The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Benjamin W.; Barber, Kathryn R.; Shilton, Brian H.

2015-03-23

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show thatmore » both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.« less

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

PubMed

Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

2018-01-15

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability*

PubMed Central

Xiao, Zhenyu; Chang, Jer-Gung; Hendriks, Ivo A.; Sigurðsson, Jón Otti; Olsen, Jesper V.; Vertegaal, Alfred C.O.

2015-01-01

Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the

Mutant Potential Ubiquitination Sites in Dur3p Enhance the Urea and Ethyl Carbamate Reduction in a Model Rice Wine System.

PubMed

Zhang, Peng; Du, Guocheng; Zou, Huijun; Xie, Guangfa; Chen, Jian; Shi, Zhongping; Zhou, Jingwen

2017-03-01

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3p K556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract-peptone-dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources.

Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

PubMed Central

Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

2016-01-01

Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

Acceptor Ionization Energies in GaN*

NASA Astrophysics Data System (ADS)

Wang, Hao; Ban Chen, An

2001-03-01

The k.p Hamiltonian and a model potential are used to deduce the acceptor ionization energies in GaN from a systematic study of the chemical trend in GaAs, GaP, and InP. The acceptors studied include Be, Mg, Ca, Zn, and Cd on the cation sites and C, Si, and Ge on the anion sites. Our calculated acceptor ionization energies are estimated to be accurate to better than ten percent across the board. The ionization energies of C and Be (152 and 187 meV respectively) in wurtzite GaN are found to be lower than that of Mg (224 meV). The C was found to behave like the hydrogenic acceptor in all systems and it has the smallest ionization energy among all the acceptors studied.

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

PubMed Central

Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

2015-01-01

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

Lysine ubiquitination and acetylation of human cardiac 20S proteasomes.

PubMed

Zong, Nobel; Ping, Peipei; Lau, Edward; Choi, Howard Jh; Ng, Dominic Cm; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie Py

2014-08-01

Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.

2011-03-15

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

Ubiquitinated Proteome: Ready for Global?*

PubMed Central

Shi, Yi; Xu, Ping; Qin, Jun

2011-01-01

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms. PMID:21339389

Calculations of acceptor ionization energies in GaN

NASA Astrophysics Data System (ADS)

Wang, H.; Chen, A.-B.

2001-03-01

The k.p Hamiltonian and a model potential are used to deduce the acceptor ionization energies in GaN from a systematic study of the chemical trend in GaAs, GaP, and InP. The acceptors studied include Be, Mg, Ca, Zn, and Cd on the cation sites and C, Si, and Ge on the anion sites. Our calculated acceptor ionization energies are estimated to be accurate to better than 10% across the board. The ionization energies of C and Be (152 and 187 meV, respectively) in wurtzite GaN are found to be lower than that of Mg (224 meV). The C was found to behave like the hydrogenic acceptor in all systems and it has the smallest ionization energy among all the acceptors studied.

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

PubMed Central

Smit, Judith J; Monteferrario, Davide; Noordermeer, Sylvie M; van Dijk, Willem J; van der Reijden, Bert A; Sixma, Titia K

2012-01-01

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains. PMID:22863777

Mass spectrometry techniques for studying the ubiquitin system.

PubMed

Heap, Rachel E; Gant, Megan S; Lamoliatte, Frederic; Peltier, Julien; Trost, Matthias

2017-10-15

Post-translational control of proteins through covalent attachment of ubiquitin plays important roles in all eukaryotic cell functions. The ubiquitin system in humans consists of 2 E1, 35 E2 and >600 E3 ubiquitin ligases as well as hundreds of deubiquitylases, which reverse ubiquitin attachment. Moreover, there are hundreds of proteins with ubiquitin-binding domains that bind one of the eight possible polyubiquitin chains. Dysfunction of the ubiquitin system is associated with many diseases such as cancer, autoimmunity and neurodegeneration, demonstrating the importance of ubiquitylation. Therefore, enzymes of the ubiquitin system are considered highly attractive drug targets. In recent years, mass spectrometry (MS)-based techniques have become increasingly important in the deciphering of the ubiquitin system. This short review addresses the state-of-the-art MS techniques for the identification of ubiquitylated proteins and their ubiquitylation sites. We also discuss the identification and quantitation of ubiquitin chain topologies and highlight how the activity of enzymes in the ubiquitin pathway can be measured. Finally, we present current MS tools that can be used for drug discovery in the ubiquitin space. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

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

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

PubMed

Nguyen, Thanh; Ho, Minh; Ghosh, Ambarnil; Kim, Truc; Yun, Sun Il; Lee, Seung Seo; Kim, Kyeong Kyu

2016-10-07

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Site preference of Mg acceptors and improvement of p-type doping efficiency in nitride alloys.

PubMed

Park, Ji-Sang; Chang, K J

2013-06-19

We perform first-principles density functional calculations to investigate the effect of Al and In on the formation energy and acceptor level of Mg in group-III nitride alloys. Our calculations reveal a tendency for the Mg dopants to prefer to occupy the lattice sites surrounded with Al atoms, whereas hole carriers are generated in In- or Ga-rich sites. The separation of the Mg dopants and hole carriers is energetically more favourable than a random distribution of dopants, being attributed to the local bonding effect of weak In and strong Al potentials in alloys. As a consequence, the Mg acceptor level, which represents the activation energy of Mg, tends to decrease with increasing numbers of Al next-nearest neighbours, whereas it increases as the number of In next-nearest neighbours increases. Based on the results, we suggest that the incorporation of higher Al and lower In compositions will improve the p-type doping efficiency in quaternary alloys, in comparison with GaN or AlGaN ternary alloys with similar band gaps.

Site preference of Mg acceptors and improvement of p-type doping efficiency in nitride alloys

NASA Astrophysics Data System (ADS)

Park, Ji-Sang; Chang, K. J.

2013-06-01

We perform first-principles density functional calculations to investigate the effect of Al and In on the formation energy and acceptor level of Mg in group-III nitride alloys. Our calculations reveal a tendency for the Mg dopants to prefer to occupy the lattice sites surrounded with Al atoms, whereas hole carriers are generated in In- or Ga-rich sites. The separation of the Mg dopants and hole carriers is energetically more favourable than a random distribution of dopants, being attributed to the local bonding effect of weak In and strong Al potentials in alloys. As a consequence, the Mg acceptor level, which represents the activation energy of Mg, tends to decrease with increasing numbers of Al next-nearest neighbours, whereas it increases as the number of In next-nearest neighbours increases. Based on the results, we suggest that the incorporation of higher Al and lower In compositions will improve the p-type doping efficiency in quaternary alloys, in comparison with GaN or AlGaN ternary alloys with similar band gaps.

Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

PubMed Central

Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

2013-01-01

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.

PubMed

Wilkie-Grantham, Rachel P; Matsuzawa, Shu-Ichi; Reed, John C

2013-05-03

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

PubMed

Wright, Joshua D; Mace, Peter D; Day, Catherine L

2016-11-01

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthetic and semi-synthetic strategies to study ubiquitin signaling.

PubMed

van Tilburg, Gabriëlle Ba; Elhebieshy, Angela F; Ovaa, Huib

2016-06-01

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself. Copyright © 2016 Elsevier Ltd. All rights reserved.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.

PubMed

Shoji, Shisako; Hanada, Kazuharu; Ohsawa, Noboru; Shirouzu, Mikako

2017-09-07

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation

PubMed Central

Hanada, Kazuharu; Ohsawa, Noboru

2017-01-01

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. PMID:28768733

New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1

PubMed Central

Mora, Mireia; Hanzu, Felicia A.; Pradas-Juni, Marta; Aranda, Gloria B.; Halperin, Irene; Puig-Domingo, Manuel; Aguiló, Sira; Fernández-Rebollo, Eduardo

2014-01-01

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases. PMID:24988226

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1▿

PubMed Central

Ning, Shunbin; Campos, Alex D.; Darnay, Bryant G.; Bentz, Gretchen L.; Pagano, Joseph S.

2008-01-01

We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6−/− mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IκB kinase ɛ. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor. PMID:18710948

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. Copyright © 2013 Elsevier B.V. All rights reserved.

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy.

