D did not induce P-gp ubiquitination. UBE2R1-WT or -T162A downregulated P-gp expression and upregulated rhodamine 123 level and sensitivity to vincristine and doxorubicin. However, UBE2R1-T162D did not confer any change in P-gp expression, rhodamine 123 accumulation and sensitivity to the drugs. These results suggest that RSK1 protects P-gp against ubiquitination by reducing UBE2R1 stability. PMID:27786305

  11. Using Protein Motion to Read, Write, and Erase Ubiquitin Signals*

    PubMed Central

    Phillips, Aaron H.; Corn, Jacob E.

    2015-01-01

    Eukaryotes use a tiny protein called ubiquitin to send a variety of signals, most often by post-translationally attaching ubiquitins to substrate proteins and to each other, thereby forming polyubiquitin chains. A combination of biophysical, biochemical, and biological studies has shown that complex macromolecular dynamics are central to many aspects of ubiquitin signaling. This review focuses on how equilibrium fluctuations and coordinated motions of ubiquitin itself, the ubiquitin conjugation machinery, and deubiquitinating enzymes enable activity and regulation on many levels, with implications for how such a tiny protein can send so many signals. PMID:26354440

  12. 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 ...

  13. Influence of the enzyme phosphorylation state and the substrate on PKA enzyme dynamics.

    PubMed

    Montenegro, Manuel; Masgrau, Laura; González-Lafont, Angels; Lluch, José M; Garcia-Viloca, Mireia

    2012-02-01

    cAMP-dependent protein kinase (PKA) is one of the simplest and best understood members of the protein kinase family. In a previous study, we have theoretically studied the complex between PKA and the heptapeptide substrate Kemptide by classical molecular dynamics. On the basis of the results obtained for Kemptide, the aim of the present work is to explore how the different conditions, such as phosphorylation state, substrate, and mutations of key residues affect the enzyme dynamics. We have built different models of the complex; particularly we have focused our attention on two crystallographic structures which main difference consists in their phosphorylation state. The first one has the residue Thr197 modified into a phospho-threonine (pThr197); the second one, in addition to the same Thr197, has also the residue Ser338 modified into a phospho-serine (pSer338). In addition, we have analyzed the effect of the choice of the substrate by building a model of the PKA-SP20 Michaelis complex. Finally, we have theoretically studied the effect of the mutation of the highly conserved residue Asp166 that, experimentally, leads to a decrease of the reaction rate. The results of this study give insight into the dynamical states of the enzyme and their relationship with different elements of the model, which correspond to different natural or human guided situations of the active biological system.

  14. Regulation of the Polycomb protein RING1B ubiquitination by USP7.

    PubMed

    de Bie, Prim; Zaaroor-Regev, Daphna; Ciechanover, Aaron

    2010-09-24

    The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

    PubMed Central

    Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

    2016-01-01

    In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases. PMID:27827840

  16. USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase.

    PubMed

    Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

    2016-11-02

    In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

  17. Backbone assignments of the 26 kDa neuron-specific ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1).

    PubMed

    Andersson, Fredrik I; Jackson, Sophie E; Hsu, Shang-Te Danny

    2010-04-01

    UCH-L1 is a member of the family of ubiquitin C-terminal hydrolases whose primary role is to hydrolyze small C-terminal adducts of ubiquitin to generate free ubiquitin monomers. Expression of UCH-L1 is highly specific to neurons and point mutations in this enzyme are associated with a hereditary form of Parkinson's disease. Herein, we present the NMR backbone assignments of human UCH-L1, thus enabling future solution-state NMR spectroscopic studies on the structure and function of this important protein.

  18. Conformation Types of Ubiquitin [M+8H]8+ Ions from Water:Methanol Solutions: Evidence for the N and A States in Aqueous Solution

    PubMed Central

    Shi, Huilin; Pierson, Nicholas A.; Valentine, Stephen J.; Clemmer, David E.

    2012-01-01

    Ion mobility and mass spectrometry measurements are used to examine the gas-phase populations of [M+8H]8+ ubiquitin ions formed upon electrospraying 20 different solutions: from 100:0 to 5:95 water:methanol that are maintained at pH = 2.0. Over this range of solution conditions, mobility distributions for the +8 charge state show substantial variations. Here we develop a model that treats the combined measurements as one data set. By varying the relative abundances of a discrete set of conformation types, it is possible to represent distributions obtained from any solution. For solutions that favor the well-known A-state ubiquitin, it is possible to represent the gas-phase distributions with seven conformation types. Aqueous conditions that favor the native structure require four more structural types to represent the distribution. This analysis provides the first direct evidence for trace amounts of the A state under native conditions. The method of analysis presented here should help illuminate how solution populations evolve into new gas-phase structures as solvent is removed. Evidence for trace quantities of previously unknown states under native solution conditions may provide insight about the relationship of dynamics to protein function as well as misfolding and aggregation phenomena. PMID:22315998

  19. 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

  20. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  1. [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.

  2. 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

  3. Structural determinants of ubiquitin conjugation in Entamoeba histolytica.

    PubMed

    Bosch, Dustin E; Siderovski, David P

    2013-01-25

    Ubiquitination is important for numerous cellular processes in most eukaryotic organisms, including cellular proliferation, development, and protein turnover by the proteasome. The intestinal parasite Entamoeba histolytica harbors an extensive ubiquitin-proteasome system. Proteasome inhibitors are known to impair parasite proliferation and encystation, suggesting the ubiquitin-proteasome pathway as a viable therapeutic target. However, no functional studies of the E. histolytica ubiquitination enzymes have yet emerged. Here, we have cloned and characterized multiple E. histolytica ubiquitination components, spanning ubiquitin and its activating (E1), conjugating (E2), and ligating (E3) enzymes. Crystal structures of EhUbiquitin reveal a clustering of unique residues on the α1 helix surface, including an eighth surface lysine not found in other organisms, which may allow for a unique polyubiquitin linkage in E. histolytica. EhUbiquitin is activated by and forms a thioester bond with EhUba1 (E1) in vitro, in an ATP- and magnesium-dependent fashion. EhUba1 exhibits a greater maximal initial velocity of pyrophosphate:ATP exchange than its human homolog, suggesting different kinetics of ubiquitin activation in E. histolytica. EhUba1 engages the E2 enzyme EhUbc5 through its ubiquitin-fold domain to transfer the EhUbiquitin thioester. However, EhUbc5 has a >10-fold preference for EhUba1∼Ub compared with unconjugated EhUba1. A crystal structure of EhUbc5 allowed prediction of a noncovalent "backside" interaction with EhUbiquitin and E3 enzymes. EhUbc5 selectively engages EhRING1 (E3) to the exclusion of two HECT family E3 ligases, and mutagenesis indicates a conserved mode of E2/RING-E3 interaction in E. histolytica.

  4. Linear ubiquitination in immunity.

    PubMed

    Shimizu, Yutaka; Taraborrelli, Lucia; Walczak, Henning

    2015-07-01

    Linear ubiquitination is a post-translational protein modification recently discovered to be crucial for innate and adaptive immune signaling. The function of linear ubiquitin chains is regulated at multiple levels: generation, recognition, and removal. These chains are generated by the linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin E3 capable of forming the linear ubiquitin linkage de novo. LUBAC is not only relevant for activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in various signaling pathways, but importantly, it also regulates cell death downstream of immune receptors capable of inducing this response. Recognition of the linear ubiquitin linkage is specifically mediated by certain ubiquitin receptors, which is crucial for translation into the intended signaling outputs. LUBAC deficiency results in attenuated gene activation and increased cell death, causing pathologic conditions in both, mice, and humans. Removal of ubiquitin chains is mediated by deubiquitinases (DUBs). Two of them, OTULIN and CYLD, are constitutively associated with LUBAC. Here, we review the current knowledge on linear ubiquitination in immune signaling pathways and the biochemical mechanisms as to how linear polyubiquitin exerts its functions distinctly from those of other ubiquitin linkage types.

  5. Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A

    PubMed Central

    Pulvino, Mary; Liang, Yue; Oleksyn, David; DeRan, Michael; Van Pelt, Elise; Shapiro, Joel; Sanz, Ignacio; Chen, Luojing

    2012-01-01

    Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell–like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell–like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents. PMID:22791293

  6. Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.

    PubMed

    Pulvino, Mary; Liang, Yue; Oleksyn, David; DeRan, Michael; Van Pelt, Elise; Shapiro, Joel; Sanz, Ignacio; Chen, Luojing; Zhao, Jiyong

    2012-08-23

    Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.

  7. 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 (3.3-22 μM) values compared to TZ9.

  8. 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

  9. Minireview: Ubiquitination-regulated G Protein-Coupled Receptor Signaling and Trafficking

    PubMed Central

    Alonso, Verónica

    2013-01-01

    G protein-coupled receptors (GPCRs) are the largest and most diverse superfamily of membrane proteins and mediate most cellular responses to hormones and neurotransmitters. Posttranslational modifications are considered the main regulators of all GPCRs. In addition to phosphorylation, glycosylation, and palmitoylation, increasing evidence as reviewed here reveals that ubiquitination also regulates the magnitude and temporospatial aspects of GPCR signaling. Posttranslational protein modification by ubiquitin is a key molecular mechanism governing proteins degradation. Ubiquitination mediates the covalent conjugation of ubiquitin, a highly conserved polypeptide of 76 amino acids, to protein substrates. This process is catalyzed by 3 enzymes acting in tandem: an E1, ubiquitin-activating enzyme; an E2, ubiquitin-carrying enzyme; and an E3, ubiquitin ligase. Ubiquitination is counteracted by deubiquitinating enzymes that deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. Although ubiquitination is known to target many GPCRs for lysosomal or proteasomal degradation, emerging findings define novel roles for the basal status of ubiquitination and for rapid deubiquitination and transubiquitination controlling cell surface expression and cellular responsiveness of some GPCRs. In this review, we highlight the classical and novel roles of ubiquitin in the regulation of GPCR function, signaling, and trafficking. PMID:23471539

  10. 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.

  11. 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.

  12. 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.)

  13. Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin.

    PubMed

    Pai, Ming-Tao; Tzeng, Shiou-Ru; Kovacs, Jeffrey J; Keaton, Mignon A; Li, Shawn S-C; Yao, Tso-Pang; Zhou, Pei

    2007-07-06

    The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here, we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisting of 12 residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the alpha 3 helix and the central beta-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76(Ub) carboxylate group either by a peptide or isopeptide bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as "sensors" of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes.

  14. Solution Structure of the Ubp-M BUZ Domain, a Highly Specific Protein Module That Recognizes the C-terminal Tail of Free Ubiquitin

    PubMed Central

    Pai, Ming-Tao; Tzeng, Shiou-Ru; Kovacs, Jeffrey J.; Keaton, Mignon A.; Li, Shawn S.-C.; Yao, Tso-Pang; Zhou, Pei

    2010-01-01

    Summary The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisted of twelve residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the α3 helix and the central β-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76Ub carboxylate group either by a peptide- or isopeptide-bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as “sensors” of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes. PMID:17512543

  15. Identification of the Major Ubiquitin-binding Domain of the Pseudomonas aeruginosa ExoU A2 Phospholipase*

    PubMed Central

    Anderson, David M.; Feix, Jimmy B.; Monroe, Andrew L.; Peterson, Francis C.; Volkman, Brian F.; Haas, Arthur L.; Frank, Dara W.

    2013-01-01

    Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex. PMID:23908356

  16. 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

  17. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

    PubMed

    Altun, Mikael; Walter, Thomas S; Kramer, Holger B; Herr, Patrick; Iphöfer, Alexander; Boström, Johan; David, Yael; Komsany, Alia; Ternette, Nicola; Navon, Ami; Stuart, David I; Ren, Jingshan; Kessler, Benedikt M

    2015-01-01

    Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

  18. 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

    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

    2013-05-21

    Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs); ∼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 intramolecular 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

  19. Elevated growth temperature decreases levels of the PEX5 peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4 ubiquitin-conjugating enzyme.

    PubMed

    Kao, Yun-Ting; Bartel, Bonnie

    2015-09-16

    Peroxisomes house critical metabolic reactions. For example, fatty acid β-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants β-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. We conclude that growth at elevated temperature increases

  20. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase.

    PubMed

    Faesen, Alex C; Dirac, Annette M G; Shanmugham, Anitha; Ovaa, Huib; Perrakis, Anastassis; Sixma, Titia K

    2011-10-07

    The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Analysis of human immunodeficiency virus type 1 Gag ubiquitination.

    PubMed

    Gottwein, Eva; Kräusslich, Hans-Georg

    2005-07-01

    Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.

  2. 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

  3. The role of E3 ligases in the ubiquitin-dependent regulation of spermatogenesis.

    PubMed

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

    2014-06-01

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation.

    PubMed

    Yamada, Yuya; Suzuki, Nobuo N; Hanada, Takao; Ichimura, Yoshinobu; Kumeta, Hiroyuki; Fujioka, Yuko; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2007-03-16

    Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.

  5. 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

  6. 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

  7. Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

    PubMed Central

    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

  8. 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

  9. Deficiency in the Ubiquitin Conjugating Enzyme UBE2A in Alzheimer’s Disease (AD) is Linked to Deficits in a Natural Circular miRNA-7 Sponge (circRNA; ciRS-7)

    PubMed Central

    Zhao, Yuhai; Alexandrov, Peter N.; Jaber, Vivian; Lukiw, Walter J.

    2016-01-01

    Our understanding of the highly specialized functions for small non-coding single-stranded RNA (ssRNA) in the transcriptome of the human central nervous system (CNS) continues to evolve. Circular RNAs (circRNAs), a recently discovered class of ssRNA enriched in the brain and retina, are extremely stable and intrinsically resilient to degradation by exonuclease. Conventional methods of ssRNA, microRNA (miRNA), or messenger RNA (mRNA) detection and quantitation requiring free ribonucleotide ends may have considerably underestimated the quantity and significance of CNS circRNA in the CNS. Highly-specific small ssRNAs, such as the ~23 nucleotide (nt) Homo sapien microRNA-7 (hsa-miRNA-7; chr 9q21.32), are not only abundant in the human limbic system but are, in addition, associated with a ~1400 nt circRNA for miRNA-7 (ciRS-7) in the same anatomical region. Structurally, ciRS-7 contains about ~70 tandem anti-miRNA-7 sequences and acts as an endogenous, anti-complementary miRNA-7 “sponge” that attracts, binds, and, hence, quenches, natural miRNA-7 functions. Using a combination of DNA and miRNA array technologies, enhanced LED-Northern and Western blot hybridization, and the magnesium-dependent exoribonuclease and circRNA-sensitive probe RNaseR, here we provide evidence of a significantly misregulated ciRS-7-miRNA-7-UBE2A circuit in sporadic Alzheimer’s disease (AD) neocortex (Brodmann A22) and hippocampal CA1. Deficits in ciRS-7-mediated “sponging events”, resulting in excess ambient miRNA-7 appear to drive the selective down-regulation in the expression of miRNA-7-sensitive mRNA targets, such as that encoding the ubiquitin conjugating enzyme E2A (UBE2A; chr Xq24). UBE2A, which normally serves as a central effector in the ubiquitin-26S proteasome system, coordinates the clearance of amyloid peptides via proteolysis, is known to be depleted in sporadic AD brain and, hence, contributes to amyloid accumulation and the formation of senile plaque deposits

  10. Deficiency in the Ubiquitin Conjugating Enzyme UBE2A in Alzheimer's Disease (AD) is Linked to Deficits in a Natural Circular miRNA-7 Sponge (circRNA; ciRS-7).

    PubMed

    Zhao, Yuhai; Alexandrov, Peter N; Jaber, Vivian; Lukiw, Walter J

    2016-12-05

    Our understanding of the highly specialized functions for small non-coding single-stranded RNA (ssRNA) in the transcriptome of the human central nervous system (CNS) continues to evolve. Circular RNAs (circRNAs), a recently discovered class of ssRNA enriched in the brain and retina, are extremely stable and intrinsically resilient to degradation by exonuclease. Conventional methods of ssRNA, microRNA (miRNA), or messenger RNA (mRNA) detection and quantitation requiring free ribonucleotide ends may have considerably underestimated the quantity and significance of CNS circRNA in the CNS. Highly-specific small ssRNAs, such as the ~23 nucleotide (nt) Homo sapien microRNA-7 (hsa-miRNA-7; chr 9q21.32), are not only abundant in the human limbic system but are, in addition, associated with a ~1400 nt circRNA for miRNA-7 (ciRS-7) in the same anatomical region. Structurally, ciRS-7 contains about ~70 tandem anti-miRNA-7 sequences and acts as an endogenous, anti-complementary miRNA-7 "sponge" that attracts, binds, and, hence, quenches, natural miRNA-7 functions. Using a combination of DNA and miRNA array technologies, enhanced LED-Northern and Western blot hybridization, and the magnesium-dependent exoribonuclease and circRNA-sensitive probe RNaseR, here we provide evidence of a significantly misregulated ciRS-7-miRNA-7-UBE2A circuit in sporadic Alzheimer's disease (AD) neocortex (Brodmann A22) and hippocampal CA1. Deficits in ciRS-7-mediated "sponging events", resulting in excess ambient miRNA-7 appear to drive the selective down-regulation in the expression of miRNA-7-sensitive mRNA targets, such as that encoding the ubiquitin conjugating enzyme E2A (UBE2A; chr Xq24). UBE2A, which normally serves as a central effector in the ubiquitin-26S proteasome system, coordinates the clearance of amyloid peptides via proteolysis, is known to be depleted in sporadic AD brain and, hence, contributes to amyloid accumulation and the formation of senile plaque deposits. Dysfunction of circ

  11. 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

  12. 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

  13. Characterizing substrate selectivity of ubiquitin C-terminal hydrolase-L3 using engineered α-linked ubiquitin substrates.

    PubMed

    Navarro, Mario F; Carmody, Lisa; Romo-Fewell, Octavio; Lokensgard, Melissa E; Love, John J

    2014-12-30

    The ubiquitin-proteasome system (UPS) is highly complex and entails the concerted actions of many enzymes that function to ubiquitinate proteins targeted to the proteasome as well as enzymes that remove and recycle ubiquitin for additional rounds of proteolysis. Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a human cytosolic deubiquitinase whose precise biological function is not known. It is believed to hydrolyze small peptides or chemical adducts from the C-terminus of ubiquitin that may be remnant from proteasomal processing. In addition, UCH-L3 is a highly effective biotechnological tool that is used to produce small or unstable peptides/proteins recalcitrant to production in Escherichia coli expression systems. Previous research, which explored the substrate selectivity of UCH-L3, demonstrated a substrate size limitation for proteins/peptides expressed as α-linked C-terminal fusions to ubiquitin and also suggested that an additional substrate property may affect UCH-L3 hydrolysis [ Larsen , C. N. et al. (1998) Biochemistry 37 , 3358 - 3368 ]. Using a series of engineered protein substrates, which are similar in size yet differ in secondary structure, we demonstrate that thermal stability is a key factor that significantly affects UCH-L3 hydrolysis. In addition, we show that the thermal stabilities of the engineered substrates are not altered by fusion to ubiquitin and offer a possible mechanism as to how ubiquitin affects the structural and unfolding properties of natural in vivo targets.