PubMed

Kwon, Yong Tae; Ciechanover, Aaron

2017-11-01

The conjugation of the 76 amino acid protein ubiquitin to other proteins can alter the metabolic stability or non-proteolytic functions of the substrate. Once attached to a substrate (monoubiquitination), ubiquitin can itself be ubiquitinated on any of its seven lysine (Lys) residues or its N-terminal methionine (Met1). A single ubiquitin polymer may contain mixed linkages and/or two or more branches. In addition, ubiquitin can be conjugated with ubiquitin-like modifiers such as SUMO or small molecules such as phosphate. The diverse ways to assemble ubiquitin chains provide countless means to modulate biological processes. We overview here the complexity of the ubiquitin code, with an emphasis on the emerging role of linkage-specific degradation signals (degrons) in the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (hereafter autophagy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Phospho-ubiquitin: upending the PINK–Parkin–ubiquitin cascade

PubMed Central

Matsuda, Noriyuki

2016-01-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson’s disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1–Parkin–ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. PMID:26839319

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

PubMed Central

Lin, Yu-Chen; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. PMID:28536288

Activation of duck RIG-I by TRIM25 is independent of anchored ubiquitin.

PubMed

Miranzo-Navarro, Domingo; Magor, Katharine E

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

PubMed Central

Miranzo-Navarro, Domingo; Magor, Katharine E.

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

PubMed

Matsuda, Noriyuki

2016-04-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

In silico modeling of the cryptic E2∼ubiquitin-binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.

PubMed

Ronchi, Virginia P; Kim, Elizabeth D; Summa, Christopher M; Klein, Jennifer M; Haas, Arthur L

2017-11-03

To understand the mechanism for assembly of Lys 48 -linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys 820 ∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model in silico UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys 820 nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in trans was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys 820 before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys 820 -linked 125 I-polyubiquitin thioester intermediate. Other studies suggest that Glu 550 serves as a general base to generate the Cys 820 thiolate within the low dielectric binding interface and Arg 506 functions to orient Glu 550 and to stabilize the incipient anionic transition state during thioester exchange. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

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 inhibitedmore » 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.« less

A Perturbed Ubiquitin Landscape Distinguishes Between Ubiquitin in Trafficking and in Proteolysis*

PubMed Central

Ziv, Inbal; Matiuhin, Yulia; Kirkpatrick, Donald S.; Erpapazoglou, Zoi; Leon, Sebastien; Pantazopoulou, Marina; Kim, Woong; Gygi, Steven P.; Haguenauer-Tsapis, Rosine; Reis, Noa; Glickman, Michael H.; Kleifeld, Oded

2011-01-01

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. PMID:21427232

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misaghi, S.; Galardy, P.J.; Meester, W.J.

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 structuremore » 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.« less

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

PubMed

Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

2016-02-01

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

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

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

PubMed Central

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

2014-01-01

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

Crystal Structure of a Ube2S-Ubiquitin Conjugate

PubMed Central

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

2016-01-01

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

Ubiquitin phosphorylated at Ser57 hyper-activates parkin.

PubMed

George, Susanna; Wang, Sabrina M; Bi, Yumin; Treidlinger, Margot; Barber, Kathryn R; Shaw, Gary S; O'Donoghue, Patrick

2017-11-01

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUb S65 ). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUb S65 -stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUb S12 . Strikingly, pUb S57 hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUb S57 chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUb S65 and more efficiently with pUb S57 chains. Parkin hyper-activation by pUb S57 demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

PubMed

Schotte, A; Rostène, W; Laduron, P M

1988-04-01

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

Ubiquitin-dependent Regulation of Phospho-AKT Dynamics by the Ubiquitin E3 Ligase, NEDD4-1, in the Insulin-like Growth Factor-1 Response*

PubMed Central

Fan, Chuan-Dong; Lum, Michelle A.; Xu, Chao; Black, Jennifer D.; Wang, Xinjiang

2013-01-01

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner. PMID:23195959

Human Papillomavirus Type 16 E6 Induces Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase

PubMed Central

Kao, Wynn H.; Beaudenon, Sylvie L.; Talis, Andrea L.; Huibregtse, Jon M.; Howley, Peter M.

2000-01-01

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells. PMID:10864652

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

Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin

PubMed Central

Lee, Sora; Tumolo, Jessica M; Ehlinger, Aaron C; Jernigan, Kristin K; Qualls-Histed, Susan J; Hsu, Pi-Chiang; McDonald, W Hayes; Chazin, Walter J

2017-01-01

Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole. PMID:29130884

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Artavanis-Tsakonas, Katerina; Weihofen, Wilhelm A.; Antos, John M.

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 themore » 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.« less

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-08-23

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

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence.

PubMed

Kim, Dong Seon; Hahn, Yoonsoo

2012-11-13

Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.

PubMed

Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y; Distl, Ottmar

2015-01-01

Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation

PubMed Central

Han, Cong; Pao, Kuan-Chuan; Kazlauskaite, Agne; Muqit, Miratul M K; Virdee, Satpal

2015-01-01

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation. PMID:26010437

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2011-05-10

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

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

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses.

PubMed

Sun, Xiaoqiang; Xian, Huifang; Tian, Shuo; Sun, Tingzhe; Qin, Yunfei; Zhang, Shoutao; Cui, Jun

2016-07-08

RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.

A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

PubMed Central

Sun, Xiaoqiang; Xian, Huifang; Tian, Shuo; Sun, Tingzhe; Qin, Yunfei; Zhang, Shoutao; Cui, Jun

2016-01-01

RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses. PMID:27387525

A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

NASA Astrophysics Data System (ADS)

Sun, Xiaoqiang; Xian, Huifang; Tian, Shuo; Sun, Tingzhe; Qin, Yunfei; Zhang, Shoutao; Cui, Jun

2016-07-01

RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

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

PubMed

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

2016-08-17

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The emerging complexity of ubiquitin architecture.

PubMed

Ohtake, Fumiaki; Tsuchiya, Hikaru

2017-02-01

Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

PubMed

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

2009-04-17

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

Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell-mimicking crowded solution.

PubMed

Munari, Francesca; Bortot, Andrea; Zanzoni, Serena; D'Onofrio, Mariapina; Fushman, David; Assfalg, Michael

2017-04-01

Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on the formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity toward the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and the ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites. © 2017 Federation of European Biochemical Societies.

How localized acceptors limit p-type conductivity in GaN

NASA Astrophysics Data System (ADS)

Lyons, John L.

2013-03-01

Despite the impressive development of GaN as an optoelectronic material, p-type conductivity is still limited. Only a single acceptor impurity, magnesium, is known to lead to p-type GaN. But Mg is far from a well-behaved acceptor. Hydrogen is known to passivate Mg, necessitating a post-growth anneal for acceptor activation. In addition, the ionization energy is quite large (~ 200 meV in GaN), meaning only a few percent of Mg acceptors are ionized at room temperature. Thus, hole conductivity is limited, and high concentrations of Mg are required to achieve moderately p-type GaN. Other acceptor impurities have not proven to be effective p-type dopants, for reasons that are still unresolved. Using advanced first-principles calculations based on a hybrid functional, we investigate the electrical and optical properties of the isolated Mg acceptor and its complexes with hydrogen in GaN, InN, and AlN.[2] We employ a technique that overcomes the band-gap-problem of traditional density functional theory, and allows for quantitative predictions of acceptor ionization energies and optical transition energies. Our results allow us to explain the deep or shallow nature of the Mg acceptor and its relation to the optical signals observed in Mg-doped GaN. We also revisit the properties of other group-II acceptors in GaN. We find that all cation-site acceptors show behavior similar to MgGa, and lead to highly localized holes. The ZnGa and BeGa acceptors have ionization energies that are even larger than that of Mg, making them ineffective dopants. All acceptors cause large lattice distortions in their neutral charge state, in turn leading to deep, broad luminescence signals that can serve as a means of experimentally verifying the deep nature of these acceptors. This work was performed in collaboration with Audrius Alkauskas, Anderson Janotti, and Chris G. Van de Walle. It was supported by the NSF and by the Solid State Lighting and Energy Center at UCSB.

Ubiquitination in Periodontal Disease: A Review.

PubMed

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

2017-07-10

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.