  14. 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

  15. 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

  16. 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

  17. 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

  18. Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads

    USDA-ARS?s Scientific Manuscript database

    The ubiquitin/proteasome pathway is the principal system for degradation of proteins in eukaryotes. Ubiquitin is a highly conserved polypeptide that covalently attaches to target proteins through the combined action ofubiquitin-activating enzyme (E1), conjugating enzyme (E2) and a protein ligase (E...

  19. 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

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

    PubMed Central

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

    2016-01-01

    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

  1. Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

    PubMed

    Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2008-07-16

    Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

  2. Ubiquitination and filamentous structure of cytidine triphosphate synthase

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  3. Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

    PubMed

    Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

    2016-11-01

    A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

  4. 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.

  5. 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

  6. Ubiquitin becomes ubiquitous in cancer: emerging roles of ubiquitin ligases and deubiquitinases in tumorigenesis and as therapeutic targets.

    PubMed

    Shi, Dingding; Grossman, Steven R

    2010-10-15

    By virtue of its ability to regulate both protein turnover and non-proteolytic signalling functions, ubiquitin protein conjugation has been implicated in the control of multiple cellular processes, including protein localization, cell cycle control, transcription regulation, DNA damage repair, and endocytosis. Ubiquitin metabolism enzymes have been identified as either oncogenes or tumor suppressors in a variety of cancers. Given that ubiquitin metabolism is governed by enzymes--E1, E2, E3, E4, deubiquitinases (DUBs), and the proteasome- the system as a whole is ripe for target and drug discovery in cancer. Of the ubiquitin/proteasome system components, the E3's and DUBs can recognize substrates with the most specificity, and are thus of key interest as drug targets in cancer. This review examines the molecular role in cancer, relevant substrates, and potential for pharmacologic development, of E3's and DUBs that have been associated thus far with human malignancies as oncogenes or tumor suppressors.

  7. Core signalling motif displaying multistability through multi-state enzymes

    PubMed Central

    Feng, Song; Sáez, Meritxell; Wiuf, Carsten; Feliu, Elisenda

    2016-01-01

    Bistability, and more generally multistability, is a key system dynamics feature enabling decision-making and memory in cells. Deciphering the molecular determinants of multistability is thus crucial for a better understanding of cellular pathways and their (re)engineering in synthetic biology. Here, we show that a key motif found predominantly in eukaryotic signalling systems, namely a futile signalling cycle, can display bistability when featuring a two-state kinase. We provide necessary and sufficient mathematical conditions on the kinetic parameters of this motif that guarantee the existence of multiple steady states. These conditions foster the intuition that bistability arises as a consequence of competition between the two states of the kinase. Extending from this result, we find that increasing the number of kinase states linearly translates into an increase in the number of steady states in the system. These findings reveal, to our knowledge, a new mechanism for the generation of bistability and multistability in cellular signalling systems. Further the futile cycle featuring a two-state kinase is among the smallest bistable signalling motifs. We show that multi-state kinases and the described competition-based motif are part of several natural signalling systems and thereby could enable them to implement complex information processing through multistability. These results indicate that multi-state kinases in signalling systems are readily exploited by natural evolution and could equally be used by synthetic approaches for the generation of multistable information processing systems at the cellular level. PMID:27733693

  8. New insights into the role of the ubiquitin-proteasome pathway in the regulation of apoptosis.

    PubMed

    Liu, Cui-Hua; Goldberg, Alfred L; Qiu, Xiao-Bo

    2007-01-01

    The ubiquitin-proteasome pathway (UPP) is the major system responsible for degradation of intracellular proteins in eukaryotes. By controlling the levels of key proteins, it regulates almost all of the cellular activities, including cell cycle progression, DNA replication and repair, transcription, protein quality control, immune response, and apoptosis. UPP is composed of the ubiquitination system that marks proteins for degradation and the proteasome which degrades the ubiquitinated proteins. The 26S proteasome is a 2400 kDa complex consisting of more than 40 subunits. Following ubiquitination catalyzed by the ubiquitin activating enzyme (El), a ubiquitin-carrier protein (E2), and one of the cell's many ubiquitin-protein ligases (E3s), the protein substrates are targeted to the proteasome for degradation into small peptides. E3s regulate the degradation of protein substrates indirectly by determining both the specificity and timing of substrate ubiquitination, whereas the deubiquitinating enzymes can inhibit this process by releasing ubiquitin from substrates. In this review, we attempt to highlight the recent progress in research on UPP and its role in the regulation of apoptosis by focusing on several of its important components, including the ubiqutin ligase Nrdp 1, which regulates ErbB/EGFR family of receptor tyrosine kinases, the ubiquitin-carrier protein BRUCE/Apollon (an Inhibitor of Apoptosis Protein), and the novel proteasome subunit hRpnl3 (a binding site for the deubiquitinating enzyme, UCH37).

  9. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  10. Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.

    PubMed

    van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

    2013-03-01

    The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

  11. Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1*

    PubMed Central

    van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

    2013-01-01

    The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1. PMID:23300075

  12. New insights to the ubiquitin-proteasome pathway (UPP) mechanism during spermatogenesis.

    PubMed

    Hou, Cong-Cong; Yang, Wan-Xi

    2013-04-01

    Spermatogenesis is a complicated and highly ordered process which begins with the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. After meiosis, several morphological changes occur during spermatogenesis. During spermatogenesis, many proteins and organelles are degraded, and the ubiquitin-proteasome pathway (UPP) plays a key role in the process which facilitates the formation of condensed sperm. UPP contains various indispensable components: ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase enzyme E3 and proteasomes. At some key stages of spermatogenesis, such as meiosis, acrosome biogenesis, and spermatozoa maturation, the ubiquitin-related components (including deubiquitination enzymes) exert positive and active functions. Generally speaking, deficient UPP will block spermatogenesis which may induce infertility at various degrees. Although ubiquitination during spermatogenesis has been widely investigated, further detailed aspects such as the mechanism of ubiquitination during the formation of midpiece and acrosome morphogenesis still remains unknown. The present review will overview current progress on ubiquitination during spermatogenesis, and will provide some suggestions for future studies on the functions of UPP components during spermatogenesis.

  13. The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

    PubMed Central

    Li, Jie; Chai, Qi-Yao; Liu, Cui Hua

    2016-01-01

    The ubiquitin system comprises enzymes that are responsible for ubiquitination and deubiquitination, as well as ubiquitin receptors that are capable of recognizing and deciphering the ubiquitin code, which act in coordination to regulate almost all host cellular processes, including host–pathogen interactions. In response to pathogen infection, the host innate immune system launches an array of distinct antimicrobial activities encompassing inflammatory signaling, phagosomal maturation, autophagy and apoptosis, all of which are fine-tuned by the ubiquitin system to eradicate the invading pathogens and to reduce concomitant host damage. By contrast, pathogens have evolved a cohort of exquisite strategies to evade host innate immunity by usurping the ubiquitin system for their own benefits. Here, we present recent advances regarding the ubiquitin system-mediated modulation of host–pathogen interplay, with a specific focus on host innate immune defenses and bacterial pathogen immune evasion. PMID:27524111

  14. pH modulation of transient state kinetics of enzymes. II. Transient state kinetics of plant cell wall acid phosphatase.

    PubMed

    Crasnier, M; Ricard, J

    1984-03-01

    The pre-steady-state kinetics of plant cell wall acid phosphatase has been investigated at different pH values. The approach of the steady stale lasts about 1 or 2 s and may be fitted with two exponential terms. For certain pH values the approach to the steady state exhibits damped oscillations. Plotting the sum and the product of the two time constants of these exponentials as a function of substrate concentration yields two straight lines. From the slopes and intercepts of these lines one may determine the values of rate and ionization constants involved in the reaction scheme. The results obtained are consistent with the view that the binding of the substrate to the enzyme does not induce a 'slow' conformation change of the enzyme. The enzyme reacts with its substrate while being mostly in its ionized form. Release of p-nitrophenol is also favoured by this ionized form of the enzyme. However, the hydrolysis of the phosphoryl-enzyme complex mostly occurs from the protonated form of the enzyme. The ionization constants of the free enzyme and of the various enzyme-ligand complexes are very similar.

  15. 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.

  16. Role of ubiquitin ligases and the proteasome in oncogenesis: novel targets for anticancer therapies.

    PubMed

    Micel, Lindsey N; Tentler, John J; Smith, Peter G; Eckhardt, Gail S

    2013-03-20

    The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS.

  17. Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies

    PubMed Central

    Micel, Lindsey N.; Tentler, John J.; Smith, Peter G.; Eckhardt, Gail S.

    2013-01-01

    The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS. PMID:23358974

  18. 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

  19. 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

    2015-07-03

    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.

  20. E1-E2 interactions in ubiquitin and Nedd8 ligation pathways.

    PubMed

    Tokgöz, Zeynep; Siepmann, Thomas J; Streich, Frederick; Kumar, Brajesh; Klein, Jennifer M; Haas, Arthur L

    2012-01-02

    Initial rates of E1-catalyzed E2 transthiolation have been used as a reporter function to probe the mechanism of 125I-ubiquitin transfer between activation and ligation half-reactions of ubiquitin conjugation. A functional survey of 11 representative human E2 paralogs reveals similar Km for binding to human Uba1 ternary complex (Km(ave)=121±72 nm) and kcat for ubiquitin transfer (kcat(ave)=4.0±1.2 s(-1)), suggesting that they possess a conserved binding site and transition state geometry and that they compete for charging through differences in intracellular concentration. Sequence analysis and mutagenesis localize this binding motif to three basic residues within Helix 1 of the E2 core domain, confirmed by transthiolation kinetics. Partial conservation of the motif among E2 paralogs not recognized by Uba1 suggests that another factor(s) account for the absolute specificity of cognate E2 binding. Truncation of the Uba1 carboxyl-terminal β-grasp domain reduces cognate Ubc2b binding by 31-fold and kcat by 3.5×10(4)-fold, indicating contributions to E2 binding and transition state stabilization. Truncation of the paralogous domain from the Nedd8 activating enzyme has negligible effect on cognate Ubc12 transthiolation but abrogates E2 specificity toward non-cognate carrier proteins. Exchange of the β-grasp domains between ubiquitin and Nedd8 activating enzymes fails to reverse the effect of truncation. Thus, the conserved Helix 1 binding motif and the β-grasp domain direct general E2 binding, whereas the latter additionally serves as a specificity filter to exclude charging of non-cognate E2 paralogs in order to maintain the fidelity of downstream signaling.

  1. Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.

    PubMed

    Laub, M; Jennissen, H P

    1997-06-27

    Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse

  2. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

    PubMed Central

    Gilon, T; Chomsky, O; Kulka, R G

    1998-01-01

    Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

  3. 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

  4. 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.

  5. Ubiquitin-dependent and independent roles of SUMO in proteostasis

    PubMed Central

    Liebelt, Frauke

    2016-01-01

    Cellular proteomes are continuously undergoing alterations as a result of new production of proteins, protein folding, and degradation of proteins. The proper equilibrium of these processes is known as proteostasis, implying that proteomes are in homeostasis. Stress conditions can affect proteostasis due to the accumulation of misfolded proteins as a result of overloading the degradation machinery. Proteostasis is affected in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and multiple polyglutamine disorders including Huntington's disease. Owing to a lack of proteostasis, neuronal cells build up toxic protein aggregates in these diseases. Here, we review the role of the ubiquitin-like posttranslational modification SUMO in proteostasis. SUMO alone contributes to protein homeostasis by influencing protein signaling or solubility. However, the main contribution of SUMO to proteostasis is the ability to cooperate with, complement, and balance the ubiquitin-proteasome system at multiple levels. We discuss the identification of enzymes involved in the interplay between SUMO and ubiquitin, exploring the complexity of this crosstalk which regulates proteostasis. These enzymes include SUMO-targeted ubiquitin ligases and ubiquitin proteases counteracting these ligases. Additionally, we review the role of SUMO in brain-related diseases, where SUMO is primarily investigated because of its role during formation of aggregates, either independently or in cooperation with ubiquitin. Detailed understanding of the role of SUMO in these diseases could lead to novel treatment options. PMID:27335169

  6. Ubiquitination of the common cytokine receptor {gamma}{sub c} and regulation of expression by an ubiquitination/deubiquitination machinery

    SciTech Connect

    Gesbert, Franck; Malarde, Valerie; Dautry-Varsat, Alice . E-mail: adautry@pasteur.fr

    2005-08-26

    The common cytokine receptor {gamma}{sub c} is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of {gamma}{sub c} receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that {gamma}{sub c} is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that {gamma}{sub c} is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces {gamma}{sub c} down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on {gamma}{sub c} expression. We show that an increase in DUB-2 expression correlates with an increased {gamma}{sub c} half-life, resulting in the up-regulation of the receptor. Altogether, we show that {gamma}{sub c} is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2.

  7. E3 ubiquitin ligases and abscisic acid signaling

    PubMed Central

    Liu, Hongxia

    2011-01-01

    The ubiquitin proteasome system is involved in the regulation of nearly every aspect of plant growth and development. Protein ubiquitination involves the covalent attachment of ubiquitin to target proteins through a cascade catalyzed by three enzymes known as E1, E2 and E3. E3s are of particular interest as they confer substrate specificity during ubiquitination through their diverse substrate recognition domains. Recently, a number of E3s have been identified that actively participate in abscisic acid hormone biology, including regulation of biosynthesis, de-repression or activation of abscisic acid response and degradation of signaling components. In this review, we summarize recent exciting studies of the different types of E3s that target specific mediators of abscisic acid signaling or affect the plants response to the hormone. PMID:21364320

  8. Assessing the Influence of Adsorbed-State Conformation on the Bioactivity of Adsorbed Enzyme Layers

    PubMed Central

    Fears, Kenan P.; Latour, Robert A.

    2013-01-01

    Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for the fabrication of stable, reusable substrates with highly specific functionality. The performance of these systems is greatly dependent upon the orientation and conformation of the adsorbed enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces using circular dichroism (CD) spectroscopy and evaluate their bioactivity using colorimetric assays. These combined measurements provide molecular-level insights regarding whether observed changes in adsorbed enzyme bioactivity are due to the adsorbed orientation of an enzyme or adsorption-induced changes in its conformation. Using this approach, we investigated the adsorption behavior of lysozyme (HEWL), xylanase (XYL), and glucose oxidase (GOx) on OH-, CH3-, NH2-, and COOH-terminated alkanethiol self-assembled monolayer (SAM) surfaces. The bioactivities of the small enzymes, HEWL and XYL, had pronounced variations between the different SAM surfaces despite their structural stability, highlighting the role of adsorbed orientation on bioactivity. In contrast, GOx, which is a much larger enzyme, exhibited wide variations in both its structure and bioactivity after adsorption, with adsorption-induced conformational changes actually enhancing its bioactivity. These results provide new insights into protein-surface interactions at the molecular level and demonstrate that adsorption can either promote or inhibit bioactivity depending on how the surface chemistry influences the orientation and conformational state of the enzyme on the surface. PMID:19499935

  9. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

    PubMed

    Beers, E P; Moreno, T N; Callis, J

    1992-08-05

    Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.

  11. Dengue Virus Genome Uncoating Requires Ubiquitination.

    PubMed

    Byk, Laura A; Iglesias, Néstor G; De Maio, Federico A; Gebhard, Leopoldo G; Rossi, Mario; Gamarnik, Andrea V

    2016-06-28

    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. Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade

  12. Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3.

    PubMed

    Alpi, Arno F; Chaugule, Viduth; Walden, Helen

    2016-10-15

    Ubiquitin signalling is a fundamental eukaryotic regulatory system, controlling diverse cellular functions. A cascade of E1, E2, and E3 enzymes is required for assembly of distinct signals, whereas an array of deubiquitinases and ubiquitin-binding modules edit, remove, and translate the signals. In the centre of this cascade sits the E2-conjugating enzyme, relaying activated ubiquitin from the E1 activating enzyme to the substrate, usually via an E3 ubiquitin ligase. Many disease states are associated with dysfunction of ubiquitin signalling, with the E3s being a particular focus. However, recent evidence demonstrates that mutations or impairment of the E2s can lead to severe disease states, including chromosome instability syndromes, cancer predisposition, and immunological disorders. Given their relevance to diseases, E2s may represent an important class of therapeutic targets. In the present study, we review the current understanding of the mechanism of this important family of enzymes, and the role of selected E2s in disease.

  13. Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3

    PubMed Central

    Alpi, Arno F.; Chaugule, Viduth; Walden, Helen

    2016-01-01

    Ubiquitin signalling is a fundamental eukaryotic regulatory system, controlling diverse cellular functions. A cascade of E1, E2, and E3 enzymes is required for assembly of distinct signals, whereas an array of deubiquitinases and ubiquitin-binding modules edit, remove, and translate the signals. In the centre of this cascade sits the E2-conjugating enzyme, relaying activated ubiquitin from the E1 activating enzyme to the substrate, usually via an E3 ubiquitin ligase. Many disease states are associated with dysfunction of ubiquitin signalling, with the E3s being a particular focus. However, recent evidence demonstrates that mutations or impairment of the E2s can lead to severe disease states, including chromosome instability syndromes, cancer predisposition, and immunological disorders. Given their relevance to diseases, E2s may represent an important class of therapeutic targets. In the present study, we review the current understanding of the mechanism of this important family of enzymes, and the role of selected E2s in disease. PMID:27729585

  14. Insights into enzyme catalysis from QM/MM modelling: transition state stabilization in chorismate mutase

    NASA Astrophysics Data System (ADS)

    Ranaghan, Kara E.; Ridder, Lars; Szefczyk, Borys; Sokalski, W. Andrzej; Hermann, Johannes C.; Mulholland, Adrian J.

    Chorismate mutase provides an important test of theories of enzyme catalysis, and of modelling methods. The Claisen rearrangement of chorismate to prephenate in the enzyme has been modelled here by a combined quantum mechanics/molecular mechanics (QM/MM) method. Several pathways have been calculated. The sensitivity of the results to details of model preparation and pathway calculation is tested, and the results are compared in detail to previous similar studies and experiments. The potential energy barrier for the enzyme reaction is estimated at 24.5-31.6 kcal mol-1 (AM1/CHARMM), and 2.7-11.9 kcal mol-1 with corrections (e.g. B3LYP/6-31+G(d)). In agreement with previous studies, the present analysis of the calculated paths provides unequivocal evidence of significant transition state stabilization by the enzyme, indicating that this is central to catalysis by the enzyme. The active site is exquisitely complementary to the transition state, stabilizing it more than the substrate, so reducing the barrier to reaction. A number of similar pathways for reaction exist in the protein, as expected. Small structural differences give rise to differences in energetic contributions. Major electrostatic contributions to transition state stabilization come in all cases from Arg90, Arg7, one or two water molecules, and Glu78 (Glu78 destabilizes the transition state less than the substrate), while Arg63 contributes significantly in one model.

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

    USDA-ARS?s Scientific Manuscript database

    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....

  16. [Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression].

    PubMed

    Yu, Juan; Chen, Wei-lin

    2015-03-01

    By regulating the ubiquitination and deubiquitination of key proteins, ubiquitin-proteasome system mediates a variety of cellular activities. Ubiquitin C-terminal hydrolase (UCH) is a deubiquitinating enzyme which can remove ubiquitin chains at the end of ubiquited proteins. The abnormal expression of UCH has been found in a variety of tumor tissues, indicating that it participates in the process of tumor development. Here we review the characteristics of UCH members and current understanding about the role of UCH in tumor development, and the potential target for cancer treatment.

  17. Cyclophilin A inhibition: targeting transition-state-bound enzyme conformations for structure-based drug design.

    PubMed

    Nagaraju, Mulpuri; McGowan, Lauren C; Hamelberg, Donald

    2013-02-25

    Human Cyclophilin A (CypA) catalyzes cis-trans isomerization of the prolyl peptide ω-bond in proteins and is involved in many subcellular processes. CypA has, therefore, been identified as a potential drug target in many diseases, and the development of potent inhibitors with high selectivity is a key objective. In computer-aided drug design, selectivity is improved by taking into account the inherent flexibility of the receptor. However, the relevant receptor conformations to focus on in order to develop highly selective inhibitors are not always obvious from available X-ray crystal structures or ensemble of conformations generated using molecular dynamics simulations. Here, we show that the conformation of the active site of CypA varies as the substrate configuration changes during catalytic turnover. We have analyzed the principal modes of the active site dynamics of CypA from molecular dynamics simulations to show that similar ensembles of enzyme conformations recognize diverse inhibitors and bind the different configurations of the peptide substrate. Small nonpeptidomimetic inhibitors with varying activity are recognized by enzyme ensembles that are similar to those that tightly bind the transition state and cis configurations of the substrate. Our results suggest that enzyme-substrate ensembles are more relevant in structure-based drug design for CypA than free enzyme. Of the vast conformational space of the free enzyme, the enzyme conformations of the tightly bound enzyme-substrate complexes are the most important for catalysis. Therefore, functionalizing lead compounds to optimize their interactions with the enzyme's conformational ensemble bound to the substrate in the cis or the transition state could lead to more potent inhibitors.

  18. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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, ...

  20. Ubiquitin — EDRN Public Portal

    Cancer.gov

    The ubiquitin protein was named due to its ubiquitous presence in the human body. This small protein, comprised of 76 amino acids, is coded for by a family of genes whose translation products are fusion proteins. The four genes that are known to code for ubiquitin are UBB, UBC, UBA52, and RPS27A. Ubiquitin molecules are often bound to other proteins as a marker for degradation.

  1. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

    PubMed Central

    Niimi, Atsuko; Brown, Stephanie; Sabbioneda, Simone; Kannouche, Patricia L.; Scott, Andrew; Yasui, Akira; Green, Catherine M.; Lehmann, Alan R.

    2008-01-01

    After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA. PMID:18845679

  2. Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?

    PubMed

    Ma, B; Kumar, S; Tsai, C J; Hu, Z; Nussinov, R

    2000-04-21

    Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view

  3. Chemoenzymatic synthesis of bifunctional polyubiquitin substrates for monitoring ubiquitin chain remodeling.

    PubMed

    Trang, Vivian H; Rodgers, Margaret L; Boyle, Kevin J; Hoskins, Aaron A; Strieter, Eric R

    2014-07-21

    Covalent attachment of ubiquitin to target proteins is one of the most pervasive post-translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We have used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.

  4. 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…

  5. 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…

  6. Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

    PubMed

    Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

    2017-06-06

    Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2(VHL) complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  7. Characterization of the Arabidopsis thaliana E3 Ubiquitin-Ligase AtSINAL7 and Identification of the Ubiquitination Sites

    PubMed Central

    Peralta, Diego A.; Araya, Alejandro; Nardi, Cristina F.; Busi, Maria V.; Gomez-Casati, Diego F.

    2013-01-01

    Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV–B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes. PMID:24015288

  8. A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

    PubMed Central

    Yoshida, Yukiko; Saeki, Yasushi; Murakami, Arisa; Kawawaki, Junko; Tsuchiya, Hikaru; Yoshihara, Hidehito; Shindo, Mayumi; Tanaka, Keiji

    2015-01-01

    The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein. PMID:25827227

  9. Systematic exploration of ubiquitin sequence, E1 activation efficiency, and experimental fitness in yeast

    PubMed Central

    Roscoe, Benjamin P.; Bolon, Daniel N. A.

    2014-01-01

    The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) as well as overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by two fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation. PMID:24862281

  10. The ubiquitin-proteasome pathway and synaptic plasticity

    PubMed Central

    Hegde, Ashok N.

    2010-01-01

    Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for degradation by a multisubunit complex called the proteasome. Linkage of ubiquitin to protein substrates is highly specific and occurs through a series of well-orchestrated enzymatic steps. The UPP regulates neurotransmitter receptors, protein kinases, synaptic proteins, transcription factors, and other molecules critical for synaptic plasticity. Accumulating evidence indicates that the operation of the UPP in neurons is not homogeneous and is subject to tightly managed local regulation in different neuronal subcompartments. Investigations on both invertebrate and vertebrate model systems have revealed local roles for enzymes that attach ubiquitin to substrate proteins, as well as for enzymes that remove ubiquitin from substrates. The proteasome also has been shown to possess disparate functions in different parts of the neuron. Here I give a broad overview of the role of the UPP in synaptic plasticity and highlight the local roles and regulation of the proteolytic pathway in neurons. PMID:20566674

  11. 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.

  12. Production of Aspergillus niger pectolytic enzymes by solid state bioprocessing of apple pomace.

    PubMed

    Berovic, M; Ostroversnik, H

    1997-02-28

    The aim of this work was to develop a low cost process for apple pomace utilisation. Accordingly this production of pectynolitic enzymes based on solid state bioprocessing of this actual waste, was developed. Production of pectolytic enzymes of Aspergillus niger, pectinesterase and polygalacturonase as well as the activity of pectolytic enzymatic complex by solid state bioprocessing were studied. The results of preliminary substrate optimization, on open trays in laboratory scale experiments, were transferred to 15 1 horizontal solid state stirred tank reactor (HSS STR). In situ sterilization of solid substrate with periodical mixing was used. Secondary raw material, apple pomace the waste from food and agriculture industry combined with soya flour, wheat bran and simple mineral salts was utilised. Various substrate moistures were studied. Process parameters such as inoculation, influence of mixing, aeration, temperature and moisture content on pectolytic enzymes production were studied. Maximal amounts of 15 g kg-1 of solid medium of polygalacturonase, 200 mg kg-1 pectinesterase at activity up to 900 AJDA U ml-1 of enzyme mixture was obtained on average.

  13. Ubiquitin makes its mark on immune regulation

    PubMed Central

    Malynn, Barbara A.; Ma, Averil

    2011-01-01

    Ubiquitination, the covalent attachment of ubiquitin molecules to proteins, is emerging as a widely utilized mechanism for rapidly regulating cell signaling. Recent studies indicate that ubiquitination plays potent roles in regulating a variety of signals in both innate and adaptive immune cells. Here, we will review recent studies of ubiquitin ligases, ubiquitin chain linkages and ubiquitin binding proteins that highlight the diversity and specificity of ubiquitin dependent functions in immune cells. We will also review studies that shed light on how ubiquitination signals are integrated in cell-type specific fashion to regulate the immune system in vivo. PMID:21168777

  14. Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase superfamily.

    PubMed

    Hou, Guanhua; Cui, Qiang

    2013-07-17

    The first step for the hydrolysis of a phosphate monoester (pNPP(2-)) 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 bimetallic 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.

  15. Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools

    PubMed Central

    Chang, Chia-En A.; Huang, Yu-Ming M.; Mueller, Leonard J.; You, Wanli

    2016-01-01

    This review discusses the use of molecular modeling tools, together with existing experimental findings, to provide a complete atomic-level description of enzyme dynamics and function. We focus on functionally relevant conformational dynamics of enzymes and the protonation states of substrates. The conformational fluctuations of enzymes usually play a crucial role in substrate recognition and catalysis. Protein dynamics can be altered by a tiny change in a molecular system such as different protonation states of various intermediates or by a significant perturbation such as a ligand association. Here we review recent advances in applying atomistic molecular dynamics (MD) simulations to investigate allosteric and network regulation of tryptophan synthase (TRPS) and protonation states of its intermediates and catalysis. In addition, we review studies using quantum mechanics/molecular mechanics (QM/MM) methods to investigate the protonation states of catalytic residues of β-Ketoacyl ACP synthase I (KasA). We also discuss modeling of large-scale protein motions for HIV-1 protease with coarse-grained Brownian dynamics (BD) simulations. PMID:27885336

  16. 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.

  17. Cystein-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

    PubMed

    Schwartzkopff, Benjamin; Platta, Harald W; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-05-14

    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 to 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 to 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 deubiquitination 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.

  18. 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

  19. 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.

  20. Incorporating distant sequence features and radial basis function networks to identify ubiquitin conjugation sites.

    PubMed

    Lee, Tzong-Yi; Chen, Shu-An; Hung, Hsin-Yi; Ou, Yu-Yen

    2011-03-09

    Ubiquitin (Ub) is a small protein that consists of 76 amino acids about 8.5 kDa. In ubiquitin conjugation, the ubiquitin is majorly conjugated on the lysine residue of protein by Ub-ligating (E3) enzymes. Three major enzymes participate in ubiquitin conjugation. They are E1, E2 and E3 which are responsible for activating, conjugating and ligating ubiquitin, respectively. Ubiquitin conjugation in eukaryotes is an important mechanism of the proteasome-mediated degradation of a protein and regulating the activity of transcription factors. Motivated by the importance of ubiquitin conjugation in biological processes, this investigation develops a method, UbSite, which uses utilizes an efficient radial basis function (RBF) network to identify protein ubiquitin conjugation (ubiquitylation) sites. This work not only investigates the amino acid composition but also the structural characteristics, physicochemical properties, and evolutionary information of amino acids around ubiquitylation (Ub) sites. With reference to the pathway of ubiquitin conjugation, the substrate sites for E3 recognition, which are distant from ubiquitylation sites, are investigated. The measurement of F-score in a large window size (-20∼+20) revealed a statistically significant amino acid composition and position-specific scoring matrix (evolutionary information), which are mainly located distant from Ub sites. The distant information can be used effectively to differentiate Ub sites from non-Ub sites. As determined by five-fold cross-validation, the model that was trained using the combination of amino acid composition and evolutionary information performs best in identifying ubiquitin conjugation sites. The prediction sensitivity, specificity, and accuracy are 65.5%, 74.8%, and 74.5%, respectively. Although the amino acid sequences around the ubiquitin conjugation sites do not contain conserved motifs, the cross-validation result indicates that the integration of distant sequence features of Ub

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

    PubMed

    Karaczyn, Aldona A; Golebiowski, Filip; Kasprzak, Kazimierz S

    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.

  2. 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.

  3. Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

    PubMed

    Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L

    2010-04-09

    DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

  4. 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

  5. Regulation of steady-state beta-amyloid levels in the brain by neprilysin and endothelin-converting enzyme but not angiotensin-converting enzyme.

    PubMed

    Eckman, Elizabeth A; Adams, Stephanie K; Troendle, Frederick J; Stodola, Becky A; Kahn, Murad A; Fauq, Abdul H; Xiao, Hong D; Bernstein, Kenneth E; Eckman, Christopher B

    2006-10-13

    The deposition of beta-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of beta-amyloid is prevented in part by the activities of several degradative enzymes, including the endothelin-converting enzymes, neprilysin, insulin-degrading enzyme, and plasmin. Recent reports indicate that another metalloprotease, angiotensin-converting enzyme (ACE), can degrade beta-amyloid in vitro and in cellular overexpression experiments. In addition, ACE gene variants are linked to AD risk in several populations. Angiotensin-converting enzyme, neprilysin and endothelin-converting enzyme function as vasopeptidases and are the targets of drugs designed to treat cardiovascular disorders, and ACE inhibitors are commonly prescribed. We investigated the potential physiological role of ACE in regulating endogenous brain beta-amyloid levels for two reasons: first, to determine whether beta-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block beta-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed beta-amyloid accumulation in brains from ACE-deficient mice and in mice treated with ACE inhibitors and found that ACE deficiency did not alter steady-state beta-amyloid concentration. In contrast, beta-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in beta-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of beta-amyloid in the brain but confirm roles for endothelin-converting enzyme and neprilysin and indicate that reductions in these enzymes result in additive increases in brain amyloid beta-peptide levels.

  6. 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

  7. Role of Ubiquitin Carboxy Terminal Hydrolase-L1 in Neural Cell Apoptosis Induced by Ischemic Retinal Injury in Vivo

    PubMed Central

    Harada, Takayuki; Harada, Chikako; Wang, Yu-Lai; Osaka, Hitoshi; Amanai, Kazuhito; Tanaka, Kohichi; Takizawa, Shuichi; Setsuie, Rieko; Sakurai, Mikako; Sato, Yae; Noda, Mami; Wada, Keiji

    2004-01-01

    Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed ∼70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo. PMID:14695319

  8. Control of cullin-ring ubiquitin ligase activity by nedd8.

    PubMed

    Deshaies, Raymond J; Emberley, Ethan D; Saha, Anjanabha

    2010-01-01

    The Cullin-RING ubiquitin ligase (CRL) family, which may number as many as 350 different enzymes, has an enormous impact on cellular regulation. CRL enzymes regulate cell biology by conjugating ubiquitin onto target proteins that are involved in a multitude of processes. In most cases this leads to degradation of the target, but in some cases CRL-dependent ubiquitination acts as a switch to activate or repress target function. The ubiquitin ligase activity of CRLs is controlled by cycles of attachment and removal of the ubiquitin-like protein Nedd8. Conjugation of Nedd8 onto the cullin subunit of CRLs promotes assembly of an intact CRL complex and switches on ubiquitin ligase activity. Conversely, removal of Nedd8 switches off ubiquitin ligase activity and initiates CRL disassembly. Continuous maintenance of CRL function in vivo requires the activities of both the Nedd8-conjugating and deconjugating enzymes, pointing to a critical role of complex dynamics in CRL function. Here, we review how the Nedd8 cycle controls CRL activity and how perturbations of this cycle can lead to disease.

  9. RNF111-dependent neddylation activates DNA damage-induced ubiquitination

    PubMed Central

    Ma, Teng; Chen, Yibin; Zhang, Feng; Yang, Chao-Yie; Wang, Shaomeng; Yu, Xiaochun

    2013-01-01

    Summary Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites, and this accumulation was dependent on an E2 enzyme UBE2M and an E3 ubiquitin ligase RNF111. We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (Motif Interacting with Ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process. PMID:23394999

  10. 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.

  11. 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.

  12. 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

  13. Functional annotation of deubiquitinating enzymes using RNA interference.

    PubMed

    Dirac, Annette M G; Nijman, Sebastian M B; Brummelkamp, Thijn R; Bernards, René

    2005-01-01

    Protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of ubiquitin ligases and their antagonists, the ubiquitin-specific proteases or deubiquitinating enzymes. The family of ubiquitin ligases has been studied intensively and it is well established that their deregulation contributes to diverse disease processes, including cancer. Much less is known about the function and regulation of the large group of deubiquitinating enzymes. This chapter describes how RNA interference against deubiquitinating enzymes can be used to elucidate their function. The application of this technology will greatly improve the functional annotation of this family of proteases.

  14. [New relations for steady-state enzyme kinetics and their application to rapid equilibrium assumption].

    PubMed

    Vrzheshch, P V

    2013-01-01

    With the use of a graph theory new relations for steady-state enzyme kinetics are derived and strictly proved for the arbitrary mechanism of an enzyme-catalysed reaction containing a reversible segment. Using these relations, a general principle for rapid equilibrium assumption is formulated and proved: the reversible bound segment can be considered as an equilibrium segment only when the values of the base trees that are not proper to this segment can be neglected (within a prescribed accuracy) in relation to the values of the base trees that belong to this segment. In contrast with the foreign base trees the base trees that are proper to the segment have the following properties: the tree that is directed to the base within this segment does not contain the edges leaving this segment; and the tree that is directed to the base outside the segment contains only one edge leaving this segment. Equilibrium variations are assessed for steady-state intermediates concentrations of the equilibrium segment, numerical expressions are obtained for the accuracy of determination of the intermediates concentrations as well as for the accuracy of determination of the rate of enzyme-catalysed reaction in case of using rapid equilibrium assumption.

  15. Isoform-Specific SCFFbw7 Ubiquitination Mediates Differential Regulation of PGC-1α

    PubMed Central

    Trausch-Azar, Julie S.; Abed, Mona; Orian, Amir; Schwartz, Alan L.

    2015-01-01

    The E3 ubiquitin ligase and tumor suppressor SCFFbw7 exists as three isoforms that govern the degradation of a host of critical cell regulators, including c-Myc, cyclin E, and PGC-1α. Peroxisome proliferator activated receptor-gamma coactivator 1α (PGC-1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC-1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin-proteasome system. Yet, isoform-specific functions of SCFFbw7 are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCFFbw7, regulates cellular PGC-1α levels via two independent, isoform specific, mechanisms. The cytoplasmic isoform (SCFFbw7β) reduces cellular PGC-1α levels via accelerated ubiquitin-proteasome degradation. In contrast, the nuclear isoform (SCFFbw7α) increases cellular PGC-1α levels and protein stability via inhibition of ubiquitin-proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC-1α protein levels are reduced through accelerated ubiquitin-proteasomal degradation. We find that SCFFbw7β catalyzes high molecular weight PGC-1α-ubiquitin conjugation, whereas SCFFbw7α produces low molecular weight PGC-1α-ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC-1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC-1α levels via selective isoform-specific ubiquitination. Fbw7β reduces cellular PGC-1α via ubiquitin-mediated degradation, whereas Fbw7α increases cellular PGC-1α via ubiquitin-mediated stabilization. PMID:25204433

  16. 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.

  17. 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.