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

PubMed Central

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

2015-01-01

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

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

PubMed Central

Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights.

PubMed

Hewings, David S; Flygare, John A; Bogyo, Matthew; Wertz, Ingrid E

2017-05-01

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology. © 2017 Federation of European Biochemical Societies.

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

PubMed Central

2012-01-01

Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution. PMID:23148531

Difference between resistance degradation of fixed valence acceptor (Mg) and variable valence acceptor (Mn)-doped BaTiO3 ceramics

NASA Astrophysics Data System (ADS)

Yoon, Seok-Hyun; Randall, Clive A.; Hur, Kang-Heon

2010-09-01

The difference in the resistance degradation behavior was investigated between fixed valence acceptor (Mg) and the variable valence acceptor (Mn)-doped BaTiO3 ceramics with an increase of each acceptor concentration. Coarse-grained specimens with uniform grain sizes and different acceptor concentrations were prepared. In the case of Mg-doped BaTiO3, the time to degradation systematically decreased with the increase in Mg concentration. In contrast, there is a systematically increased time to degradation with the increase in Mn concentration in Mn-doped BaTiO3. The fast degradation by the increase in Mg concentration directly corresponded to an increase in the Warburg impedance and ionic transference number (tion) associated with an increase in oxygen vacancy concentration ([VO••]). On the other hand, no distinct Warburg impedance or ionic conduction contribution could be observed with the increase in Mn concentration. It is supposed that the increase in [VO••] is negligible in spite of the increase in acceptor Mn concentration, when it is compared to Mg-doped BaTiO3. The much lower [VO••] and more dominant electron/hole trapping effect due to multivalence nature of Mn are supposed to cause such a contrary degradation behavior between Mg and Mn-doped BaTiO3. Reoxidation in a slightly reducing atmosphere (N2) showed better resistance to degradation behavior than in a oxidizing air atmosphere in both Mg and Mn-doped BaTiO3, which is anticipated to be an increase in the electron/hole trapping sites. All these behaviors could be explained by the low temperature defect chemical model that shows difference in the defect structure between Mg and Mn-doped BaTiO3, and its dependence on the oxygen partial pressure (pO2) during reoxidation and cooling. Not only the [VO••], but also the density of electron/hole trap sites, are believed to be crucial in controlling resistance degradation.

Donor assists acceptor binding and catalysis of human α1,6-fucosyltransferase.

PubMed

Kötzler, Miriam P; Blank, Simon; Bantleon, Frank I; Wienke, Martin; Spillner, Edzard; Meyer, Bernd

2013-08-16

α1,6-Core-fucosyltransferase (FUT8) is a vital enzyme in mammalian physiological and pathophysiological processes such as tumorigenesis and progress of, among others, non-small cell lung cancer and colon carcinoma. It was also shown that therapeutic antibodies have a dramatically higher efficacy if the α1,6-fucosyl residue is absent. However, specific and potent inhibitors for FUT8 and related enzymes are lacking. Hence, it is crucial to elucidate the structural basis of acceptor binding and the catalytic mechanism. We present here the first structural model of FUT8 in complex with its acceptor and donor molecules. An unusually large acceptor, i.e., a hexasaccharide from the core of N-glycans, is required as minimal structure. Acceptor substrate binding of FUT8 is being dissected experimentally by STD NMR and SPR and theoretically by molecular dynamics simulations. The acceptor binding site forms an unusually large and shallow binding site. Binding of the acceptor to the enzyme is much faster and stronger if the donor is present. This is due to strong hydrogen bonding between O6 of the proximal N-acetylglucosamine and an oxygen atom of the β-phosphate of GDP-fucose. Therefore, we propose an ordered Bi Bi mechanism for FUT8 where the donor molecule binds first. No specific amino acid is present that could act as base during catalysis. Our results indicate a donor-assisted mechanism, where an oxygen of the β-phosphate deprotonates the acceptor. Knowledge of the mechanism of FUT8 is now being used for rational design of targeted inhibitors to address metastasis and prognosis of carcinomas.

Ubiquitin in health and disease.

PubMed

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

1991-06-13

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

Theory for electron transfer from a mixed-valence dimer with paramagnetic sites to a mononuclear acceptor

NASA Astrophysics Data System (ADS)

Bominaar, E. L.; Achim, C.; Borshch, S. A.

1999-06-01

Polynuclear transition-metal complexes, such as Fe-S clusters, are the prosthetic groups in a large number of metalloproteins and serve as temporary electron storage units in a number of important redox-based biological processes. Polynuclearity distinguishes clusters from mononuclear centers and confers upon them unique properties, such as spin ordering and the presence of thermally accessible excited spin states in clusters with paramagnetic sites, and fractional valencies in clusters of the mixed-valence type. In an earlier study we presented an effective-mode (EM) analysis of electron transfer from a binuclear mixed-valence donor with paramagnetic sites to a mononuclear acceptor which revealed that the cluster-specific attributes have an important impact on the kinetics of long-range electron transfer. In the present study, the validity of these results is tested in the framework of more detailed theories which we have termed the multimode semiclassical (SC) model and the quantum-mechanical (QM) model. It is found that the qualitative trends in the rate constant are the same in all treatments and that the semiclassical models provide a good approximation of the more rigorous quantum-mechanical description of electron transfer under physiologically relevant conditions. In particular, the present results corroborate the importance of electron transfer via excited spin states in reactions with a low driving force and justify the use of semiclassical theory in cases in which the QM model is computationally too demanding. We consider cases in which either one or two donor sites of a dimer are electronically coupled to the acceptor. In the case of multiconnectivity, the rate constant for electron transfer from a valence-delocalized (class-III) donor is nonadditive with respect to transfer from individual metal sites of the donor and undergoes an order-of-magnitude change by reversing the sign of the intradimer metal-metal resonance parameter (β). In the case of

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

PubMed

Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

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

PubMed

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

2015-10-14

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

Vaccine perception among acceptors and non-acceptors in Sokoto State, Nigeria.

PubMed

Murele, Bola; Vaz, Rui; Gasasira, Alex; Mkanda, Pascal; Erbeto, Tesfaye; Okeibunor, Joseph

2014-05-30

Vaccine perceptions among acceptors and non-acceptors of childhood vaccination were explored. Seventy-two care givers, among them, acceptors and non-acceptors were interviewed in-depth with an interview guide that assessed vaccine acceptance, social and personality factors, and health belief model (HBM) categories in relation to oral polio vaccine (perceived susceptibility, severity, cost barriers, general barriers, benefits, knowledge, and engagement in preventative health behaviours). Community leaders were purposively selected while parents were selected on the basis of availability while ensuring the different attitude to vaccines was covered. Results showed that the HBM framework was found to be appropriate for identifying and distinguishing vaccine acceptors and non-acceptors. In addition, the HBM categories of benefits and susceptibility were found to influence oral polio vaccine acceptance. Second, the opinion of family members about the oral polio vaccine moderated the relationship between number of social ties and vaccine acceptance. Further, oral polio vaccine acceptance was related to outbreaks of paralysis of any sort, but not aggregate scores of other preventative health behaviours. Implications of this study include the investigation of vaccine acceptance in a high risk population. Research was done to investigate vaccine acceptance. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Diggin’ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

PubMed Central

Rubel, Carrie E.; Schisler, Jonathan C.; Hamlett, Eric D.; DeKroon, Robert M.; Gautel, Mathias; Alzate, Oscar; Patterson, Cam

2013-01-01

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets. PMID:23695782

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

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

PubMed

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.

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

PubMed Central

Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

2015-01-01

SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin*

PubMed Central

Isogai, Shin; Morimoto, Daichi; Arita, Kyohei; Unzai, Satoru; Tenno, Takeshi; Hasegawa, Jun; Sou, Yu-shin; Komatsu, Masaaki; Tanaka, Keiji; Shirakawa, Masahiro; Tochio, Hidehito

2011-01-01

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62. PMID:21715324

Ubiquitin enzymes in the regulation of immune responses.

PubMed

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

2017-08-01

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.

Targeting ubiquitination for cancer therapies.

PubMed

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.

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.

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

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

PubMed Central

Stringer, Daniel K.

2011-01-01

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination. PMID:21242292

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

PubMed

Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

2017-03-15

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

Dynamic survey of mitochondria by ubiquitin

PubMed Central

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

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. PMID:24569520

Structural Basis for the Interaction of Mutasome Assembly Factor REV1 with Ubiquitin.

PubMed

Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges

2018-05-18

REV1 is an evolutionarily conserved translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen is thought to be the trigger for TLS activation. Here we report the solution NMR structure of a 108-residue fragment of human REV1 encompassing the two putative ubiquitin-binding motifs UBM1 and UBM2 in complex with ubiquitin. While in mammals UBM1 and UBM2 are both required for optimal association of REV1 with replication factories after DNA damage, we show that only REV1 UBM2 binds ubiquitin. Structure-guided mutagenesis in Saccharomyces cerevisiae further highlights the importance of UBM2 for REV1-mediated mutagenesis and DNA damage tolerance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Ubiquitin--conserved protein or selfish gene?

PubMed

Catic, André; Ploegh, Hidde L

2005-11-01

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

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

Lysine 206 in Arabidopsis phytochrome A is the major site for ubiquitin-dependent protein degradation.

PubMed

Rattanapisit, Kaewta; Cho, Man-Ho; Bhoo, Seong Hee

2016-02-01

Phytochrome A (phyA) is a light labile phytochrome that mediates plant development under red/far-red light condition. Degradation of phyA is initiated by red light-induced phyA-ubiquitin conjugation through the 26S proteasome pathway. The N-terminal of phyA is known to be important in phyA degradation. To determine the specific lysine residues in the N-terminal domain of phyA involved in light-induced ubiquitination and protein degradation, we aligned the amino acid sequence of the N-terminal domain of Arabidopsis phyA with those of phyA from other plant species. Based on the alignment results, phytochrome over-expressing Arabidopsis plants were generated. In particular, wild-type and mutant (substitutions of conserved lysines by arginines) phytochromes fused with GFP were expressed in phyA(-)211 Arabidopsis plants. Degradation kinetics of over-expressed phyA proteins revealed that degradation of the K206R phyA mutant protein was delayed. Delayed phyA degradation of the K206R phyA mutant protein resulted in reduction of red-light-induced phyA-ubiquitin conjugation. Furthermore, seedlings expressing the K206R phyA mutant protein showed an enhanced phyA response under far-red light, resulting in inhibition of hypocotyl elongation as well as cotyledon opening. Together, these results suggest that lysine 206 is the main lysine for rapid ubiquitination and protein degradation of Arabidopsis phytochrome A. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

PubMed Central

Wojtaszek, Jessica L.; Wang, Su; Kim, Hyungjin; Wu, Qinglin; D'Andrea, Alan D.; Zhou, Pei

2014-01-01

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ–ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20–ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20–ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair. PMID:25414354

Ubiquitin acetylation inhibits polyubiquitin chain elongation

PubMed Central

Ohtake, Fumiaki; Saeki, Yasushi; Sakamoto, Kensaku; Ohtake, Kazumasa; Nishikawa, Hiroyuki; Tsuchiya, Hikaru; Ohta, Tomohiko; Tanaka, Keiji; Kanno, Jun

2015-01-01

Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B—which we identify as an endogenous substrate of acetylated ubiquitin—and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono- and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology. PMID:25527407

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Deciphering the Ubiquitin Code.

PubMed

Dittmar, Gunnar; Selbach, Matthias

2017-03-02

In this issue of Molecular Cell, Zhang et al. (2017) systematically identify proteins interacting with all possible di-ubiquitin linkages, thus providing a catalog of readers of the ubiquitin code. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective autophagy: ubiquitin-mediated recognition and beyond.

PubMed

Kraft, Claudine; Peter, Matthias; Hofmann, Kay

2010-09-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

PubMed

Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

2017-06-01

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Electron paramagnetic resonance study of neutral Mg acceptors in β-Ga2O3 crystals

NASA Astrophysics Data System (ADS)

Kananen, B. E.; Halliburton, L. E.; Scherrer, E. M.; Stevens, K. T.; Foundos, G. K.; Chang, K. B.; Giles, N. C.

2017-08-01

Electron paramagnetic resonance (EPR) is used to directly observe and characterize neutral Mg acceptors ( M gGa0 ) in a β-Ga2O3 crystal. These acceptors, best considered as small polarons, are produced when the Mg-doped crystal is irradiated at or near 77 K with x rays. During the irradiation, neutral acceptors are formed when holes are trapped at singly ionized Mg acceptors ( M gGa- ). Unintentionally present Fe3+ (3d5) and Cr3+ (3d3) transition-metal ions serve as the corresponding electron traps. The hole is localized in a nonbonding p orbital on a threefold-coordinated oxygen ion adjacent to an Mg ion at a sixfold-coordinated Ga site. These M gGa0 acceptors (S = 1/2) have a slightly anisotropic g matrix (principal values are 2.0038, 2.0153, and 2.0371). There is also partially resolved 69Ga and 71Ga hyperfine structure resulting from unequal interactions with the two Ga ions adjacent to the hole. With the magnetic field along the a direction, hyperfine parameters are 2.61 and 1.18 mT for the 69Ga nuclei at the two inequivalent neighboring Ga sites. The M gGa0 acceptors thermally convert back to their nonparamagnetic M gGa- charge state when the temperature of the crystal is raised above approximately 250 K.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains.

PubMed

Kniss, Andreas; Schuetz, Denise; Kazemi, Sina; Pluska, Lukas; Spindler, Philipp E; Rogov, Vladimir V; Husnjak, Koraljka; Dikic, Ivan; Güntert, Peter; Sommer, Thomas; Prisner, Thomas F; Dötsch, Volker

2018-02-06

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of ubiquitin/ubiquitin-like protein modification from tandem mass spectra with various PTMs

PubMed Central

2011-01-01

Background Various solutions have been introduced for the identification of post-translational modification (PTM) from tandem mass spectrometry (MS/MS) in proteomics field but the identification of peptide modifiers, such as Ubiquitin (Ub) and ubiquitin-like proteins (Ubls), is still a challenge. The fragmentation of peptide modifier produce complex shifted ion mass patterns in combination with other PTMs, which makes it difficult to identify and locate the PTMs on a protein sequence. Currently, most PTM identification methods do not consider the complex fragmentation of peptide modifier or deals it separately from the other PTMs. Results We developed an advanced PTM identification method that inspects possible ion patterns of the most known peptide modifiers as well as other known biological and chemical PTMs to make more comprehensive and accurate conclusion. The proposed method searches all detectable mass differences of measured peaks from their theoretical values and the mass differences within mass tolerance range are grouped as mass shift classes. The most possible locations of multiple PTMs including peptide modifiers can be determined by evaluating all possible scenarios generated by the combination of the qualified mass shift classes.The proposed method showed excellent performance in the test with simulated spectra having various PTMs including peptide modifiers and in the comparison with recently developed methods such as QuickMod and SUMmOn. In the analysis of HUPO Brain Proteome Project (BPP) datasets, the proposed method could find the ubiquitin modification sites that were not identified by other conventional methods. Conclusions This work presents a novel method for identifying bothpeptide modifiers that generate complex fragmentation patternsand PTMs that are not fragmented during fragmentation processfrom tandem mass spectra. PMID:22373085

Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression.

PubMed

Liu, Juan; Zhang, Cen; Zhao, Yuhan; Yue, Xuetian; Wu, Hao; Huang, Shan; Chen, James; Tomsky, Kyle; Xie, Haiyang; Khella, Christen A; Gatza, Michael L; Xia, Dajing; Gao, Jimin; White, Eileen; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

2017-11-28

Mutations in E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Accumulating evidence suggests that Parkin is a tumor suppressor, but the underlying mechanism is poorly understood. Here we show that Parkin is an E3 ubiquitin ligase for hypoxia-inducible factor 1α (HIF-1α). Parkin interacts with HIF-1α and promotes HIF-1α degradation through ubiquitination, which in turn inhibits metastasis of breast cancer cells. Parkin downregulation in breast cancer cells promotes metastasis, which can be inhibited by targeting HIF-1α with RNA interference or the small-molecule inhibitor YC-1. We further identify lysine 477 (K477) of HIF-1α as a major ubiquitination site for Parkin. K477R HIF-1α mutation and specific cancer-associated Parkin mutations largely abolish the functions of Parkin to ubiquitinate HIF-1α and inhibit cancer metastasis. Importantly, Parkin expression is inversely correlated with HIF-1α expression and metastasis in breast cancer. Our results reveal an important mechanism for Parkin in tumor suppression and HIF-1α regulation.