  18. Binding of Ubiquitin Conjugates to Proteasomes as Visualized with Native Gels

    PubMed Central

    Elsasser, Suzanne; Shi, Yuan; Finley, Daniel

    2013-01-01

    Summary The proteasome is an ATP-dependent molecular machine that degrades proteins through the concerted activity of dozens of subunits. It is the yin to the ribosome’s yang, and together these entities mold the protein landscape of the cell. Native gels are generally superior to conventional and affinity purifications for the analytical resolution proteasomal variants, and have thus become a staple of proteasome work. Here we describe the technique of using native gels to observe proteasomes in complex with ubiquitin conjugates. We discuss the consequences of ubiquitin conjugate length and concentration on the migration of these complexes, the use of this mobility shift to evaluate the relative affinity of mutant proteasomes for ubiquitin conjugates, and the effects of deubiquitinating enzymes and competing ubiquitin binding proteins on the interactions of ubiquitin conjugates with the proteasome. PMID:22350901

  19. Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin.

    PubMed

    Haller, Jens D; Schanda, Paul

    2013-11-01

    Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps-μs motions in proteins by solid-state NMR.

  20. 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

  1. Cell cycle regulatory E3 ubiquitin ligases as anticancer targets.

    PubMed

    Pray, Todd R; Parlati, Francesco; Huang, Jianing; Wong, Brian R; Payan, Donald G; Bennett, Mark K; Issakani, Sarkiz Daniel; Molineaux, Susan; Demo, Susan D

    2002-12-01

    Disregulation of the cell cycle and proliferation play key roles in cellular transformation and tumorigenesis. Such processes are intimately tied to the concentration, localization and activity of enzymes, adapters, receptors, and structural proteins in cells. Ubiquitination of these cellular regulatory proteins, governed by specific enzymes in the ubiquitin (Ub) conjugation cascade, has profound effects on their various functions, most commonly through proteasome targeting and degradation. This review will focus on a variety of E3 Ub ligases as potential oncology drug targets, with particular emphasis on the role of these molecules in the regulation of stability, localization, and activity of key proteins such as tumor suppressors and oncoproteins. E3 ubiquitin ligases that have established roles in cell cycle and apoptosis, such as the anaphase-promoting complex (APC), the Skp-1-Cul1-F-box class, and the murine double minute 2 (MDM2) protein, in addition to more recently discovered E3 ubiquitin ligases which may be similarly important in tumorigenesis, (e.g. Smurf family, CHFR, and Efp), will be discussed. We will present evidence to support E3 ligases as good biological targets in the development of anticancer therapeutics and address challenges in drug discovery for these targets.

  2. Suramin inhibits cullin-RING E3 ubiquitin ligases

    PubMed Central

    Wu, Kenneth; Chong, Robert A.; Yu, Qing; Bai, Jin; Spratt, Donald E.; Ching, Kevin; Lee, Chan; Miao, Haibin; Tappin, Inger; Hurwitz, Jerard; Zheng, Ning; Shaw, Gary S.; Sun, Yi; Felsenfeld, Dan P.; Sanchez, Roberto; Zheng, Jun-nian; Pan, Zhen-Qiang

    2016-01-01

    Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1’s conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2–E3 interface through small-molecule modulators. PMID:27001857

  3. Suramin inhibits cullin-RING E3 ubiquitin ligases.

    PubMed

    Wu, Kenneth; Chong, Robert A; Yu, Qing; Bai, Jin; Spratt, Donald E; Ching, Kevin; Lee, Chan; Miao, Haibin; Tappin, Inger; Hurwitz, Jerard; Zheng, Ning; Shaw, Gary S; Sun, Yi; Felsenfeld, Dan P; Sanchez, Roberto; Zheng, Jun-Nian; Pan, Zhen-Qiang

    2016-04-05

    Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.

  4. 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.

  5. Transition state determination of enzyme reaction on free energy surface: Application to chorismate mutase

    NASA Astrophysics Data System (ADS)

    Higashi, Masahiro; Hayashi, Shigehiko; Kato, Shigeki

    2007-04-01

    The transition state on the free energy surface for Claisen rearrangement of chorismate in Bacillus subtilis chorismate mutase is calculated with a method based on a linear response theory. The calculated activation free energy is 16.9 kcal/mol, which is in good agreement with the experimental one. The catalytic ability of the enzyme is examined by comparing the activation barrier with that in aqueous solution and found to be mainly attributed to the conformational restriction of the substrate. We also calculate the kinetic isotope effects, which are in accord with the experimental estimates.

  6. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation

    PubMed Central

    Petrovska, Ivana; Nüske, Elisabeth; Munder, Matthias C; Kulasegaran, Gayathrie; Malinovska, Liliana; Kroschwald, Sonja; Richter, Doris; Fahmy, Karim; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2014-01-01

    One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell. DOI: http://dx.doi.org/10.7554/eLife.02409.001 PMID:24771766

  7. Production of ligninolytic enzymes by solid-state fermentation using Pleurotus eryngii.

    PubMed

    Akpinar, Merve; Urek, Raziye Ozturk

    2012-01-01

    Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ability to produce various ligninolytic enzymes such as laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) by solid-state fermentation (SSF), which was carried out using a support substrate from the fruit juice industry. The chemical content of grape waste from this industry was studied. Also, the production patterns of these extracellular enzymes were researched during the growth of the organism for a period of 20 days and the protein, reducing sugar, and nitrogen levels were monitored during the stationary cultivation. The highest Lac activity was obtained as 2247.62 ± 75 U/L on day 10 in the presence of 750 µM Mn²⁺, while the highest MnP activity was attained as 2198.44 ± 65 U/L on day 15 in the presence of 500 µM Mn²⁺. Decolorization of methyl orange and reactive red 2 azo dyes was also achieved with ligninolytic enzymes, produced in SSF of P. eryngii.

  8. 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.

  9. 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.

  10. Orthopoxviruses Require a Functional Ubiquitin-Proteasome System for Productive Replication▿

    PubMed Central

    Teale, Alastair; Campbell, Stephanie; Van Buuren, Nick; Magee, Wendy C.; Watmough, Kelly; Couturier, Brianne; Shipclark, Robyn; Barry, Michele

    2009-01-01

    Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection. PMID:19109393

  11. The ubiquitin-proteasome pathway an emerging anticancer strategy for therapeutics: a patent analysis.

    PubMed

    Jain, Chakresh K; Arora, Shivam; Khanna, Aparna; Gupta, Money; Wadhwa, Gulshan; Sharma, Sanjeev K

    2015-01-01

    The degradation of intracellular proteins is targeted by ubiquitin via non-lysosomal proteolytic pathway in the cell system. These ubiquitin molecules have been found to be conserved from yeast to humans. Ubiquitin proteasome machinery utilises ATP and other mechanisms for degrading proteins of cytosol as well as nucleus. This process of ubiquitination is regulated by activating the E3 enzyme ligase, involved in phosphorylation. In humans, proteins which regulate the cell cycle are controlled by ubiquitin; therefore the ubiquitin-proteasome pathway can be targeted for novel anti-cancer strategies. Dysregulation of the components of the ubiquitin system has been linked to many diseases like cancer and inflammation. The primary triggering mechanism (apoptosis) of these diseases can also be induced when TNF-related apoptosis-inducing ligand (TRAIL) binds to its specific receptor DR4 and DR5. In this review, the emerging prospects and importance of ubiquitin proteasome pathway as an evolving anticancer strategy have been discussed. Current challenges in the field of drug discovery have also been discussed on the basis of recent patents on cancer diagnosis and therapeutics.

  12. 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.

  13. RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

    PubMed

    Paul, Atanu; Wang, Bin

    2017-05-18

    Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6.

    PubMed

    Perrody, Elsa; Abrami, Laurence; Feldman, Michal; Kunz, Beatrice; Urbé, Sylvie; van der Goot, F Gisou

    2016-10-18

    Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle. To avoid infinite futile cycles, folded LRP6 molecules undergo palmitoylation and ER export, while unsuccessfully folded proteins are, with time, polyubiquitinated on other lysines and targeted to degradation. This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2, two poor folders involved in severe human diseases.

  15. An integrated bioinformatics platform for investigating the human E3 ubiquitin ligase-substrate interaction network.

    PubMed

    Li, Yang; Xie, Ping; Lu, Liang; Wang, Jian; Diao, Lihong; Liu, Zhongyang; Guo, Feifei; He, Yangzhige; Liu, Yuan; Huang, Qin; Liang, Han; Li, Dong; He, Fuchu

    2017-08-24

    The ubiquitination mediated by ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) cascade is crucial to protein degradation, transcription regulation, and cell signaling in eukaryotic cells. The high specificity of ubiquitination is regulated by the interaction between E3 ubiquitin ligases and their target substrates. Unfortunately, the landscape of human E3-substrate network has not been systematically uncovered. Therefore, there is an urgent need to develop a high-throughput and efficient strategy to identify the E3-substrate interaction. To address this challenge, we develop a computational model based on multiple types of heterogeneous biological evidence to investigate the human E3-substrate interactions. Furthermore, we provide UbiBrowser as an integrated bioinformatics platform to predict and present the proteome-wide human E3-substrate interaction network ( http://ubibrowser.ncpsb.org ).Protein stability modulation by E3 ubiquitin ligases is an important layer of functional regulation, but screening for E3 ligase-substrate interactions is time-consuming and costly. Here, the authors take an in silico naïve Bayesian classifier approach to integrate multiple lines of evidence for E3-substrate prediction, enabling prediction of the proteome-wide human E3 ligase interaction network.

  16. Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import.

    PubMed

    Kiel, Jan A K W; Emmrich, Kerstin; Meyer, Helmut E; Kunau, Wolf-H

    2005-01-21

    PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.

  17. Comparative Proteomic Analysis of Wild-Type and SAP Domain Mutant Foot-and-Mouth Disease Virus-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication.

    PubMed

    Zhu, Zixiang; Yang, Fan; Zhang, Keshan; Cao, Weijun; Jin, Ye; Wang, Guoqing; Mao, Ruoqing; Li, Dan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

    2015-10-02

    Leader protein (L(pro)) of foot-and-mouth disease virus (FMDV) manipulates the activities of several host proteins to promote viral replication and pathogenicity. L(pro) has a conserved protein domain SAP that is suggested to subvert interferon (IFN) production to block antiviral responses. However, apart from blocking IFN production, the roles of the SAP domain during FMDV infection in host cells remain unknown. Therefore, we identified host proteins associated with the SAP domain of L(pro) by a high-throughput quantitative proteomic approach [isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with liquid chromatography/electrospray ionization tandem mass spectrometry]. Comparison of the differentially regulated proteins in rA/FMDVΔmSAP- versus rA/FMDV-infected SK6 cells revealed 45 down-regulated and 32 up-regulated proteins that were mostly associated with metabolic, ribosome, spliceosome, and ubiquitin-proteasome pathways. The results also imply that the SAP domain has a function similar to SAF-A/B besides its potential protein inhibitor of activated signal transducer and activator of transcription (PIAS) function. One of the identified proteins UBE1 was further analyzed and displayed a novel role for the SAP domain of L(pro). Overexpression of UBE1 enhanced the replication of FMDV, and knockdown of UBE1 decreased FMDV replication. This shows that FMDV manipulates UBE1 for increased viral replication, and the SAP domain was involved in this process.

  18. 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

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

    PubMed

    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

    2014-05-15

    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.

  20. 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

  1. Dynamic ubiquitin signaling in cell cycle regulation.

    PubMed

    Gilberto, Samuel; Peter, Matthias

    2017-08-07

    The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. © 2017 Gilberto and Peter.

  2. 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

  3. A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.

    PubMed

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

    2011-05-01

    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.

  4. Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica.

    PubMed

    Haas, A L; Katzung, D J; Reback, P M; Guarino, L A

    1996-04-30

    The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.

  5. The role of deubiquitinating enzymes in spermatogenesis.

    PubMed

    Suresh, Bharathi; Lee, Junwon; Hong, Seok-Ho; Kim, Kye-Seong; Ramakrishna, Suresh

    2015-12-01

    Spermatogenesis is a complex process through which spermatogonial stem cells undergo mitosis, meiosis, and cell differentiation to generate mature spermatozoa. During this process, male germ cells experience several translational modifications. One of the major post-translational modifications in eukaryotes is the ubiquitination of proteins, which targets proteins for degradation; this enables control of the expression of enzymes and structural proteins during spermatogenesis. It has become apparent that ubiquitination plays a key role in regulating every stage of spermatogenesis starting from gonocytes to differentiated spermatids. It is understood that, where there is ubiquitination, deubiquitination by deubiquitinating enzymes (DUBs) also exists to counterbalance the ubiquitination process in a reversible manner. Normal spermatogenesis is dependent on the balanced actions of ubiquitination and deubiquitination. This review highlights the current knowledge of the role of DUBs and their essential regulatory contribution to spermatogenesis, especially during progression into meiotic phase, acrosome biogenesis, quality sperm production, and apoptosis of germ cells.

  6. Genetically Directed Production of Recombinant, Isosteric and Nonhydrolysable Ubiquitin Conjugates

    PubMed Central

    Stanley, Mathew

    2016-01-01

    Abstract We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages. PMID:27197715

  7. The new function of two ubiquitin C-terminal hydrolase isozymes as reciprocal modulators of germ cell apoptosis.

    PubMed

    Kwon, Jungkee

    2007-04-01

    Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. The two ubiquitin C-terminal hydrolase (UCH) enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. These two UCH isozymes have 52% amino acid identity and share significant structural similarity. A new function of these two closely related UCH enzymes during spermatogenesis which is associated with germ cell apoptosis has been analyzed. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. During spermatogenesis, apoptosis controls germ cell numbers and eliminates defective germ cells to facilitate testicular homeostasis. In this paper, I review the distinct function of the two UCH isozymes in the testis of gad and Uchl3 knockout mice, which are strongly but reciprocally expressed during spermatogenesis. In addition, the importance of UCHL1-dependent apoptosis for normal spermatogenesis and sperm quality control is discussed.

  8. 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

  9. 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.

  10. Characterization and structural studies of the Plasmodium falciparum ubiquitin and Nedd8 hydrolase UCHL3.

    PubMed

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

    2010-02-26

    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.

  11. Ubiquitin and Proteasomes in Transcription

    PubMed Central

    Geng, Fuqiang; Wenzel, Sabine; Tansey, William P.

    2013-01-01

    Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription—from transcription initiation through to export of mRNA from the nucleus—is influenced by the UPS and that all major arms of the system—from the first step in ubiquitin (Ub) conjugation through to the proteasome—are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power. PMID:22404630

  12. 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

  13. RNA metabolism and ubiquitin/ubiquitin-like modifications collide.

    PubMed

    Pelisch, Federico; Risso, Guillermo; Srebrow, Anabella

    2013-01-01

    Alternative splicing and post-translational modifications are key events for the generation of proteome diversity in eukaryotes. The study of the molecular mechanisms governing these processes, and every other step of gene expression, has underscored the existing interconnectedness among them. Therefore, molecules that could concertedly regulate different stages from transcription to pre-mRNA processing, translation and even protein activity have called our attention. Serine/arginine-rich proteins, initially identified as splicing regulators, are involved in diverse aspects of gene expression. Although most of the roles exerted by members of this family are related to mRNA biogenesis and metabolism, few recently uncovered ones link these proteins to other regulatory steps along gene expression, particularly the regulation of post-translational modification by conjugation of the small ubiquitin-related modifier. This along with the established link between ubiquitin, transcription and pre-mRNA processing points to a general mechanism of interaction between different cellular machineries, such as ubiquitin/ubiquitin-like conjugation pathways, transcription apparatus and the spliceosome.

  14. Linear ubiquitination signals in adaptive immune responses.

    PubMed

    Ikeda, Fumiyo

    2015-07-01

    Ubiquitin can form eight different linkage types of chains using the intrinsic Met 1 residue or one of the seven intrinsic Lys residues. Each linkage type of ubiquitin chain has a distinct three-dimensional topology, functioning as a tag to attract specific signaling molecules, which are so-called ubiquitin readers, and regulates various biological functions. Ubiquitin chains linked via Met 1 in a head-to-tail manner are called linear ubiquitin chains. Linear ubiquitination plays an important role in the regulation of cellular signaling, including the best-characterized tumor necrosis factor (TNF)-induced canonical nuclear factor-κB (NF-κB) pathway. Linear ubiquitin chains are specifically generated by an E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) and hydrolyzed by a deubiquitinase (DUB) called ovarian tumor (OTU) DUB with linear linkage specificity (OTULIN). LUBAC linearly ubiquitinates critical molecules in the TNF pathway, such as NEMO and RIPK1. The linear ubiquitin chains are then recognized by the ubiquitin readers, including NEMO, which control the TNF pathway. Accumulating evidence indicates an importance of the LUBAC complex in the regulation of apoptosis, development, and inflammation in mice. In this article, I focus on the role of linear ubiquitin chains in adaptive immune responses with an emphasis on the TNF-induced signaling pathways.

  15. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed Central

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-01-01

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis. PMID:10582239

  16. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-09-29

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

  17. Autoregulation of Parkin activity through its ubiquitin-like domain

    PubMed Central

    Chaugule, Viduth K; Burchell, Lynn; Barber, Kathryn R; Sidhu, Ateesh; Leslie, Simon J; Shaw, Gary S; Walden, Helen

    2011-01-01

    Parkin is an E3-ubiquitin ligase belonging to the RBR (RING–InBetweenRING–RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity. PMID:21694720

  18. 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

  19. 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.

  20. 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.

  1. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

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

    2015-01-01

    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 provide 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 did random non-cancer 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

  2. 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

  3. The role of conformation on electron capture dissociation of ubiquitin.

    PubMed

    Robinson, Errol W; Leib, Ryan D; Williams, Evan R

    2006-10-01

    Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 +/- 7% for ions formed from an acidified water/methanol solution to 88 +/- 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.

  4. 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.

  5. Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

    PubMed

    Barna, T M; Khan, H; Bruce, N C; Barsukov, I; Scrutton, N S; Moody, P C

    2001-07-06

    Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme. Copyright 2001 Academic Press.

  6. An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn

    PubMed Central

    Rao, Navin; Ghosh, Amiya K.; Douillard, Patrice; Andoniou, Christopher E.; Zhou, Pengcheng; Band, Hamid

    2009-01-01

    SUMMARY The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl+/+ and Cbl−/− mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases. PMID:19966925

  7. Dynamic survey of mitochondria by ubiquitin.

    PubMed

    Escobar-Henriques, Mafalda; Langer, Thomas

    2014-03-01

    Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria.