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex.

PubMed

Sánchez-Martín, Pablo; Romá-Mateo, Carlos; Viana, Rosa; Sanz, Pascual

2015-12-01

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

TUBEs-Mass Spectrometry for Identification and Analysis of the Ubiquitin-Proteome.

PubMed

Azkargorta, Mikel; Escobes, Iraide; Elortza, Felix; Matthiesen, Rune; Rodríguez, Manuel S

2016-01-01

Mass spectrometry (MS) has become the method of choice for the large-scale analysis of protein ubiquitylation. There exist a number of proposed methods for mapping ubiquitin sites, each with different pros and cons. We present here a protocol for the MS analysis of the ubiquitin-proteome captured by TUBEs and subsequent data analysis. Using dedicated software and algorithms, specific information on the presence of ubiquitylated peptides can be obtained from the MS search results. In addition, a quantitative and functional analysis of the ubiquitylated proteins and their interacting partners helps to unravel the biological and molecular processes they are involved in.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

Associative Memory Acceptors.

ERIC Educational Resources Information Center

Card, Roger

The properties of an associative memory are examined in this paper from the viewpoint of automata theory. A device called an associative memory acceptor is studied under real-time operation. The family "L" of languages accepted by real-time associative memory acceptors is shown to properly contain the family of languages accepted by one-tape,…

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

PubMed Central

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

2015-01-01

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

Ubiquitin in Influenza Virus Entry and Innate Immunity.

PubMed

Rudnicka, Alina; Yamauchi, Yohei

2016-10-24

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle.

Ubiquitin in Influenza Virus Entry and Innate Immunity

PubMed Central

Rudnicka, Alina; Yamauchi, Yohei

2016-01-01

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle. PMID:27783058

Binding to Syntenin-1 Protein Defines a New Mode of Ubiquitin-based Interactions Regulated by Phosphorylation*

PubMed Central

Rajesh, Sundaresan; Bago, Ružica; Odintsova, Elena; Muratov, Gayrat; Baldwin, Gouri; Sridhar, Pooja; Rajesh, Sandya; Overduin, Michael; Berditchevski, Fedor

2011-01-01

Syntenin-1 is a PDZ domain-containing adaptor that controls trafficking of transmembrane proteins including those associated with tetraspanin-enriched microdomains. We describe the interaction of syntenin-1 with ubiquitin through a novel binding site spanning the C terminus of ubiquitin, centered on Arg72, Leu73, and Arg74. A conserved LYPSL sequence in the N terminus, as well as the C-terminal region of syntenin-1, are essential for binding to ubiquitin. We present evidence for the regulation of this interaction through syntenin-1 dimerization. We have also established that syntenin-1 is phosphorylated downstream of Ulk1, a serine/threonine kinase that plays a critical role in autophagy and regulates endocytic trafficking. Importantly, Ulk1-dependent phosphorylation of Ser6 in the LYPSL prevents the interaction of syntenin-1 with ubiquitin. These results define an unprecedented ubiquitin-dependent pathway involving syntenin-1 that is regulated by Ulk1. PMID:21949238

A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function

PubMed Central

Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R.; Xu, Guoqiang

2015-01-01

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen-Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. PMID:26231201

Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation

USDA-ARS?s Scientific Manuscript database

The accumulation, aggregation, and precipitation of proteins are etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome p...

Activation of the Slx5–Slx8 Ubiquitin Ligase by Poly-small Ubiquitin-like Modifier Conjugates*S⃞

PubMed Central

Mullen, Janet R.; Brill, Steven J.

2008-01-01

Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5–Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5–Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5–Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5–Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5–Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5–Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain. PMID:18499666

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

PubMed

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

PubMed Central

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

2016-01-01

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation. PMID:27685940

The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax

PubMed Central

Fryrear, Kimberly A.; Guo, Xin

2012-01-01

The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)–modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB–mediated and decreased cAMP Response Element-Binding (CREB)–mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage–induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes. PMID:22106342

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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,more » 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.« less

Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

PubMed Central

Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia

2015-01-01

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity. PMID:26286193

Proteostasis regulation by the ubiquitin system.

PubMed

Bett, John S

2016-10-15

Cells have developed an evolutionary obligation to survey and maintain proteome fidelity and avoid the possible toxic consequences of protein misfolding and aggregation. Disturbances to protein homoeostasis (proteostasis) can result in severe cellular phenotypes and are closely linked with the accumulation of microscopically visible deposits of aggregated proteins. These include inclusion bodies found in AD (Alzheimer's disease), HD (Huntington's disease) and ALS (amyotrophic lateral sclerosis) patient neurons. Protein aggregation is intimately linked with the ubiquitin and ubiquitin-like post-translational modifier system, which manages cellular protein folding stress and promotes the restoration of proteostasis. This is achieved in large part through the action of the UPS (ubiquitin-proteasome system), which is responsible for directing the proteasomal destruction of misfolded and damaged proteins tagged with ubiquitin chains. There are other less well understood ways in which ubiquitin family members can help to maintain proteostasis that complement, but are independent of, the UPS. This article discusses our current understanding of how the ubiquitin family regulates the protein misfolding pathways that threaten proteome fidelity, and how this is achieved by the key players in this process. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

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

Enhanced biodegradation of cyclotetramethylenetetranitramine (HMX) under mixed electron-acceptor condition.

PubMed

Boopathy, R

2001-02-01

The biodegradation of cyclotetramethylenetetranitramine, commonly known as 'high melting explosive' (HMX), under various electron-acceptor conditions was investigated using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that the HMX was biodegraded under sulfate reducing, nitrate reducing, fermenting, methanogenic, and mixed electron accepting conditions. However, the rates of degradation varied among the various conditions studied. The fastest removal of HMX (from 22 ppm on day 0 to < 0.05 ppm on day 11) was observed under mixed electron-acceptor conditions, followed in order by sulfate reducing, fermenting, methanogenic, and nitrate reducing conditions. Under aerobic conditions, HMX was not biodegraded, which indicated that HMX degradation takes place under anaerobic conditions via reduction. HMX was converted to methanol and chloroform under mixed electron-acceptor conditions. This study showed evidence for HMX degradation under anaerobic conditions in a mixed microbial population system similar to any contaminated field sites, where a heterogeneous population exists.

Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

PubMed Central

Noor, Natassya M.; Møllgård, Kjeld; Wheaton, Benjamin J.; Steer, David L.; Truettner, Jessie S.; Dziegielewska, Katarzyna M.; Dietrich, W. Dalton; Smith, A. Ian; Saunders, Norman R.

2013-01-01

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets. PMID:23626776

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Acceptor and Excitation Density Dependence of the Ultrafast Polaron Absorption Signal in Donor-Acceptor Organic Solar Cell Blends.

PubMed

Zarrabi, Nasim; Burn, Paul L; Meredith, Paul; Shaw, Paul E

2016-07-21

Transient absorption spectroscopy on organic semiconductor blends for solar cells typically shows efficient charge generation within ∼100 fs, accounting for the majority of the charge carriers. In this Letter, we show using transient absorption spectroscopy on blends containing a broad range of acceptor content (0.01-50% by weight) that the rise of the polaron signal is dependent on the acceptor concentration. For low acceptor content (<10% by weight), the polaron signal rises gradually over ∼1 ps with most polarons generated after 200 fs, while for higher acceptor concentrations (>10%) most polarons are generated within 200 fs. The rise time in blends with low acceptor content was also found to be sensitive to the pump fluence, decreasing with increasing excitation density. These results indicate that the sub-100 fs rise of the polaron signal is a natural consequence of both the high acceptor concentrations in many donor-acceptor blends and the high excitation densities needed for transient absorption spectroscopy, which results in a short average distance between the exciton and the donor-acceptor interface.

Acceptors in ZnO

DOE PAGES

Mccluskey, Matthew D.; Corolewski, Caleb; Lv, Jinpeng; ...