  8. Trypanosoma cruzi ubiquitin as an antigen in the differential diagnosis of Chagas disease and leishmaniasis.

    PubMed

    Telles, Senobia; Abate, Teresa; Slezynger, Thelma; Henriquez, Diana A

    2003-06-10

    In the present report we describe Trypanosoma cruzi ubiquitin as an antigen to be utilized in the differential diagnosis of Chagas disease and leishmaniasis. Initially, recombinant T. cruzi ubiquitin was evaluated against a panel of sera by phage dot immunoassay, showing a good performance against chagasic sera. However, the presence of a carboxy-terminal tail region encoding a ribosomal protein homologous to a related protein present in the genome of Leishmania sp. gave significant cross-reactivity with leishmanial sera. Therefore, ubiquitin was purified by a simple biochemical protocol and its immunoreactivity was studied by enzyme-linked immunosorbent assay. Analysis of 104 sera indicates that the response to ubiquitin is very sensitive towards chronic chagasic sera (98%) and, more important, highly species-specific, presenting better performance compared to the use of the recombinant protein or the total epimastigote extracts when tested against a panel of leishmanial sera, where out of a total of 70 sera tested, only five sera from the mucocutaneous form of the disease reacted with T. cruzi ubiquitin. On the other hand, Leishmania ubiquitin was not recognized by chagasic sera, but was recognized by sera from different forms of leishmaniasis. These results make ubiquitin an excellent candidate to be used in the differential diagnosis of these two parasitic diseases. The molecular basis for this highly species-specific response is discussed.

  9. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    PubMed

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  10. Mechanism of Polyubiquitination by Human Anaphase-Promoting Complex: RING Repurposing for Ubiquitin Chain Assembly

    DOE PAGES

    Brown, Nicholas G.; Watson, Edmond R.; Weissmann, Florian; ...

    2014-10-09

    Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here in this paper we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms.more » During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.« less

  11. Mechanism of polyubiquitination by human Anaphase Promoting Complex: RING repurposing for ubiquitin chain assembly

    PubMed Central

    Brown, Nicholas G.; Watson, Edmond R.; Weissmann, Florian; Jarvis, Marc A.; VanderLinden, Ryan; Grace, Christy R. R.; Frye, Jeremiah J.; Qiao, Renping; Dube, Prakash; Petzold, Georg; Cho, Shein Ei; Alsharif, Omar; Bao, Ju; Davidson, Iain F.; Zheng, Jie; Nourse, Amanda; Kurinov, Igor; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A.

    2014-01-01

    Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including Anaphase Promoting Complex/Cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2~ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain elongating E2 UBE2S in ways that differ completely from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal unexpectedly diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation. PMID:25306923

  12. 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

  13. Identification of conjugation specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2.

    PubMed

    Huang, Danny T; Zhuang, Min; Ayrault, Olivier; Schulman, Brenda A

    2008-03-01

    Ubiquitin-like proteins (UBLs) modify targets via related E1-E2-E3 cascades. How is UBL conjugation fidelity established? Here we report the basis for UBL selection by UBL conjugating enzyme 12 (Ubc12), which is specific for the neural precursor cell expressed, developmentally down-regulated protein 8 (NEDD8), and does not form a thioester-linked conjugate with ubiquitin. We systematically identified Ubc12 surfaces impeding Ubc12 approximately ubiquitin conjugate formation and found that several structurally dispersed E1 binding elements, rather than UBL-interacting surfaces, determine E2 approximately UBL specificity. In addition to roles for conserved E1 and E2 domains, unique structures contribute UBL specificity to the NEDD8 and ubiquitin pathways. By removing surface elements, without substituting corresponding sequences from ubiquitin E2s, we unmasked Ubc12's vestigial preference for ubiquitin over NEDD8 by approximately 10(10)-fold. This has implications for the evolution of specific functions among ubiquitin E2s. We also find that Ubc12 sequences dictating UBL selection map to the E3 binding site, thus providing a molecular mechanism preventing inappropriate modification of targets.

  14. 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.

  15. Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

    PubMed Central

    Hao, Yi-Heng; Doyle, Jennifer M.; Ramanathan, Saumya; Gomez, Timothy S.; Jia, Da; Xu, Ming; Chen, Zhijian J.; Billadeau, Daniel D.; Rosen, Michael K.; Potts, Patrick Ryan

    2013-01-01

    SUMMARY Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex. PMID:23452853

  16. Muscle wasting in a rat model of long-lasting sepsis results from the activation of lysosomal, Ca2+ -activated, and ubiquitin-proteasome proteolytic pathways.

    PubMed Central

    Voisin, L; Breuillé, D; Combaret, L; Pouyet, C; Taillandier, D; Aurousseau, E; Obled, C; Attaix, D

    1996-01-01

    We studied the alterations in skeletal muscle protein breakdown in long lasting sepsis using a rat model that reproduces a sustained and reversible catabolic state, as observed in humans. Rats were injected intravenously with live Escherichia coli; control rats were pair-fed to the intake of infected rats. Rats were studied in an acute septic phase (day 2 postinfection), in a chronic septic phase (day 6), and in a late septic phase (day 10). The importance of the lysosomal, Ca2+ -dependent, and ubiquitin-proteasome proteolytic processes was investigated using proteolytic inhibitors in incubated epitrochlearis muscles and by measuring mRNA levels for critical components of these pathways. Protein breakdown was elevated during the acute and chronic septic phases (when significant muscle wasting occurred) and returned to control values in the late septic phase (when wasting was stopped). A nonlysosomal and Ca2+ -independent process accounted for the enhanced proteolysis, and only mRNA levels for ubiquitin and subunits of the 20 S proteasome, the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates, paralleled the increased and decreased rates of proteolysis throughout. However, increased mRNA levels for the 14-kD ubiquitin conjugating enzyme E2, involved in substrate ubiquitylation, and for cathepsin B and m-calpain were observed in chronic sepsis. These data clearly support a major role for the ubiquitin-proteasome dependent proteolytic process during sepsis but also suggest that the activation of lysosomal and Ca2+ -dependent proteolysis may be important in the chronic phase. PMID:8601625

  17. Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7.

    PubMed

    Zhang, Yingnan; Zhou, Lijuan; Rouge, Lionel; Phillips, Aaron H; Lam, Cynthia; Liu, Peter; Sandoval, Wendy; Helgason, Elizabeth; Murray, Jeremy M; Wertz, Ingrid E; Corn, Jacob E

    2013-01-01

    Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

  18. Reversible monoubiquitination regulates the Parkinson disease-associated ubiquitin hydrolase UCH-L1.

    PubMed

    Meray, Robin K; Lansbury, Peter T

    2007-04-06

    Deubiquitinating enzymes (DUBs) are negative regulators of protein ubiquitination and play an important role in ubiquitin-dependent processes. Recent studies have found that diverse cellular mechanisms are employed to control the activity of DUBs. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a highly expressed neuronal DUB linked to Parkinson disease; however, little is known about its specific functions or modes of regulation. Here, we demonstrate that UCH-L1 is post-translationally modified by monoubiquitin in cells, at lysine residues near the active site. This modification restricts enzyme activity by preventing binding to ubiquitinated targets, and permanent monoubiquitination, as mimicked by a ubiquitin-UCH-L1 fusion, inhibits UCH-L1 in its capacity to increase free ubiquitin levels in cells. Interestingly, UCH-L1 catalyzes its own deubiquitination in an intramolecular manner, thereby regulating the lifetime of this modification. Our results illustrate monoubiquitination as a reversible regulatory mechanism for DUB activity involving auto-deubiquitination.

  19. E3 Ubiquitin Ligases: Ubiquitous Actors in Plant Development and Abiotic Stress Responses.

    PubMed

    Shu, Kai; Yang, Wenyu

    2017-09-01

    Understanding the precise regulatory mechanisms of plant development and stress responses at the post-translational level is currently a topic of intensive research. Protein ubiquitination, including the sequential performances of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes, is a refined post-translational modification ubiquitous in all eukaryotes. Plants are an integral part of our ecosystem and, as sessile organisms, the ability to perceive internal and external signals and to adapt well to various environmental challenges is crucial for their survival. Over recent decades, extensive studies have demonstrated that protein ubiquitination plays key roles in multiple plant developmental stages (e.g. seed dormancy and germination, root growth, flowering time control, self-incompatibility and chloroplast development) and several abiotic stress responses (e.g. drought and high salinity), by regulating the abundance, activities or subcellular localizations of a variety of regulatory polypeptides and enzymes. Importantly, diverse E3 ligases are involved in these regulatory pathways by mediating phytohormone and light signaling or other pathways. In this updated review, we mainly summarize recent advances in our understanding of the regulatory roles of protein ubiquitination in plant development and plant-environment interactions, and primarily focus on different types of E3 ligases because they play critical roles in determining substrate specificity. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Estrogen modification of human glutamate dehydrogenases is linked to enzyme activation state.

    PubMed

    Borompokas, Nikolas; Papachatzaki, Maria-Martha; Kanavouras, Konstantinos; Mastorodemos, Vasileios; Zaganas, Ioannis; Spanaki, Cleanthe; Plaitakis, Andreas

    2010-10-08

    Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC(50) = 0.1-0.3 μM) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC(50) = 0.08 ± 0.01 μM) and with ∼18-fold lower affinity hGDH1 (IC(50) = 1.67 ± 0.06 μM; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC(50) = 1.53 ± 0.24 μM) than for hGDH1 (IC(50) = 26.94 ± 1.07 μM; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.

  1. 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.

  2. 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

  3. A Bacterial Inhibitor of Host Programmed Cell Death Defenses is an E3 Ubiquitin Ligase

    SciTech Connect

    Janjusevic,R.; Abramovitch, R.; Martin, G.; Stebbins, C.

    2005-01-01

    The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

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

    PubMed Central

    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.

    2011-01-01

    Summary 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

  5. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2

    PubMed Central

    Mikawa, Takumi; Maruyama, Takeshi; Okamoto, Koji; Nakagama, Hitoshi; Lleonart, Matilde E.; Tsusaka, Takeshi; Hori, Kousuke; Murakami, Itsuo; Izumi, Taisuke; Takaori-Kondo, Akifumi; Yokode, Masayuki; Peters, Gordon; Beach, David

    2014-01-01

    Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage–induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM. PMID:24567357

  6. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2.

    PubMed

    Mikawa, Takumi; Maruyama, Takeshi; Okamoto, Koji; Nakagama, Hitoshi; Lleonart, Matilde E; Tsusaka, Takeshi; Hori, Kousuke; Murakami, Itsuo; Izumi, Taisuke; Takaori-Kondo, Akifumi; Yokode, Masayuki; Peters, Gordon; Beach, David; Kondoh, Hiroshi

    2014-03-03

    Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage-induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.

  7. Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics.

    PubMed

    Archelas, Alain; Zhao, Wei; Faure, Bruno; Iacazio, Gilles; Kotik, Michael

    2016-02-01

    A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

  8. Cell fate determination by ubiquitin-dependent regulation of translation

    PubMed Central

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  9. Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction.

    PubMed

    Ranaghan, Kara E; Ridder, Lars; Szefczyk, Borys; Sokalski, W Andrzej; Hermann, Johannes C; Mulholland, Adrian J

    2004-04-07

    To investigate fundamental features of enzyme catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within enzymes. We have modelled an important model enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the enzyme are 7.4-11.0 kcal mol(-1)(MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7-16.1 kcal mol(-1)(B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of Delta H(++)= 12.7 +/- 0.4 kcal mol(-1). The results provide unequivocal evidence of transition state (TS) stabilization by the enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol(-1)(MP2/6-31+G(d)/PCM) and 24.6 kcal mol(-1)(B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-fold rate acceleration by the enzyme. The substrate is found to be significantly distorted in the enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the enzyme, involving reaction from a 'near-attack conformer' of the substrate, indicates that adoption of this

  10. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  11. Synthesis of defined ubiquitin dimers.

    PubMed

    Eger, Silvia; Scheffner, Martin; Marx, Andreas; Rubini, Marina

    2010-11-24

    Many proteins are post-translationally modified by the attachment of poly-ubiquitin (Ub) chains. Notably, the biological function of the attached Ub chain depends on the specific lysine residue used for conjugate formation. Here, we report an easy and efficient method to synthesize site-specifically linked Ub dimers by click reaction between two artificial amino acids. In fact, we were able to synthesize all seven naturally occurring Ub connectivities, providing the first example of a method that gives access to all Ub dimers. Furthermore, these synthetic Ub dimers are recognized by the natural ubiquitination machinery and are proteolytically stable, making them optimal candidates to further investigate the function of differently linked Ub chains.

  12. It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin

    PubMed Central

    Liebl, Martina P.

    2016-01-01

    Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases. PMID:27225656

  13. Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste.

    PubMed

    Romo Sánchez, Sheila; Gil Sánchez, Irene; Arévalo-Villena, María; Briones Pérez, Ana

    2015-03-01

    The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. Castilla-La Mancha produces around three million tons of waste coming from olive oil and wine industries, both of which have a pivotal role in the economy of this region. For this reason, this study reports on the exploitation of grape skins and olive pomaces for the production of lignocellulosic enzymes, which are able to deconstruct the agroindustrial waste and, therefore, reuse them in future industrial processes. To this end, solid-state fermentation was carried out using two local fungal strains (Aspergillus niger-113 N and Aspergillus fumigatus-3). In some trials, a wheat supplementation with a 1:1 ratio was used to improve the growth conditions, and the particle size of the substrates was altered through milling. Separate fermentations were run and collected after 2, 4, 6, 8, 10 and 15 days to monitor enzymatic activity (xylanase, cellulase, β-glucosidase, pectinase). The highest values were recorded after 10 and 15 days of fermentation. The use of A. niger on unmilled grape skin yielded the best outcomes (47.05 U xylanase/g by-product). The multi-enzymatic extracts obtained were purified, freeze dried, and immobilized on chitosan by adsorption to assess the possible advantages provided by the different techniques.

  14. Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state.

    PubMed

    Lien, T; Schreiner, O; Steine, M

    1975-03-28

    Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.

  15. Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by Trichoderma spp.

    PubMed

    da Silva, Luis C A; Honorato, Talita L; Cavalcante, Rosane S; Franco, Telma T; Rodrigues, Sueli

    2012-03-01

    Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40-50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).

  16. Cellulolytic Enzymes Production via Solid-State Fermentation: Effect of Pretreatment Methods on Physicochemical Characteristics of Substrate

    PubMed Central

    Brijwani, Khushal; Vadlani, Praveen V.

    2011-01-01

    We investigated the effect of pretreatment on the physicochemical characteristics—crystallinity, bed porosity, and volumetric specific surface of soybean hulls and production of cellulolytic enzymes in solid-state fermentation of Trichoderma reesei and Aspergillus oryzae cultures. Mild acid and alkali and steam pretreatments significantly increased crystallinity and bed porosity without significant change inholocellulosic composition of substrate. Crystalline and porous steam-pretreated soybean hulls inoculated with T. reesei culture had 4 filter paper units (FPU)/g-ds, 0.6 IU/g-ds β-glucosidase, and 45 IU/g-ds endocellulase, whereas untreated hulls had 0.75 FPU/g-ds, 0.06 IU/g-ds β-glucosidase, and 7.29 IU/g-ds endocellulase enzyme activities. In A. oryzae steam-pretreated soybean hulls had 47.10 IU/g-ds endocellulase compared to 30.82 IU/g-ds in untreated soybean hulls. Generalized linear statistical model fitted to enzyme activity data showed that effects of physicochemical characteristics on enzymes production were both culture and enzyme specific. The paper shows a correlation between substrate physicochemical properties and enzyme production. PMID:21755043

  17. Rapid enzyme production and mycelial growth in solid-state fermentation using the non-airflow box.

    PubMed

    Ito, Kazunari; Gomi, Katsuya; Kariyama, Masahiro; Miyake, Tsuyoshi

    2013-11-01

    Solid-state fermentation (SSF) has become an attractive alternative to submerged fermentation (SMF) for the production of enzymes, organic acids, and secondary metabolites, while there are many problems during the culture of SSF. We recently created a SSF system using a non-airflow box (NAB) in order to resolve the problems, which enabled the uniform culture in the whole substrate and high yield of many enzymes. In this paper, further characterization of SSF using the NAB was carried out to obtain other advantages. The NAB culture under the fixed environmental condition exhibited a rapid increase in enzyme production at earlier phase during the culture compared with conventional SSF. Total mycelial growth also exhibited the same trend as enzyme production. Thus, the increase in the rate of the enzyme production was thought to mainly be attributed to that of the growth. To support it, it was suggested that the NAB culture resulted in most optimal water activity for the growth just at the log phase. In addition, the NAB culture was able to achieve high reproducibility of enzyme production, derived from uniform condition of the substrate during the culture. The results indicate that the NAB culture has many benefits for SSF.

  18. Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities.

    PubMed

    Neumann, Piotr; Tittmann, Kai

    2014-12-01

    Although general principles of enzyme catalysis are fairly well understood nowadays, many important details of how exactly the substrate is bound and processed in an enzyme remain often invisible and as such elusive. In fortunate cases, structural analysis of enzymes can be accomplished at true atomic resolution thus making possible to shed light on otherwise concealed fine-structural traits of bound substrates, intermediates, cofactors and protein groups. We highlight recent structural studies of enzymes using ultrahigh-resolution X-ray protein crystallography showcasing its enormous potential as a tool in the elucidation of enzymatic mechanisms and in unveiling fundamental principles of enzyme catalysis. We discuss the observation of seemingly hyper-reactive, physically distorted cofactors and intermediates with elongated scissile substrate bonds, the detection of 'hidden' conformational and chemical equilibria and the analysis of protonation states with surprising findings. In delicate cases, atomic resolution is required to unambiguously disclose the identity of atoms as demonstrated for the metal cluster in nitrogenase. In addition to the pivotal structural findings and the implications for our understanding of enzyme catalysis, we further provide a practical framework for resolution enhancement through optimized data acquisition and processing.

  19. The pineal gland: A model for adrenergic modulation of ubiquitin ligases

    PubMed Central

    Liu, Wenjun; Reiter, Russel J.

    2017-01-01

    Introduction A recent study of the pineal gland of the rat found that the expression of more than 3000 genes showed significant day/night variations (The Hartley dataset). The investigators of this report made available a supplemental table in which they tabulated the expression of many genes that they did not discuss, including those coding for components of the ubiquitin proteasome system. Herein we identify the genes of the ubiquitin proteasome system whose expression were significantly influenced by environmental lighting in the Hartley dataset, those that were stimulated by DBcAMP in pineal glands in culture, and those that were stimulated by norepinephrine. Purpose Using the Ubiquitin and Ubiquitin-like Conjugation Database (UUCA) we identified ubiquitin ligases and conjugases, and deubiquitinases in the Hartley dataset for the purpose of determining whether expression of genes of the ubiquitin proteasome pathway were significantly influenced by day/night variations and if these variations were regulated by autonomic innervation of the pineal gland from the superior cervical ganglia. Methods In the Hartley experiments pineal glands groups of rats sacrificed during the day and groups sacrificed during the night were examined for gene expression. Additional groups of rats had their superior cervical ganglia removed surgically or surgically decentralized and the pineal glands likewise examined for gene expression. Results The genes with at least a 2-fold day/night significant difference in expression included genes for 5 ubiquitin conjugating enzymes, genes for 58 ubiquitin E3 ligases and genes for 6 deubiquitinases. A 35-fold day/night difference was noted in the expression of the gene Sik1, which codes for a protein containing both an ubiquitin binding domain (UBD) and an ubiquitin-associated (UBA) domain. Most of the significant differences in these genes were prevented by surgical removal, or disconnection, of the superior cervical ganglia, and most were

  20. Ubiquitin conjugation to Gag is essential for ESCRT-mediated HIV-1 budding

    PubMed Central

    2013-01-01

    Background HIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag. Results Expressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV’s, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites. Conclusions These studies demonstrate a necessary and

  1. Production of manganese peroxidase and laccase in a solid-state bioreactor and modeling of enzyme production kinetics.

    PubMed

    Moilanen, Ulla; Winquist, Erika; Mattila, Tuomas; Hatakka, Annele; Eerikäinen, Tero

    2015-01-01

    Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

  2. Deubiquitinating enzymes as novel anticancer targets

    PubMed Central

    Nicholson, Benjamin; Marblestone, Jeffrey G; Butt, Tauseef R; Mattern, Michael R

    2008-01-01

    Tagging proteins with mono- or poly-ubiquitin is now recognized as a multifaceted and universal means of regulating cell growth and physiology. It does so by controlling the cellular lifetime of nearly all eukaryotic proteins and the cellular localization of many critical proteins. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases [DUBs]) ubiquitin tags to or from their target proteins in a selective fashion. Similarly to the kinases and their corresponding phosphatases, ubiquitin ligases and DUBs have become actively studied molecular oncology targets for drug discovery. Approximately 79 functional DUBs exist in the human proteome, suggesting that selective intervention is a reasonable therapeutic objective, with the goal of downregulating or ablating oncogene products or, alternatively, upregulating or sparing tumor suppressors. In the following review, this fascinating class of regulatory enzymes will be described, and specific examples of DUBs that are viable targets for anticancer therapy will be considered. PMID:17381419

  3. Multiple Ionization of Free Ubiquitin Molecular Ions in Extreme Ultraviolet Free-Electron Laser Pulses.

    PubMed

    Schlathölter, Thomas; Reitsma, Geert; Egorov, Dmitrii; Gonzalez-Magaña, Olmo; Bari, Sadia; Boschman, Leon; Bodewits, Erwin; Schnorr, Kirsten; Schmid, Georg; Schröter, Claus Dieter; Moshammer, Robert; Hoekstra, Ronnie

    2016-08-26

    The fragmentation of free tenfold protonated ubiquitin in intense 70 femtosecond pulses of 90 eV photons from the FLASH facility was investigated. Mass spectrometric investigation of the fragment cations produced after removal of many electrons revealed fragmentation predominantly into immonium ions and related ions, with yields increasing linearly with intensity. Ionization clearly triggers a localized molecular response that occurs before the excitation energy equilibrates. Consistent with this interpretation, the effect is almost unaffected by the charge state, as fragmentation of sixfold deprotonated ubiquitin leads to a very similar fragmentation pattern. Ubiquitin responds to EUV multiphoton ionization as an ensemble of small peptides.

  4. Cardiac Arrest Alters Regional Ubiquitin Levels in Association with the Blood-Brain Barrier Breakdown and Neuronal Damages in the Porcine Brain.

    PubMed

    Sharma, Hari S; Patnaik, Ranjana; Sharma, Aruna; Lafuente, José Vicente; Miclescu, Adriana; Wiklund, Lars

    2015-10-01

    The possibility that ubiquitin expression is altered in cardiac arrest-associated neuropathology was examined in a porcine model using immunohistochemical and biochemical methods. Our observations show that cardiac arrest induces progressive increase in ubiquitin expression in the cortex and hippocampus in a selective and specific manner as compared to corresponding control brains using enzyme-linked immunoassay technique (enzyme-linked immunosorbent assay (ELISA)). Furthermore, immunohistochemical studies showed ubiquitin expression in the neurons exhibiting immunoreaction in the cytoplasm and karyoplasm of distorted or damaged cells. Separate Nissl and ubiquitin staining showed damaged and distorted neurons and in the same cortical region ubiquitin expression indicating that ubiquitin expression after cardiac arrest represents dying neurons. The finding that methylene blue treatment markedly induced neuroprotection following identical cardiac arrest and reduced ubiquitin expression strengthens this view. Taken together, our observations are the first to show that cardiac arrest enhanced ubiquitin expression in the brain that is related to the magnitude of neuronal injury and the finding that methylene blue reduced ubiquitin expression points to its role in cell damage, not reported earlier.

  5. Unfolding dynamics of the protein ubiquitin: insight from simulation.

    PubMed

    Dastidar, Shubhra Ghosh; Mukhopadhyay, Chaitali

    2005-11-01

    The temperature-induced unfolding pathway of ubiquitin has been investigated by molecular dynamics simulation at four different temperatures. It has been observed that the sequences of the unfolding events are same at all the temperatures. However, the time scale of the dynamics at different temperatures are different. The transition states at various temperatures also possess similar secondary structural elements. The intermediate conformations visited by the protein at different temperatures can help determination of the transition states. The well known " state" of ubiquitin, hitherto found to be stable only in methanol water mixture, have been observed to be a common transient intermediate conformation in the unfolding path of the protein in water. Our observation about the similarities of the unfolding process at different temperatures strongly recommend for a defined pathway for the unfolding process.

  6. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. UBIQUITIN-SPECIFIC PROTEASE16 interacts with a HEAVY METAL ASSOCIATED ISOPRENYLATED PLANT PROTEIN27 and modulates cadmium tolerance.

    PubMed

    Zhao, Jinfeng; Zhou, Huapeng; Li, Yueyong

    2013-10-01

    Protein ubiquitination and deubiquitination are two reversible processes catalyzed by ubiquitin ligases and deubiquitinating enzymes, respectively. In Arabidopsis, lots of substrates of ubiquitin ligases were found, whereas only a few targets of deubiquitinating enzymes were identified. Recently, we reported that a functional UBIQUITIN-SPECIFIC PROTEASE16 (UBP16) was involved in salt tolerance through positively regulating plasma membrane Na+/H+ antiport activity and at least partially modulating SERINE HYDROXYMETHYLTRANSFERASE1 (SHM 1) stability and activity. Here, we report that UBP16 interacts with HEAVY META L ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27 (HI PP27), a metallochaperone containing a predicted heavy-metal-associated domain, which has been reported to play an important role in cadmium detoxification. Meanwhile, the ubp16 mutant showed more sensitive to cadmium than wild-type. Taken together, HI PP27 may be another target of UBP16 in cadmium response.

  8. A 'random steady-state' model for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase enzyme complexes

    NASA Astrophysics Data System (ADS)

    Najdi, T. S.; Hatfield, G. W.; Mjolsness, E. D.

    2010-03-01

    The multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, involved in the central metabolism of Escherichia coli consist of multiple copies of three different enzymes, E1, E2 and E3, that cooperate to channel substrate intermediates between their active sites. The E2 components form the core of the complex, while a mixture of E1 and E3 components binds to the core. We present a random steady-state model to describe catalysis by such multienzyme complexes. At a fast time scale, the model describes the enzyme catalytic mechanisms of substrate channeling at a steady state, by polynomially approximating the analytic solution of a biochemical master equation. At a slower time scale, the structural organization of the different enzymes in the complex and their random binding/unbinding to the core is modeled using methods from equilibrium statistical mechanics. Biologically, the model describes the optimization of catalytic activity by substrate sharing over the entire enzyme complex. The resulting enzymatic models illustrate the random steady state (RSS) for modeling multienzyme complexes in metabolic pathways.

  9. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  10. Dysregulation of ubiquitin homeostasis and β-catenin signaling promote spinal muscular atrophy

    PubMed Central

    Wishart, Thomas M.; Mutsaers, Chantal A.; Riessland, Markus; Reimer, Michell M.; Hunter, Gillian; Hannam, Marie L.; Eaton, Samantha L.; Fuller, Heidi R.; Roche, Sarah L.; Somers, Eilidh; Morse, Robert; Young, Philip J.; Lamont, Douglas J.; Hammerschmidt, Matthias; Joshi, Anagha; Hohenstein, Peter; Morris, Glenn E.; Parson, Simon H.; Skehel, Paul A.; Becker, Thomas; Robinson, Iain M.; Becker, Catherina G.; Wirth, Brunhilde; Gillingwater, Thomas H.

    2014-01-01

    The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA. PMID:24590288

  11. Ubiquitination of tissue transglutaminase is modulated by interferon alpha in human lung cancer cells.

    PubMed Central

    Esposito, Carla; Marra, Monica; Giuberti, Gaia; D'Alessandro, Anna Maria; Porta, Raffaele; Cozzolino, Anna; Caraglia, Michele; Abbruzzese, Alberto

    2003-01-01

    The addition of 2500 i.u./ml interferon alpha (IFNalpha) for 48 h induced apoptosis, and caused an approx. 4-fold increase in the activity and expression of tissue transglutaminase (tTG), in human lung cancer H1355 cells. However, the increase in mRNA levels for tTG was just 1.6-fold. On the basis of these data, we investigated whether tTG levels may be regulated through regulation of its degradation via ubiquitination. It was found that 2500 i.u./ml IFNalpha induced a time-dependent decrease in tTG ubiquitination. On the other hand, addition of the proteasome inhibitor lactacystin led to accumulation of the ubiquitinated form of the enzyme and to a consequent increase in its expression. Treatment of the cells with the two agents combined antagonized the accumulation of the ubiquitinated isoforms of tTG induced by lactacystin and caused a potentiation of tTG expression. Moreover, the tTG inducer retinoic acid was also able to cause increased expression and ubiquitination of tTG in H1355 cells. The addition of monodansylcadaverine (a tTG inhibitor) to IFNalpha-treated H1355 cells completely antagonized growth inhibition and apoptosis induced by the cytokine. In conclusion, we demonstrate for the first time that tTG is ubiquitinated and degraded by a proteasome-dependent pathway. Moreover, IFNalpha can, at least in part, induce apoptosis through the modulation of this pathway. PMID:12401132

  12. Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

    SciTech Connect

    T Messick; N Russel; A Iwata; K Sarachan; R Shiekhattar; I Shanks; F Reyes-Turcu; K Wilkinson; R Marmorstein

    2011-12-31

    Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of {approx}130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys{sup 48} linkages, having little or no activity on Lys{sup 63}- and Lys{sup 29}-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

  13. Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs*

    PubMed Central

    Peth, Andreas; Kukushkin, Nikolay; Bossé, Marc; Goldberg, Alfred L.

    2013-01-01

    Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction. PMID:23341450

  14. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

  15. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

  16. Microsecond molecular dynamics simulation of guanidinium chloride induced unfolding of ubiquitin.

    PubMed

    Mandal, Manoj; Mukhopadhyay, Chaitali

    2014-10-21

    An all atom molecular dynamics simulation was used to explore the atomic detail mechanism of guanidinium induced unfolding of the protein ubiquitin. Ubiquitin unfolds through pre-unfolded (intermediate) states, i.e. guanidinium induced unfolding of ubiquitin appears to be a multi-step process, and loss of hydrophobic contacts of C-terminal residues is crucial for ubiquitin unfolding. Free-energy landscapes show that barrier separation between folded and unfolded basins is ∼5.0 kcal mol(-1), and both the basins are of comparable energy. It was observed that guanidinium ions interact directly with ubiquitin. Favorable electrostatic interaction is the main driving force for such accumulation of guanidinium ions near protein, but van der Waals energy also contributes. RDF plots show that accumulation of guanidinium ions near specific residues is the main cause for destabilization of intra-residue interactions crucial to maintain the three-dimensional fold of the protein. One salt-bridge interaction between Lys11 and Glu34 appears to be important to maintain the crystal structure of ubiquitin and this salt-bridge can map the unfolding process of ubiquitin.

  17. Bacteria-host relationship: ubiquitin ligases as weapons of invasion

    PubMed Central

    Maculins, Timurs; Fiskin, Evgenij; Bhogaraju, Sagar; Dikic, Ivan

    2016-01-01

    Eukaryotic cells utilize the ubiquitin (Ub) system for maintaining a balanced functioning of cellular pathways. Although the Ub system is exclusive to eukaryotes, prokaryotic bacteria have developed an armory of Ub ligase enzymes that are capable of employing the Ub systems of various hosts, ranging from plant to animal cells. These enzymes have been acquired through the evolution and can be classified into three main classes, RING (really interesting new gene), HECT (homologous to the E6-AP carboxyl terminus) and NEL (novel E3 ligases). In this review we describe the roles played by different classes of bacterial Ub ligases in infection and pathogenicity. We also provide an overview of the different mechanisms by which bacteria mimic specific components of the host Ub system and outline the gaps in our current understanding of their functions. Additionally, we discuss approaches and experimental tools for validating this class of enzymes as potential novel antibacterial therapy targets. PMID:26964724

  18. Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

    SciTech Connect

    Kozlov, G.; Nguyen, L; Lin, T; De Crescenzo, G; Park, M; Gehring, K

    2007-01-01

    EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85{angstrom} crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices {alpha}1 and {alpha}3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

  19. Functional diversity and structural disorder in the human ubiquitination pathway.

    PubMed

    Bhowmick, Pallab; Pancsa, Rita; Guharoy, Mainak; Tompa, Peter

    2013-01-01

    The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well

  20. Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

    PubMed Central

    Bhowmick, Pallab; Pancsa, Rita; Guharoy, Mainak; Tompa, Peter

    2013-01-01

    The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred – E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well

  1. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    PubMed Central

    Hurst-Kennedy, Jennifer; Chin, Lih-Shen; Li, Lian

    2012-01-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5) is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis. PMID:22811913

  2. Strategies for discovery and improvement of enzyme function: state of the art and opportunities

    PubMed Central

    Kaul, Praveen; Asano, Yasuhisa

    2012-01-01

    Summary Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. PMID:21883976

  3. Monte Carlo docking with ubiquitin.

    PubMed Central

    Cummings, M. D.; Hart, T. N.; Read, R. J.

    1995-01-01

    The development of general strategies for the performance of docking simulations is prerequisite to the exploitation of this powerful computational method. Comprehensive strategies can only be derived from docking experiences with a diverse array of biological systems, and we have chosen the ubiquitin/diubiquitin system as a learning tool for this process. Using our multiple-start Monte Carlo docking method, we have reconstructed the known structure of diubiquitin from its two halves as well as from two copies of the uncomplexed monomer. For both of these cases, our relatively simple potential function ranked the correct solution among the lowest energy configurations. In the experiments involving the ubiquitin monomer, various structural modifications were made to compensate for the lack of flexibility and for the lack of a covalent bond in the modeled interaction. Potentially flexible regions could be identified using available biochemical and structural information. A systematic conformational search ruled out the possibility that the required covalent bond could be formed in one family of low-energy configurations, which was distant from the observed dimer configuration. A variety of analyses was performed on the low-energy dockings obtained in the experiment involving structurally modified ubiquitin. Characterization of the size and chemical nature of the interface surfaces was a powerful adjunct to our potential function, enabling us to distinguish more accurately between correct and incorrect dockings. Calculations with the structure of tetraubiquitin indicated that the dimer configuration in this molecule is much less favorable than that observed in the diubiquitin structure, for a simple monomer-monomer pair. Based on the analysis of our results, we draw conclusions regarding some of the approximations involved in our simulations, the use of diverse chemical and biochemical information in experimental design and the analysis of docking results, as well as

  4. Prokaryotic Ubiquitin-Like Protein Provides a Two-Part Degron to Mycobacterium Proteasome Substrates▿ †

    PubMed Central

    Burns, Kristin E.; Pearce, Michael J.; Darwin, K. Heran

    2010-01-01

    Prokaryotic ubiquitin-like protein (Pup) is a posttranslational modifier that targets proteins for degradation by the mycobacterial proteasome. We show that the disordered amino terminus of Pup is required for degradation, while the helical carboxyl terminus mediates its attachment to proteins. Thus, Pup has distinct regions that either interact with pupylation enzymes or initiate proteasomal degradation. PMID:20233925

  5. Solid-state fermentation of new substrates for production of cellulase and other biopolymer-hydrolyzing enzymes

    SciTech Connect

    Sharma, D.K.; Tiwari, M.; Behera, B.K.

    1995-12-31

    With the realization that the use of leaded gasoline results in environmental pollution and can effect human health, interest in the use of ethanol either directly or as a gasohol is increasing. Production of ethanol from lignocellulosic biomass was commercially practiced during the world wars by acidic hydrolysis technique. However, these processes were found to be uneconomical after the war emergencies were over. An alternate route is the enzymatic hydrolysis of lignocellulosic biomass. However, the economics of the enzymatic hydrolysis process relies substantially on the cost associated with the production of cellulose enzyme. The liquid-state fermentation technique of cellulose enzyme production involves problems related to maintenance of cellulose concentration, inorganic nutrients, and pH. However, the authors feel that the solid-state fermentation (SF) technique may be more suitable for the large-scale production of cellulose enzyme. The cost of substrate and its potential for the induction and production of cellulose, along with other equally important enzymes, such as xylanase, {beta}-1,3-glucanase, chitinase, and cellobiase enzymes, should be studied by selecting different cheap substrates in the SF process. There are several latex- and resin-bearing plants that contain hydrocarbons and related compounds. These plants are called petrocrops, since these hydrocarbons and related compounds can be extracted from these plants by simple heptane extraction. In the present work, the spent residue (SR) obtained after heptane or hexane extraction was used as a substrate for the production of cellulose and related enzymes by the wheat litter decomposing fungi. The results are reported.

  6. Properties of thermostable hemicellulolytic enzymes from Thermomonospora strain 29 grown in solid state fermentation on coffee processing solid waste.

    PubMed

    Srivastava, K C

    1993-01-01

    During decaffeination of Coffee Processing Plant Solid Wastes (CPSW) by actinomycetes, Thermomonospora, Strain 29 exhibited high titers of cellulase and xylanase. This organism, originally isolated on soybean seed coat was grown in solid state fermentation on CPSW supplemented with mineral salts. Enzymes recovered were arabinosidase, xylanase, and beta-D-xylosidase. Higher activity of the former two enzymes was in the extracellular broth, whereas the beta-D-xylosidase activity was highest in the cell fraction. The enzymes were characterized after precipitation with (NH(4))(2)SO(4), dialysis, and gel filtration. Production of all three enzymes was inhibited by monomeric sugars and sugar alcohols but not by arabinoxylan, xylans, or xylan containing water insoluble carbohydrates. The optimum pH for the activity was 6.5, 7.0, and 7.5 for beta-xylosidase, xylanase and arabinosidase (alpha-L-arabinofuranosidase, alpha-arabinosidase, alpha-L-arabinosidase) respectively. These enzymes were stable in the pH range of 6.5 to 8.0. All three enzymes were thermostable up to 80 degrees C. At 55 degrees C, arabinosidase had the longest half life of 120 h. However, at 40 degrees C, xylanase had the longest half life (504 h). At either temperature, beta-D-xylosidase had the shortest half life. The molecular weights (kDa), and Kms (mM) were estimated to be 95, 0.27; 45, 12.4; and 106, 0.67 for arbinosidase, xylanase, and beta-xylosidase respectively. Step wise addition of the three enzymes showed higher saccharification of lignocellulosics.