2015-03-21

Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence shows that these point defects have acceptor levels 3.2, 1.5, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO2 contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peakmore » in ZnO nanocrystals has been attributed to an acceptor, which may involve a zinc vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g = 2.0033 and g = 2.0075, along with an isotropic center at g = 2.0053.« less

Ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation

PubMed Central

Lau, Alan F.

2009-01-01

The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. PMID:19468686

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

PubMed Central

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

2015-01-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

PubMed

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

2015-08-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin

Ubiquitin is part of the retrovirus budding machinery

NASA Astrophysics Data System (ADS)

Patnaik, Akash; Chau, Vincent; Wills, John W.

2000-11-01

Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

Mechanism of phospho-ubiquitin induced PARKIN activation

PubMed Central

Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

2016-01-01

Summary The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy1–3. PINK1 is stabilised on the outside of depolarised mitochondria, and phosphorylates poly-ubiquitin (polyUb)4–8 as well as the PARKIN Ub-like (Ubl) domain9,10. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism4–12. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in AR-JP patients. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilisation of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory13 but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens new avenues to identify small molecule PARKIN activators. PMID:26161729

Receptor Tyrosine Kinase Ubiquitination and De-Ubiquitination in Signal Transduction and Receptor Trafficking

PubMed Central

Critchley, William R.; Pellet-Many, Caroline; Ringham-Terry, Benjamin; Zachary, Ian C.; Ponnambalam, Sreenivasan

2018-01-01

Receptor tyrosine kinases (RTKs) are membrane-based sensors that enable rapid communication between cells and their environment. Evidence is now emerging that interdependent regulatory mechanisms, such as membrane trafficking, ubiquitination, proteolysis and gene expression, have substantial effects on RTK signal transduction and cellular responses. Different RTKs exhibit both basal and ligand-stimulated ubiquitination, linked to trafficking through different intracellular compartments including the secretory pathway, plasma membrane, endosomes and lysosomes. The ubiquitin ligase superfamily comprising the E1, E2 and E3 enzymes are increasingly implicated in this post-translational modification by adding mono- and polyubiquitin tags to RTKs. Conversely, removal of these ubiquitin tags by proteases called de-ubiquitinases (DUBs) enables RTK recycling for another round of ligand sensing and signal transduction. The endocytosis of basal and activated RTKs from the plasma membrane is closely linked to controlled proteolysis after trafficking and delivery to late endosomes and lysosomes. Proteolytic RTK fragments can also have the capacity to move to compartments such as the nucleus and regulate gene expression. Such mechanistic diversity now provides new opportunities for modulating RTK-regulated cellular responses in health and disease states. PMID:29543760

Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome.

PubMed

Kattah, Michael G; Malynn, Barbara A; Ma, Averil

2017-11-10

Ubiquitin and ubiquitin-modifying enzymes play critical roles in a wide variety of intracellular signaling pathways. Inflammatory signaling cascades downstream of TNF, TLR agonists, antigen receptor cross-linking, and cytokine receptors, all rely on ubiquitination events to direct subsequent immune responses. In the past several years, inflammasome activation and subsequent signal transduction have emerged as an excellent example of how ubiquitin signals control inflammatory responses. Inflammasomes are multiprotein signaling complexes that ultimately lead to caspase activation and release of the interleukin-1 (IL-1) family members, IL-1β and IL-18. Inflammasome activation is critical for the host's defense against pathogens, but dysregulation of inflammasomes may contribute to the pathogenesis of multiple diseases. Ultimately, understanding how various ubiquitin interacting proteins control inflammatory signaling cascades could provide new pathways for therapeutic intervention. Here we review specific ubiquitin-modifying enzymes and ubiquitination events that orchestrate inflammatory responses, with an emphasis on the NLRP3 inflammasome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pathological Heterogeneity of Frontotemporal Lobar Degeneration with Ubiquitin-Positive Inclusions Delineated by Ubiquitin Immunohistochemistry and Novel Monoclonal Antibodies

PubMed Central

Sampathu, Deepak M.; Neumann, Manuela; Kwong, Linda K.; Chou, Thomas T.; Micsenyi, Matthew; Truax, Adam; Bruce, Jennifer; Grossman, Murray; Trojanowski, John Q.; Lee, Virginia M.-Y.

2006-01-01

Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) is a common neuropathological subtype of frontotemporal dementia. Although this subtype of frontotemporal dementia is defined by the presence of ubiquitin-positive but tau- and α-synuclein-negative inclusions, it is unclear whether all cases of FTLD-U have the same underlying pathogenesis. Examination of tissue sections from FTLD-U brains stained with anti-ubiquitin antibodies revealed heterogeneity in the morphological characteristics of pathological inclusions among subsets of cases. Three types of FTLD-U were delineated based on morphology and distribution of ubiquitin-positive inclusions. To address the hypothesis that FTLD-U is pathologically heterogeneous, novel monoclonal antibodies (mAbs) were generated by immunization of mice with high molecular mass (Mr > 250 kd) insoluble material prepared by biochemical fractionation of FTLD-U brains. Novel mAbs were identified that immunolabeled all of the ubiquitin-positive inclusions in one subset of FTLD-U cases, whereas other mAbs stained the ubiquitin-positive inclusions in a second subset of cases. These novel mAbs did not stain inclusions in other neurodegenerative disorders, including tauopathies and α-synucleinopathies. Therefore, ubiquitin immunohistochemistry and the immunostaining properties of the novel mAbs generated here suggest that FTLD-U is pathologically he-terogeneous. Identification of the disease proteins recognized by these mAbs will further advance understanding of molecular substrates of FTLD-U neurodegenerative pathways. PMID:17003490

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

PubMed

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Chemical ubiquitination for decrypting a cellular code.

PubMed

Stanley, Mathew; Virdee, Satpal

2016-05-15

The modification of proteins with ubiquitin (Ub) is an important regulator of eukaryotic biology and deleterious perturbation of this process is widely linked to the onset of various diseases. The regulatory capacity of the Ub signal is high and, in part, arises from the capability of Ub to be enzymatically polymerised to form polyubiquitin (polyUb) chains of eight different linkage types. These distinct polyUb topologies can then be site-specifically conjugated to substrate proteins to elicit a number of cellular outcomes. Therefore, to further elucidate the biological significance of substrate ubiquitination, methodologies that allow the production of defined polyUb species, and substrate proteins that are site-specifically modified with them, are essential to progress our understanding. Many chemically inspired methods have recently emerged which fulfil many of the criteria necessary for achieving deeper insight into Ub biology. With a view to providing immediate impact in traditional biology research labs, the aim of this review is to provide an overview of the techniques that are available for preparing Ub conjugates and polyUb chains with focus on approaches that use recombinant protein building blocks. These approaches either produce a native isopeptide, or analogue thereof, that can be hydrolysable or non-hydrolysable by deubiquitinases. The most significant biological insights that have already been garnered using such approaches will also be summarized. © 2016 Authors; published by Portland Press Limited.

Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

PubMed

Bouvier, Benjamin

2014-01-07

Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

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

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

PubMed

Aleidi, Shereen M; Yang, Alryel; Sharpe, Laura J; Rao, Geetha; Cochran, Blake J; Rye, Kerry-Anne; Kockx, Maaike; Brown, Andrew J; Gelissen, Ingrid C

2018-04-01

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated. Copyright © 2018 Elsevier B.V. All rights reserved.

Correlation between ubiquitination and defects of bull spermatozoa and removal of defective spermatozoa using anti-ubiquitin antibody-coated magnetized beads.

PubMed

Zhang, Jian; Su, Jie; Hu, Shuxiang; Zhang, Jindun; Ding, Rui; Guo, Jitong; Cao, Guifang; Li, Rongfeng; Sun, Qing-Yuan; Li, Xihe

2018-05-01

Ubiquitination is an important cellular process in spermatogenesis and involves the regulation of spermatid differentiation and spermiogenesis. In the current study, the correlation between bull sperm ubiquitination and sperm defects was analyzed, and the feasibility using anti-ubiquitin specific antibody immobilized magnetic beads to remove the spermatozoa with defects was assessed. A total of nine bulls were examined, and the amount of sperm ubiquitination ranged from 55 to 151. Correspondingly, the percentage of sperm deformity ranged from 9.3% to 28.1%. The coefficient of correlation was r = 0.92, indicating a significant correlation between the percentage of sperm deformity and the amount of ubiquitination (P < 0.05). The results from use of fluorescence staining and single-channel flow cytometry indicated there was a significant correlation between the sperm deformity and amount of ubiquitination (r = 0.86, P < 0.05). Results gained by use of the TUNEL and ubiquitination assays by double-channel flow cytometry indicated that the proportion of genetically defective spermatozoa with ubiquitination in Q3 and Q2 quartiles was markedly greater than that of spermatozoa with ubiquitination in Q1 and Q4 quartiles (82.1% compared with 17.9%). All these results confirmed that sperm ubiquitination is associated with genetic DNA defects (P < 0.01). Furthermore, nine semen samples with sperm motility of less than 50% (minimal motility), 50% to 70% (moderate motility) and greater than 70% (greatest motility) were selected for sorting defective spermatozoa using anti-ubiquitin specific antibody-coated magnetic beads. Strikingly, the percentage of sperm deformity significantly decreased from 18.8%, 19.0% and 17.1% to 11.7%, 11.0% and 11.0%, respectively (P < 0.05), suggesting that this method might be a feasible technology to improve the productivity via removal of the defective spermatozoa from bull semen. Copyright © 2018 Elsevier B.V. All rights

An Interaction Landscape of Ubiquitin Signaling.

PubMed

Zhang, Xiaofei; Smits, Arne H; van Tilburg, Gabrielle B A; Jansen, Pascal W T C; Makowski, Matthew M; Ovaa, Huib; Vermeulen, Michiel

2017-03-02

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

A Review on Ubiquitination of Neurotrophin Receptors: Facts and Perspectives

PubMed Central

Sánchez-Sánchez, Julia; Arévalo, Juan Carlos

2017-01-01

Ubiquitination is a reversible post-translational modification involved in a plethora of different physiological functions. Among the substrates that are ubiquitinated, neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR) have been studied recently. TrkA is the most studied receptor in terms of its ubiquitination, and different E3 ubiquitin ligases and deubiquitinases have been implicated in its ubiquitination, whereas not much is known about the other neurotrophin receptors aside from their ubiquitination. Additional studies are needed that focus on the ubiquitination of TrkB, TrkC, and p75NTR in order to further understand the role of ubiquitination in their physiological and pathological functions. Here we review what is currently known regarding the ubiquitination of neurotrophin receptors and its physiological and pathological relevance. PMID:28335430

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy

Ubiquitination of specific mitochondrial matrix proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lehmann, Gilad; Ziv, Tamar; Braten, Ori

2016-06-17

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinatedmore » proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.« less

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

PubMed

Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

2017-07-21

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP*

PubMed Central

Narayan, Vikram; Pion, Emmanuelle; Landré, Vivien; Müller, Petr; Ball, Kathryn L.

2011-01-01

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. PMID:20947504

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

PubMed Central

Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

Attenuation of T cell receptor signaling by serine phosphorylation-mediated lysine 30 ubiquitination of SLP-76 protein.

PubMed

Wang, Xiaohong; Li, Ju-Pi; Chiu, Li-Li; Lan, Joung-Liang; Chen, Der-Yuan; Boomer, Jonathan; Tan, Tse-Hua

2012-10-05

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein*

PubMed Central

Wang, Xiaohong; Li, Ju-Pi; Chiu, Li-Li; Lan, Joung-Liang; Chen, Der-Yuan; Boomer, Jonathan; Tan, Tse-Hua

2012-01-01

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling. PMID:22902619

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides*

PubMed Central

Mitra, Sharmistha; Traughber, C. Alicia; Brannon, Mary K.; Gomez, Stephanie; Capelluto, Daniel G. S.

2013-01-01

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. PMID:23880770

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

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

Problem-Solving Test: The Role of Ubiquitination in Epidermal Growth Factor Receptor Trafficking

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2012-01-01

Terms to be familiar with before you start to solve the test: growth factor signaling, epidermal growth factor, tyrosine protein kinase, tyrosine phosphorylation, ubiquitin, monoubiquitination, polyubiquitination, site-directed mutagenesis, transfection, expression vector, cDNA, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, Western…

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

PubMed Central

Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M.; Baker, Susan C.; Mesecar, Andrew

2014-01-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. PMID:24854014

Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3

PubMed Central

Tang, Liu-Ya; Yamashita, Motozo; Coussens, Nathan P; Tang, Yi; Wang, Xiangchun; Li, Cuiling; Deng, Chu-Xia; Cheng, Steven Y; Zhang, Ying E

2011-01-01

TGF-β signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-β signalling by attenuating the activity of Smad3 rather than promoting its degradation. PMID:22045334

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

PubMed

Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

2016-07-01

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

A SUMO and ubiquitin code coordinates protein traffic at replication factories.

PubMed

Lecona, Emilio; Fernandez-Capetillo, Oscar

2016-12-01

Post-translational modifications regulate each step of DNA replication to ensure the faithful transmission of genetic information. In this context, we recently showed that deubiquitination of SUMO2/3 and SUMOylated proteins by USP7 helps to create a SUMO-rich and ubiquitin-low environment around replisomes that is necessary to maintain the activity of replication forks and for new origin firing. We propose that a two-flag system mediates the collective concentration of factors at sites of DNA replication, whereby SUMO and Ubiquitinated-SUMO would constitute "stay" or "go" signals respectively for replisome and accessory factors. We here discuss the findings that led to this model, which have implications for the potential use of USP7 inhibitors as anticancer agents. © 2016 WILEY Periodicals, Inc.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsumoto, Hotaru; Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp; Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of themore » SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.« less

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation.

PubMed

Jaeger, Werner C; Seeber, Ruth M; Eidne, Karin A; Pfleger, Kevin D G

2014-01-01

The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX₁ and OX₂) with similar affinity, and orexin B binding to OX₂ with higher affinity than OX₁ receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX₂ receptors with greater β-arrestin-ubiquitin complex stability. The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays. Replacement of the OX₁ receptor C-terminus with that of the OX₂ receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX₂ receptor-β-arrestin-ubiquitin complex formation. This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets. © 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

PubMed Central

Ronnebaum, Sarah M.; Wu, Yaxu; McDonough, Holly

2013-01-01

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging. PMID:24043303

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

PubMed Central

Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi; Liu, Yi; Thirumaran, Ranjit K.; Brandman, Relly; Schuetz, Erin G.; Burlingame, Alma L.; Correia, Maria Almira

2012-01-01

CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may

Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases

PubMed Central

Mohapatra, Bhopal; Ahmad, Gulzar; Nadeau, Scott; Zutshi, Neha; An, Wei; Scheffe, Sarah; Dong, Lin; Feng, Dan; Goetz, Benjamin; Arya, Priyanka; Bailey, Tameka A.; Palermo, Nicholas; Borgstahl, Gloria E.O.; Natarajan, Amarnath; Raja, Srikumar M.; Naramura, Mayumi; Band, Vimla; Band, Hamid

2012-01-01

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader

Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2

PubMed Central

2017-01-01

The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate. PMID:28884161

Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

PubMed Central

Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

2014-01-01

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes. PMID:24852066

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

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encodedmore » by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.« less

Mitochondrial Ubiquitin Ligase in Cardiovascular Disorders.

PubMed

Yu, Tao; Zhang, Yinfeng; Li, Pei-Feng

2017-01-01

Mitochondrial dynamics play a critical role in cellular responses and physiological process. However, their dysregulation leads to a functional degradation, which results in a diverse array of common disorders, including cardiovascular disease. In this background, the mitochondrial ubiquitin ligase has been attracting substantial research interest in recent years. Mitochondrial ubiquitin ligase is localized in the mitochondrial outer membrane, where it plays an essential role in the regulation of mitochondrial dynamics and apoptosis. In this chapter, we provide a comprehensive overview of the functions of mitochondrial ubiquitin ligases identified hitherto, with a special focus on cardiovascular disorders.

Atomic-level description of ubiquitin folding

PubMed Central

Piana, Stefano; Lindorff-Larsen, Kresten; Shaw, David E.

2013-01-01

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. PMID:23503848

Histone ubiquitination: a tagging tail unfolds?