  7. Deubiquitylation of hepatitis B virus X protein (HBx) by ubiquitin-specific peptidase 15 (USP15) increases HBx stability and its transactivation activity

    PubMed Central

    Su, Zhi-Jun; Cao, Jia-Shou; Wu, Yan-Fang; Chen, Wan-Nan; Lin, Xinjian; Wu, Yun-Li; Lin, Xu

    2017-01-01

    Hepatitis B virus X protein (HBx) plays important roles in viral replication and the development of hepatocellular carcinoma. HBx is a rapid turnover protein and ubiquitin-proteasome pathway has been suggested to influence HBx stability as treatment with proteasome inhibitors increases the levels of HBx protein and causes accumulation of the polyubiquitinated forms of HBx. Deubiquitinases (DUBs) are known to act by removing ubiquitin moieties from proteins and thereby reverse their stability and/or activity. However, no information is available regarding the involvement of DUBs in regulation of ubiquitylation-dependent proteasomal degradation of HBx protein. This study identified the deubiquitylating enzyme USP15 as a critical regulator of HBx protein level. USP15 was found to directly interact with HBx via binding to the HBx region between amino acid residues 51 and 80. USP15 increased HBx protein levels in a dose-dependent manner and siRNA-mediated knockdown of endogenous USP15 reduced HBx protein levels. Increased HBx stability and steady-state level by USP15 were attributable to reduced HBx ubiquitination and proteasomal degradation. Importantly, the transcriptional transactivation function of HBx is enhanced by overexpression of USP15. These results suggest that USP15 plays an essential role in stabilizing HBx and subsequently affects the biological function of HBx. PMID:28074857

  8. Cellulolytic enzymes production by utilizing agricultural wastes under solid state fermentation and its application for biohydrogen production.

    PubMed

    Saratale, Ganesh D; Kshirsagar, Siddheshwar D; Sampange, Vilas T; Saratale, Rijuta G; Oh, Sang-Eun; Govindwar, Sanjay P; Oh, Min-Kyu

    2014-12-01

    Phanerochaete chrysosporium was evaluated for cellulase and hemicellulase production using various agricultural wastes under solid state fermentation. Optimization of various environmental factors, type of substrate, and medium composition was systematically investigated to maximize the production of enzyme complex. Using grass powder as a carbon substrate, maximum activities of endoglucanase (188.66 U/gds), exoglucanase (24.22 U/gds), cellobiase (244.60 U/gds), filter paperase (FPU) (30.22 U/gds), glucoamylase (505.0 U/gds), and xylanase (427.0 U/gds) were produced under optimized conditions. The produced crude enzyme complex was employed for hydrolysis of untreated and mild acid pretreated rice husk. The maximum amount of reducing sugar released from enzyme treated rice husk was 485 mg/g of the substrate. Finally, the hydrolysates of rice husk were used for hydrogen production by Clostridium beijerinckii. The maximum cumulative H2 production and H2 yield were 237.97 mL and 2.93 mmoL H2/g of reducing sugar, (or 2.63 mmoL H2/g of cellulose), respectively. Biohydrogen production performance obtained from this work is better than most of the reported results from relevant studies. The present study revealed the cost-effective process combining cellulolytic enzymes production under solid state fermentation (SSF) and the conversion of agro-industrial residues into renewable energy resources.

  9. Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.

    PubMed

    Nakatsukasa, Kunio; Kamura, Takumi

    2016-01-01

    During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.

  10. Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function.

    PubMed

    Lee, Shirley Y; Pullen, Lester; Virgil, Daniel J; Castañeda, Carlos A; Abeykoon, Dulith; Bolon, Daniel N A; Fushman, David

    2014-04-03

    Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Ubiquitin fusion technology: bioprocessing of peptides.

    PubMed

    Pilon, A; Yost, P; Chase, T E; Lohnas, G; Burkett, T; Roberts, S; Bentley, W E

    1997-01-01

    Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli. Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996). There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production.

  12. Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity.

    PubMed Central

    Evans, Paul C; Ovaa, Huib; Hamon, Maureen; Kilshaw, Peter J; Hamm, Svetlana; Bauer, Stefan; Ploegh, Hidde L; Smith, Trevor S

    2004-01-01

    Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which 'trim' polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-kappaB (nuclear factor kappaB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IkappaBalpha (inhibitor of kappaB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKgamma (IkappaB kinase gamma)/NEMO (NF-kappaB essential modifier) signal adaptors relies on their modification with 'nonclassical' forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-kappaB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown. PMID:14748687

  13. Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads.

    PubMed

    Jain, Jagrati; Jain, Surendra K; Walker, Larry A; Tekwani, Babu L

    2017-06-02

    Protein ubiquitylation is an important post-translational regulation, which has been shown to be necessary for life cycle progression and survival of Plasmodium falciparum. Ubiquitin is a highly conserved 76 amino acid polypeptide, which attaches covalently to target proteins through combined action of three classes of enzymes namely, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). Ubiquitin E1 and E2 are highly conserved within eukaryotes. However, the P. falciparum E3 ligase is substantially variable and divergent compared to the homologs from other eukaryotes, which make the E3 ligase a parasite-specific target. A set of selected E3 ubiquitin ligase inhibitors was tested in vitro against a chloroquine-sensitive P. falciparum D6 strain (PfD6) and a chloroquine-resistant P. falciparum W2 strain (PfW2). The inhibitors were also tested against Vero and transformed THP1 cells for cytotoxicity. The lead antimalarial E3 ubiquitin ligase inhibitors were further evaluated for the stage-specific antimalarial action and effects on cellular development of P. falciparum in vitro. Statistics analysis was done by two-way ANOVA followed by Tukey and Sidak multiple comparison test using GraphPad Prism 6. E3 ligase inhibitors namely, JNJ 26854165, HLI 373 and Nutlin 3 showed prominent antimalarial activity against PfD6 and PfW2. These inhibitors were considerably less cytotoxic to mammalian Vero cells. JNJ 26854165, HLI 373 and Nutlin 3 blocked the development of P. falciparum parasite at the trophozoite and schizont stages, resulting in accumulation of distorted trophozoites and immature schizonts. Interruption of trophozoites and schizont maturation by the antimalarial E3 ligase inhibitors suggest the role of ubiquitin/proteasome functions in the intraerythrocytic development of malaria parasite. The ubiquitin/proteasome functions may be critical for schizont maturation. Further investigations on the lead E3 ligase

  14. Allosteric Activation of the RNF146 Ubiquitin Ligase by a Poly(ADP-ribosyl)ation Signal

    PubMed Central

    DaRosa, Paul A.; Wang, Zhizhi; Jiang, Xiaomo; Pruneda, Jonathan N.; Cong, Feng; Klevit, Rachel E.; Xu, Wenqing

    2014-01-01

    Protein poly(ADP-ribosyl)ation (PARylation) plays a role in diverse cellular processes such as DNA repair, transcription, Wnt signaling, and cell death1–6. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several critical regulatory proteins, including Axin and 3BP2 (refs 7–9). The RING-type E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is responsible for PARylation-dependent ubiquitination (PARdU)10–12. Here we provide a structural basis for RNF146 catalyzed PARdU and how PARdU specificity is achieved. First, we show that iso-ADPr, the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to efficiently bind and activate an E2. Binding of PAR/iso-ADPr induces a major conformational change that creates a functional RING structure. Thus RNF146 represents a new mechanistic class of RING E3 ligases whose activities are regulated by non-covalent ligand binding, which may provide a template for designing inducible protein-degradation systems. Second, we found that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146/TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalyzed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell. PMID:25327252

  15. RBR E3 ubiquitin ligases: new structures, new insights, new questions

    PubMed Central

    Spratt, Donald E.; Walden, Helen; Shaw, Gary S.

    2014-01-01

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. PMID:24576094

  16. Enzyme catalysis: a new definition accounting for noncovalent substrate- and product-like states.

    PubMed

    Purich, D L

    2001-07-01

    Biological catalysis frequently causes changes in noncovalent bonding. By building on Pauling's assertion that any long-lived, chemically distinct interaction is a chemical bond, this article redefines enzyme catalysis as the facilitated making and/or breaking of chemical bonds, not just of covalent bonds. It is also argued that nearly every ATPase or GTPase is misnamed as a hydrolase and actually belongs to a distinct class of enzymes, termed here 'energases'. By transducing covalent bond energy into mechanical work, energases mediate such fundamental processes as protein folding, self-assembly, G-protein interactions, DNA replication, chromatin remodeling and even active transport.

  17. Solution Dependence of the Collisional Activation of Ubiquitin [M+7H]7+ Ions

    PubMed Central

    Shi, Huilin; Atlasevich, Natalya; Merenbloom, Samuel I.; Clemmer, David E.

    2014-01-01

    The solution dependence of gas-phase unfolding for ubiquitin [M+7H]7+ ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas, activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed. PMID:24658799

  18. Chemical systems modeling the d(1) Mo(V) states of molybdenum enzymes.

    PubMed

    Young, Charles G

    2016-09-01

    This review focuses on the synthesis, properties and electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and electron-nuclear double resonance (ENDOR) spectroscopy of mononuclear d(1) oxo- and sulfido-Mo(V) complexes relevant to the understanding of the EPR-active Mo(V) forms of pterin-containing molybdenum enzymes. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Marine enzymes.

    PubMed

    Debashish, Ghosh; Malay, Saha; Barindra, Sana; Joydeep, Mukherjee

    2005-01-01

    Marine enzyme biotechnology can offer novel biocatalysts with properties like high salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and ease in large-scale cultivation. This review deals with the research and development work done on the occurrence, molecular biology, and bioprocessing of marine enzymes during the last decade. Exotic locations have been accessed for the search of novel enzymes. Scientists have isolated proteases and carbohydrases from deep sea hydrothermal vents. Cold active metabolic enzymes from psychrophilic marine microorganisms have received considerable research attention. Marine symbiont microorganisms growing in association with animals and plants were shown to produce enzymes of commercial interest. Microorganisms isolated from sediment and seawater have been the most widely studied, proteases, carbohydrases, and peroxidases being noteworthy. Enzymes from marine animals and plants were primarily studied for their metabolic roles, though proteases and peroxidases have found industrial applications. Novel techniques in molecular biology applied to assess the diversity of chitinases, nitrate, nitrite, ammonia-metabolizing, and pollutant-degrading enzymes are discussed. Genes encoding chitinases, proteases, and carbohydrases from microbial and animal sources have been cloned and characterized. Research on the bioprocessing of marine-derived enzymes, however, has been scanty, focusing mainly on the application of solid-state fermentation to the production of enzymes from microbial sources.

  20. The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors

    PubMed Central

    Grou, Cláudia P.; Pinto, Manuel P.; Mendes, Andreia V.; Domingues, Pedro; Azevedo, Jorge E.

    2015-01-01

    Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co- and post-translational processing. Using an unbiased biochemical approach we found that UCHL3, USP9X, USP7, USP5 and Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway. PMID:26235645

  1. Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines

    PubMed Central

    Lee, Joon No; Gong, Yi; Zhang, Xiangyu; Ye, Jin

    2006-01-01

    Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH2-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination. PMID:16549805

  2. The ubiquitin hybrid gene UBA52 regulates ubiquitination of ribosome and sustains embryonic development

    PubMed Central

    Kobayashi, Masanori; Oshima, Shigeru; Maeyashiki, Chiaki; Nibe, Yoichi; Otsubo, Kana; Matsuzawa, Yu; Nemoto, Yasuhiro; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

    2016-01-01

    Ubiquitination is a crucial post-translational modification; however, the functions of ubiquitin-coding genes remain unclear. UBA52 encodes a fusion protein comprising ubiquitin at the N-terminus and ribosomal protein L40 (RPL40) at the C-terminus. Here we showed that Uba52-deficient mice die during embryogenesis. UBA52-deficient cells exhibited normal levels of total ubiquitin. However, UBA52-deficient cells displayed decreased protein synthesis and cell-cycle arrest. The overexpression of UBA52 ameliorated the cell-cycle arrest caused by UBA52 deficiency. Surprisingly, RPL40 expression itself is insufficient to regulate cyclin D expression. The cleavage of RPL40 from UBA52 was required for maintaining protein synthesis. Furthermore, we found that RPL40 formed a ribosomal complex with ubiquitin cleaved from UBA52. UBA52 supplies RPL40 and ubiquitin simultaneously to the ribosome. Our study demonstrated that the ubiquitin-coding gene UBA52 is not just an ubiquitin supplier to the ubiquitin pool but is also a regulator of the ribosomal protein complex. These findings provide novel insights into the regulation of ubiquitin-dependent translation and embryonic development. PMID:27829658

  3. Secondary Structures of Ubiquitin Ions Soft-Landed onto Self-Assembled Monolayer Surfaces

    SciTech Connect

    Hu, Qichi; Laskin, Julia

    2016-06-09

    The secondary structures of multiply charged ubiquitin ions soft-landed onto self-assembled monolayer (SAM) surfaces were studied using in situ infrared reflection-absorption spectroscopy (IRRAS). Two charge states of ubiquitin, 5+ and 13+, were mass selected separately from a mixture of different charge states produced by electrospray ionization (ESI). The low 5+ charge state represents a native-like folded state of ubiquitin, while the high 13+ charge state assumes an extended, almost linear conformation. Each of the two charge states was soft-landed onto a CH3- and COOH-terminated SAM of alkylthiols on gold (HSAM and COOH-SAM). HSAM is a hydrophobic surface known to stabilize helical conformations of soft-landed protonated peptides, whereas COOH-SAM is a hydrophilic surface that preferentially stabilizes β-sheet conformations. IRRAS spectra of the soft-landed ubiquitin ions were acquired as a function of time during and after ion soft-landing. Similar to smaller peptide ions, helical conformations of ubiquitin are found to be more abundant on HSAM, while the relative abundance of β-sheet conformations increases on COOH-SAM. The initial charge state of ubiquitin also has a pronounced effect on its conformation on the surface. Specifically, on both surfaces, a higher relative abundance of helical conformations and lower relative abundance of β-sheet conformations is observed for the 13+ charge state compared to the 5+ charge state. Time-resolved experiments indicate that the α-helical band in the spectrum of the 13+ charge state slowly increases with time on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results further support the preference of the hydrophobic HSAM surface toward helical conformations and demonstrate that soft-landed protein ions may undergo slow conformational changes during and after deposition.

  4. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

    PubMed Central

    Sudakin, V; Ganoth, D; Dahan, A; Heller, H; Hershko, J; Luca, F C; Ruderman, J V; Hershko, A

    1995-01-01

    The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles. Images PMID:7787245

  5. Peroxynitrite-dependent zinc release and inactivation of guanosine 5'-triphosphate cyclohydrolase 1 instigate its ubiquitination in diabetes.

    PubMed

    Zhao, Yu; Wu, Jiliang; Zhu, Huaiping; Song, Ping; Zou, Ming-Hui

    2013-12-01

    Aberrant degradation of guanosine 5'-triphosphate cyclohydrolase 1 (GTPCH1) with consequent deficiency of tetrahydrobiopterin is considered the primary cause for endothelial dysfunction in diabetes. How GTPCH1 becomes susceptible to the degradation remains unknown. We hypothesized that oxidation and release of the zinc ion by peroxynitrite (ONOO(-)), a potent oxidant generated by nitric oxide and superoxide anions, instigates GTPCH1 ubiquitination and degradation. Zinc contents, GTPCH1 ubiquitination, and GTPCH1 activity were assayed in purified GTPCH1, endothelial cells, and hearts from diabetic mice. Exogenous ONOO(-) dose-dependently released zinc, inhibited its activity, and increased the ubiquitin binding affinity of GTPCH1 in vitro and in endothelial cells. Consistently, high glucose (30 mmol/L) inhibited GTPCH1 activity with increased ubiquitination, which was inhibited by antioxidants. Furthermore, mutation of the zinc-binding cysteine (141) (C141R or C141A) significantly reduced GTPCH1 activity and reduced its half-life but increased GTPCH1 ubiquitination, indicating an essential role of the zinc ion in maintaining the catalytic activity and stability of GTPCH1. Finally, GTPCH1 ubiquitination and degradation markedly increased in parallel with decreased GTPCH1 activity in the aortas and hearts of diabetic mice, both of which were attenuated by the inhibitors of ONOO(-) in mice in vivo. Taken together, we conclude that ONOO(-) releases zinc and inhibits GTPCH1, resulting in its ubiquitination and degradation of the enzyme.

  6. Determination of the pKa of the N-terminal amino group of ubiquitin by NMR

    PubMed Central

    Oregioni, Alain; Stieglitz, Benjamin; Kelly, Geoffrey; Rittinger, Katrin; Frenkiel, Tom

    2017-01-01

    Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains. PMID:28252051

  7. Peroxynitrite-Dependent Zinc Release and Inactivation of Guanosine 5′-Triphosphate Cyclohydrolase 1 Instigate Its Ubiquitination in Diabetes

    PubMed Central

    Zhao, Yu; Wu, Jiliang; Zhu, Huaiping; Song, Ping; Zou, Ming-Hui

    2013-01-01

    Aberrant degradation of guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) with consequent deficiency of tetrahydrobiopterin is considered the primary cause for endothelial dysfunction in diabetes. How GTPCH1 becomes susceptible to the degradation remains unknown. We hypothesized that oxidation and release of the zinc ion by peroxynitrite (ONOO−), a potent oxidant generated by nitric oxide and superoxide anions, instigates GTPCH1 ubiquitination and degradation. Zinc contents, GTPCH1 ubiquitination, and GTPCH1 activity were assayed in purified GTPCH1, endothelial cells, and hearts from diabetic mice. Exogenous ONOO− dose-dependently released zinc, inhibited its activity, and increased the ubiquitin binding affinity of GTPCH1 in vitro and in endothelial cells. Consistently, high glucose (30 mmol/L) inhibited GTPCH1 activity with increased ubiquitination, which was inhibited by antioxidants. Furthermore, mutation of the zinc-binding cysteine (141) (C141R or C141A) significantly reduced GTPCH1 activity and reduced its half-life but increased GTPCH1 ubiquitination, indicating an essential role of the zinc ion in maintaining the catalytic activity and stability of GTPCH1. Finally, GTPCH1 ubiquitination and degradation markedly increased in parallel with decreased GTPCH1 activity in the aortas and hearts of diabetic mice, both of which were attenuated by the inhibitors of ONOO− in mice in vivo. Taken together, we conclude that ONOO− releases zinc and inhibits GTPCH1, resulting in its ubiquitination and degradation of the enzyme. PMID:23974923

  8. Determination of the pKa of the N-terminal amino group of ubiquitin by NMR.

    PubMed

    Oregioni, Alain; Stieglitz, Benjamin; Kelly, Geoffrey; Rittinger, Katrin; Frenkiel, Tom

    2017-03-02

    Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains.

  9. The ubiquitin system, disease, and drug discovery

    PubMed Central

    Petroski, Matthew D

    2008-01-01

    The ubiquitin system of protein modification has emerged as a crucial mechanism involved in the regulation of a wide array of cellular processes. As our knowledge of the pathways in this system has grown, so have the ties between the protein ubiquitin and human disease. The power of the ubiquitin system for therapeutic benefit blossomed with the approval of the proteasome inhibitor Velcade in 2003 by the FDA. Current drug discovery activities in the ubiquitin system seek to (i) expand the development of new proteasome inhibitors with distinct mechanisms of action and improved bioavailability, and (ii) validate new targets. This review summarizes our current understanding of the role of the ubiquitin system in various human diseases ranging from cancer, viral infection and neurodegenerative disorders to muscle wasting, diabetes and inflammation. I provide an introduction to the ubiquitin system, highlight some emerging relationships between the ubiquitin system and disease, and discuss current and future efforts to harness aspects of this potentially powerful system for improving human health. Republished from Current BioData's Targeted Proteins database (TPdb; ). PMID:19007437

  10. Ubiquitin recognition by the proteasome.

    PubMed

    Saeki, Yasushi

    2017-02-01

    The 26S proteasome is a 2.5-MDa complex responsible for the selective, ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Substrates in hundreds cellular pathways are timely ubiquitylated and converged to the proteasome by direct recognition or by multiple shuttle factors. Engagement of substrate protein triggers conformational changes of the proteasome, which drive substrate unfolding, deubiquitylation and translocation of substrates to proteolytic sites. Recent studies have challenged the previous paradigm that Lys48-linked tetraubiquitin is a minimal degradation signal: in addition, monoubiquitylation or multiple short ubiquitylations can serve as the targeting signal for proteasomal degradation. In this review, I highlight recent advances in our understanding of the proteasome structure, the ubiquitin topology in proteasome targeting, and the cellular factors that regulate proteasomal degradation. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. Mimicking Heme Enzymes in the Solid State: Metal-Organic Materials with Selectively Encapsulated Heme

    SciTech Connect

    Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

    2011-06-13

    To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal–organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a “ship-in-a-bottle” fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

  12. The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme.

    PubMed Central

    Davisson, V J; Schulz, A R

    1985-01-01

    The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrate describes this inhibition. The apparent kinetic parameters for this reaction are Ka(NADH) = 239 microM and Vr = 1.1 mumol/min per mg at 23 degrees C. The steady-state product-inhibition patterns for pyruvate and NADH indicate a sequential binding of the substrates: NAD+ followed by L-malate. These data also indicate that NADH is the last product released. A steady-state kinetic model is proposed that incorporates NADH-enzyme dead-end complexes. PMID:3977837

  13. Ubc13: the Lys63 ubiquitin chain building machine

    PubMed Central

    Hodge, Curtis D.; Spyracopoulos, Leo; Glover, J. N. Mark

    2016-01-01

    Ubc13 is an ubiquitin E2 conjugating enzyme that participates with many different E3 ligases to form lysine 63-linked (Lys63) ubiquitin chains that are critical to signaling in inflammatory and DNA damage response pathways. Recent studies have suggested Ubc13 as a potential therapeutic target for intervention in various human diseases including several different cancers, alleviation of anti-cancer drug resistance, chronic inflammation, and viral infections. Understanding a potential therapeutic target from different angles is important to assess its usefulness and potential pitfalls. Here we present a global review of Ubc13 from its structure, function, and cellular activities, to its natural and chemical inhibition. The aim of this article is to review the literature that directly implicates Ubc13 in a biological function, and to integrate structural and mechanistic insights into the larger role of this critical E2 enzyme. We discuss observations of multiple Ubc13 structures that suggest a novel mechanism for activation of Ubc13 that involves conformational change of the active site loop. PMID:27486774

  14. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

    PubMed Central

    Chowdary, D R; Dermody, J J; Jha, K K; Ozer, H L

    1994-01-01

    The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway. Images PMID:8114731

  15. Identification of TRIM22 as a RING finger E3 ubiquitin ligase

    SciTech Connect

    Duan Zhijian; Gao Bo; Xu Wei; Xiong Sidong

    2008-09-26

    TRIM22, a member of the TRIM family proteins which contain RING finger, B-box, and coiled-coil domains, has been reported as a transcriptional regulator and involved in various cellular processes. In this study, the E3 ubiquitin ligase activity, a novel property of TRIM22, was demonstrated. It was found that TRIM22 underwent self-ubiquitylation in vitro in combination with the E2 enzyme UbcH5B and the ubiquitylation was dependent on its RING finger domain. Further evidences showed that TRIM22 could also be self-ubiquitylated in vivo. Importantly, TRIM22 was conjugated with poly-ubiquitin chains and stabilized by the proteasome inhibitor in 293T cells, suggesting that TRIM22 targeted itself for proteasomal degradation through the poly-ubiquitylation. We also found that TRIM22 was located in the nucleus, indicating that TRIM22 might function as a nuclear E3 ubiquitin ligase.

  16. Human Liver Cytochrome P450 3A4 Ubiquitination

    PubMed Central

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J.; Liu, Yi; Burlingame, A. L.; Correia, Maria Almira

    2015-01-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  17. The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

    PubMed Central

    Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

    2009-01-01

    Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

  18. Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins.

    PubMed

    Teo, Hsiangling; Veprintsev, Dmitry B; Williams, Roger L

    2004-07-02

    The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.

  19. The unique Morgue ubiquitination protein is conserved in a diverse but restricted set of invertebrates.

    PubMed

    Zhou, Ying; Carpenter, Zachary W; Brennan, Gregory; Nambu, John R

    2009-10-01

    Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways.

  20. Twists and turns in ubiquitin-like protein conjugation cascades†

    PubMed Central

    Schulman, Brenda A

    2011-01-01

    Post-translational modification by ubiquitin-like proteins (UBLs) is a predominant eukaryotic regulatory mechanism. The vast reach of this form of regulation extends to virtually all eukaryotic processes that involve proteins. UBL modifications play critical roles in controlling the cell cycle, transcription, DNA repair, stress responses, signaling, immunity, plant growth, embryogenesis, circadian rhythms, and a plethora of other pathways. UBLs dynamically modulate target protein properties including enzymatic activity, conformation, half-life, subcellular localization, and intermolecular interactions. Moreover, the enzymatic process of UBL ligation to proteins is itself dynamic, with the UBL moving between multiple enzyme active sites and ultimately to a target. This review highlights our work on how the dynamic conformations of selected enzymes catalyzing UBL ligation help mediate this fascinating form of protein regulation. PMID:22012881

  1. Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation

    PubMed Central

    Lindsten, Kristina; de Vrij, Femke M.S.; Verhoef, Lisette G.G.C.; Fischer, David F.; van Leeuwen, Fred W.; Hol, Elly M.; Masucci, Maria G.; Dantuma, Nico P.

    2002-01-01

    Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins. PMID:11980917

  2. Saccharification and hydrolytic enzyme production of alkali pre-treated wheat bran by Trichoderma virens under solid state fermentation.

    PubMed

    El-Shishtawy, Reda M; Mohamed, Saleh A; Asiri, Abdullah M; Gomaa, Abu-Bakr M; Ibrahim, Ibrahim H; Al-Talhi, Hasan A

    2015-05-28

    In continuation of our previously interest in the saccharification of agriculture wastes by Bacillus megatherium in solid state fermentation (SSF), we wish to report an investigation and comparative evaluation among Trichoderma sp. for the saccharification of four alkali-pretreated agricultural residues and production of hydrolytic enzymes, carboxymethyl cellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase) in SSF. The optimization of the physiological conditions of production of hydrolytic enzymes and saccharification content from Trichoderma virens using alkali-pretreated wheat bran was the last goal. The physico-chemical parameters of SSF include incubation time, incubation temperature, moisture content of the substrate, incubation pH, supplementation with carbon and nitrogen sources were optimized. Saccharification of different solid state fermentation sources wheat bran, date's seeds, grass and palm leaves, were tested for the production of fermentable sugar by Trichoderma sp. The maximum production of hydrolytic enzymes CMCase, FPase, PGase and Xylase and saccharification content were obtained on wheat bran. Time course, moisture content, optimum temperature, optimum pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of the hydrolytic enzymes, protein and total carbohydrate of T. virens using alkali pre-treated wheat bran. The maximum production of CMCase, FPase, PGase, Xylase, protein and carbohydrate content was recorded at 72 h of incubation, 50-70 % moisture, temperature 25-35 °C and pH 5. The influence of supplementary carbon and nitrogen sources was studied. While lactose and sucrose enhanced the activity of PGase from 79.2 to 582.9 and 632.6 U/g, starch inhibited all other enzymes. This was confirmed by maximum saccharification content. Among the nitrogen sources, yeast extract and urea enhanced the saccharification content and CMCase, PGase and Xylase. The results of

  3. Alternative ubiquitin activation/conjugation cascades interact with N-end rule ubiquitin ligases to control degradation of RGS proteins.

    PubMed

    Lee, Peter C W; Sowa, Mathew E; Gygi, Steven P; Harper, J Wade

    2011-08-05

    Vertebrates express two enzymes for activation of ubiquitin-UBA1, which is responsible for activation of the vast majority of E2 conjugating enzymes, and UBA6, which uses the dedicated E2, USE1. However, targets and E3s for UBA6-USE1 are unknown. Here, we demonstrate that UBA6-USE1 functions with the UBR1-3 subfamily of N-recognin E3s to degrade the N-end rule substrates RGS4, RGS5, and Arg (R)-GFP. This pathway functions in the cytoplasm in parallel with the UBA1-UBE2A/B-UBR2 cascade, which promotes turnover of nuclear RGS4/5 proteins and an apparently phenotypically distinct pool of cytoplasmic RGS4/5. UBR2 promotes Lys48 (K48)-specific ubiquitin discharge from, and RGS4 ubiquitylation by, both USE1 and UBE2A in vitro. This work provides insight into the machinery employed by the UBA6-USE1 cascade to promote protein turnover and suggests that the UBA6 and UBA1 pathways can function in parallel with the same E3 to degrade the same targets in a spatially distinct manner. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Alternative Ubiquitin Activation/Conjugation Cascades Interact with N-end rule Ubiquitin Ligases to Control Degradation of RGS Proteins

    PubMed Central

    Lee, Peter C.W.; Sowa, Mathew E.; Gygi, Steven P.; Harper, J. Wade

    2011-01-01

    Vertebrates express two enzymes for activation of ubiquitin – UBA1, which is responsible for activation of the vast majority of E2 conjugating enzymes, and UBA6, which uses the dedicated E2, USE1. However, targets and E3s for UBA6-USE1 are unknown. Here, we demonstrate that UBA6-USE1 functions with the UBR1-3 subfamily of N-recognin E3s to degrade the N-end rule substrates RGS4, RGS5, and Arg(R)-GFP. This pathway functions in the cytoplasm in parallel with the UBA1-UBE2A/B-UBR2 cascade, which promotes turnover of nuclear RGS4/5 proteins and an apparently phenotypically distinct pool of cytoplasmic RGS4/5. UBR2 promotes Lys48 (K48)-specific ubiquitin discharge from, and RGS4 ubiquitylation by, both USE1 and UBE2A in vitro. This work provides insight into the machinery employed by the UBA6-USE1 cascade to promote protein turnover, and suggests that the UBA6 and UBA1 pathways can function in parallel with the same E3 to degrade the same targets in a spatially distinct manner. PMID:21816346

  5. Control of BACE1 degradation and APP processing by ubiquitin carboxyl-terminal hydrolase L1.

    PubMed

    Zhang, Mingming; Deng, Yu; Luo, Yawen; Zhang, Shuting; Zou, Haiyan; Cai, Fang; Wada, Keiji; Song, Weihong

    2012-03-01

    Deposition of amyloid β protein (Aβ) in the brain is the hallmark of Alzheimer's disease (AD) pathogenesis. Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the β-secretase in vivo essential for generation of Aβ. Previously we demonstrated that BACE1 is ubiquitinated and the degradation of BACE1 is mediated by the ubiquitin-proteasome pathway (UPP). However the mechanism underlying regulation of BACE1 degradation by UPP remains elusive. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme highly specific to neuron, catalyzing the hydrolysis of ubiquitin conjugates from ubiquitinated substrates. UCHL1 regulates ubiquitin-dependent protein degradation. However, whether UCHL1 is particularly involved in the proteasomal degradation of BACE1 and what is the role of UCHL1 in AD pathogenesis remain elusive. To investigate the effect of UCHL1 on BACE1 degradation, HUCH cells, a UCHL1 stably over-expressed HEK293 cell line, was established. We found that inhibition of UCHL1 significantly increased BACE1 protein level in a time-dependent manner. Half life of BACE1 was reduced in HUCH cells compared with HEK. Over-expression of UCHL1 decreased APP C-terminal fragment C99 and Aβ levels in HUCH cells. Moreover, disruption of Uchl1 gene significantly elevated levels of endogenous BACE1, C99 and Aβ in the Uchl1-null gad mice. These results demonstrated that UCHL1 accelerates BACE1 degradation and affects APP processing and Aβ production. This study suggests that potentiation of UCHL1 might be able to reduce the level of BACE1 and Aβ in brain, which makes it a novel target for AD drug development.

  6. Atrophy, hypertrophy, and hypoxemia induce transcriptional regulators of the ubiquitin proteasome system in the rat heart.

    PubMed

    Razeghi, Peter; Baskin, Kedryn K; Sharma, Saumya; Young, Martin E; Stepkowski, Stanislaw; Essop, M Faadiel; Taegtmeyer, Heinrich

    2006-04-07

    In skeletal muscle, transcript levels of proteins regulating the ubiquitin proteasome system (UPS) increase with atrophy and decrease with hypertrophy. Whether the same is true for heart muscle is not known. We set out to characterize the transcriptional profile of regulators of the UPS during atrophy-, hypertrophy-, and hypoxia-induced remodeling of the heart. Cardiac atrophy was induced by heterotopic transplantation of the rat heart. Left ventricular hypertrophy was induced by banding of the ascending aorta in rats. To study the effects of hypoxemia on the left ventricle, rats were exposed to hypobaric hypoxia. Transcript levels of six known regulators of the UPS, ubiquitin B (UbB), the ubiquitin conjugating enzymes UbcH2 and E2-14kDa, the ubiquitin ligases Mafbx/Atrogin-1 and MuRF-1, and the proteasomal subunit PSMB4 were measured using quantitative RT-PCR. Unloading-induced atrophy increased mRNA levels of UbB and decreased levels of both ubiquitin ligases. Transcript levels of all UPS genes investigated increased in the hypertrophied and hypoxic heart (with the exception of E2-14kDa). Cardiac atrophy, hypertrophy, and hypoxemia all increase myocardial UbB expression, suggesting that UbB is a transcriptional marker for load-induced and hypoxia-mediated cardiac remodeling.

  7. E3Miner: a text mining tool for ubiquitin-protein ligases.

    PubMed

    Lee, Hodong; Yi, Gwan-Su; Park, Jong C

    2008-07-01

    Ubiquitination is a regulatory process critically involved in the degradation of >80% of cellular proteins, where such proteins are specifically recognized by a key enzyme, or a ubiquitin-protein ligase (E3). Because of this important role of E3s, a rapidly growing body of the published literature in biology and biomedical fields reports novel findings about various E3s and their molecular mechanisms. However, such findings are neither adequately retrieved by general text-mining tools nor systematically made available by such protein databases as UniProt alone. E3Miner is a web-based text mining tool that extracts and organizes comprehensive knowledge about E3s from the abstracts of journal articles and the relevant databases, supporting users to have a good grasp of E3s and their related information easily from the available text. The tool analyzes text sentences to identify protein names for E3s, to narrow down target substrates and other ubiquitin-transferring proteins in E3-specific ubiquitination pathways and to extract molecular features of E3s during ubiquitination. E3Miner also retrieves E3 data about protein functions, other E3-interacting partners and E3-related human diseases from the protein databases, in order to help facilitate further investigation. E3Miner is freely available through http://e3miner.biopathway.org.

  8. Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2.

    PubMed

    Yamanaka, Koji; Ishikawa, Haruto; Megumi, Yuzuru; Tokunaga, Fuminori; Kanie, Masato; Rouault, Tracey A; Morishima, Isao; Minato, Nagahiro; Ishimori, Koichiro; Iwai, Kazuhiro

    2003-04-01

    The ubiquitin system is involved in several basic cellular functions. Ubiquitination is carried out by a cascade of three reactions catalysed by the E1, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs. Although RING finger proteins constitute an important family of E3 ligases, only a few post-transcriptional modifications, including phosphorylation, proline hydroxylation and glycosylation, are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation. Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.

  9. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    PubMed

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  10. Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) delays Alzheimer's progression in vivo.

    PubMed

    Zhang, Mingming; Cai, Fang; Zhang, Shuting; Zhang, Si; Song, Weihong

    2014-12-03

    Deposition of amyloid β protein (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aβ is produced by β- and γ-cleavages of amyloid β precursor protein (APP). Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme that cleaves ubiquitin at its carboxyl terminal. Dysfunction of UCHL1 has been reported in neurodegenerative diseases. However, whether UCHL1 affects Aβ production and AD progression remains unknown. Here we report that UCHL1 interacts with APP and regulates Aβ production. UCHL1 increases free ubiquitin level and accelerates the lysosomal degradation of APP by promoting its ubiquitination. Furthermore, we demonstrate that overexpression of UCHL1 by intracranial injection of UCHL1-expressing rAAV reduces Aβ production, inhibits neuritic plaque formation and improves memory deficits in AD transgenic model mice. Our study suggests that UCHL1 may delay Alzheimer's progression by regulating APP degradation in a long-term fashion, and that overexpression of UCHL1 may be a safe and effective disease-modifying strategy to treat AD.

  11. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.

    PubMed

    Zhuang, Min; Guan, Shenheng; Wang, Haopeng; Burlingame, Alma L; Wells, James A

    2013-01-24

    Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein comp