PubMed

Jason, Laure J M; Moore, Susan C; Lewis, John D; Lindsey, George; Ausió, Juan

2002-02-01

Despite the fact that histone H2A ubiquitination affects about 10-15% of this histone in most eukaryotic cells, histone ubiquitination is among one of the less-well-characterized post-translational histone modifications. Nevertheless, some important observations have been made in recent years. Whilst several enzymes had been known to ubiquitinate histones in vitro, recent studies in yeast have led to the unequivocal identification of the enzyme responsible for this post-translational modification in this organism. A strong functional co-relation to meiosis and spermiogenesis has also now been well documented, although its participation in other functional aspects of chromatin metabolism, such as transcription or DNA repair, still remains rather speculative and controversial. Because of its nature, histone ubiquitination represents the most bulky structural change to histones and as such it would be expected to exert an important effect on chromatin structure. Past and recent structural studies, however, indicate a surprising lack of effect of (H2A/H2B) ubiquitination on nucleosome architecture and of uH2A on chromatin folding. These results suggest that this modification may serve as a signal for recognition by functionally relevant trans-acting factors and/or operate synergistically in conjunction with other post-translational modifications such as for instance acetylation. Copyright 2002 Wiley Periodicals, Inc.

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

PubMed Central

Hoe, Nicholas; Huang, Chung M.; Landis, Gary; Verhage, Marian; Ford, Daniel; Yang, Junsheng; van Leeuwen, Fred W.; Tower, John

2011-01-01

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb+1 were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb+1, was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb+1 caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects. PMID:21415465

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.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

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

2013-01-01

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. PMID:23649778

Phosphorylated ubiquitin chain is the genuine Parkin receptor

PubMed Central

Okatsu, Kei; Koyano, Fumika; Kimura, Mayumi; Kosako, Hidetaka; Saeki, Yasushi

2015-01-01

PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria. PMID:25847540

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

NASA Technical Reports Server (NTRS)

Nealson, K. H.; Moser, D. P.; Saffarini, D. A.

1995-01-01

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

Cre-lox Univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-Like protein phosphatases

PubMed Central

Jia, Fan; Gampala, Srinivas S.L.; Mittal, Amandeep; Luo, Qingjun; Rock, Christopher D.

2009-01-01

The 14,200 available full length Arabidopsis thaliana cDNAs in the Universal Plasmid System (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a “functional map-space” of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities. PMID:19499346

A novel pathogenic splice acceptor site germline mutation in intron 14 of the APC gene in a Chinese family with familial adenomatous polyposis.

PubMed

Wang, Dan; Liang, Shengyun; Zhang, Zhao; Zhao, Guoru; Hu, Yuan; Liang, Shengran; Zhang, Xipeng; Banerjee, Santasree

2017-03-28

Familial adenomatous polyposis (FAP) is an autosomal dominant precancerous condition, clinically characterized by the presence of multiple colorectal adenomas or polyps. Patients with FAP has a high risk of developing colorectal cancer (CRC) from these colorectal adenomatous polyps by the mean age of diagnosis at 40 years. Germline mutations of the APC gene cause familial adenomatous polyposis (FAP). Colectomy has recommended for the FAP patients with significant polyposis. Here, we present a clinical molecular study of a four generation Chinese family with FAP. Clinical diagnosis of FAP has been done according to the phenotype, family history and medical records. Patient's blood samples were collected and genomic DNA was extracted. In order to identify the pathogenic mutation underlying the disease phenotype targeted next-generation sequencing and confirmatory sanger sequencing has undertaken. Targeted next generation sequencing identified a novel heterozygous splice-acceptor site mutation [c.1744-1G>A] in intron 14 of APC gene, which is co-segregated with the FAP phenotypes in the proband and amongst all the affected family members. This mutation is not present in unaffected family members and in normal healthy controls of same ethnic origin. According to the LOVD database for Chinese colorectal cancer patients, in Chinese population, 60% of the previously reported APC gene mutations causes FAP, are missense mutations. This novel splice-acceptor site mutation causing FAP in this Chinese family expands the germline mutation spectrum of the APC gene in the Chinese population.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition.

PubMed

Matsumoto, Hotaru; Saitoh, Hisato

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. Copyright © 2016 Elsevier Inc. All rights reserved.

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent, FBXW7-mediated Ubiquitination and Proteasomal Degradation*

PubMed Central

Koo, Junghui; Wu, Xiaoyun; Mao, Zixu; Khuri, Fadlo R.; Sun, Shi-Yong

2015-01-01

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation. PMID:25897075

The canonical wnt signal restricts the glycogen synthase kinase 3/fbw7-dependent ubiquitination and degradation of eya1 phosphatase.

PubMed

Sun, Ye; Li, Xue

2014-07-01

Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1(+/-); Wnt9b(+/-) mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Non-fullerene electron acceptors for organic photovoltaic devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenekhe, Samson A.; Li, Haiyan; Earmme, Taeshik

Non-fullerene electron acceptors for highly efficient organic photovoltaic devices are described. The non-fullerene electron acceptors have an extended, rigid, .pi.-conjugated electron-deficient framework that can facilitate exciton and charge derealization. The non-fullerene electron acceptors can physically mix with a donor polymer and facilitate improved electron transport. The non-fullerene electron acceptors can be incorporated into organic electronic devices, such as photovoltaic cells.

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

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

PubMed

Boomsma, Wouter; Nielsen, Sofie V; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus; Ellgaard, Lars

2016-01-01

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is

Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

PubMed

Michel, Martin A; Swatek, Kirby N; Hospenthal, Manuela K; Komander, David

2017-10-05

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

PubMed Central

Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

2014-01-01

Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

Ubiquitin proteasome system in Parkinson's disease: a keeper or a witness?

PubMed

Martins-Branco, Diogo; Esteves, Ana R; Santos, Daniel; Arduino, Daniela M; Swerdlow, Russell H; Oliveira, Catarina R; Januario, Cristina; Cardoso, Sandra M

2012-12-01

The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models. We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations. We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

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

The roles of ubiquitin modifying enzymes in neoplastic disease.

PubMed

Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam

2017-12-01

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

Fullerene-bisadduct acceptors for polymer solar cells.

PubMed

Li, Yongfang

2013-10-01

Polymer solar cells (PSCs) have drawn great attention in recent years for their simple device structure, light weight, and low-cost fabrication in comparison with inorganic semiconductor solar cells. However, the power-conversion efficiency (PCE) of PSCs needs to be increased for their future application. The key issue for improving the PCE of PSCs is the design and synthesis of high-efficiency conjugated polymer donors and fullerene acceptors for the photovoltaic materials. For the acceptor materials, several fullerene-bisadduct acceptors with high LUMO energy levels have demonstrated excellent photovoltaic performance in PSCs with P3HT as a donor. In this Focus Review, recent progress in high-efficiency fullerene-bisadduct acceptors is discussed, including the bisadduct of PCBM, indene-C60 bisadduct (ICBA), indene-C70 bisadduct (IC70BA), DMPCBA, NCBA, and bisTOQC. The LUMO levels and photovoltaic performance of these bisadduct acceptors with P3HT as a donor are summarized and compared. In addition, the applications of an ICBA acceptor in new device structures and with other conjugated polymer donors than P3HT are also introduced and discussed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure

PubMed Central

Debelouchina, Galia T.; Gerecht, Karola; Muir, Tom W.

2016-01-01

Ubiquitylation of histone H2B, associated with gene activation, leads to chromatin decompaction through an unknown mechanism. We used a hydrogen-deuterium exchange strategy coupled with nuclear magnetic resonance spectroscopy to map the ubiquitin surface responsible for its structural effects on chromatin. Our studies revealed that a previously uncharacterized acidic patch on ubiquitin comprising residues Glu16 and Glu18 is essential for decompaction. These residues mediate promiscuous electrostatic interactions with the basic histone proteins, potentially positioning the ubiquitin moiety as a dynamic “wedge” that prevents the intimate association of neighboring nucleosomes. Using two independent cross-linking strategies and an oligomerization assay, we also showed that ubiquitin-ubiquitin contacts occur in the chromatin environment and are important for the solubilization of the chromatin polymers. Our work highlights a novel, chromatin-related aspect of the “ubiquitin code”, and sheds light on how the information rich ubiquitin modification can orchestrate different biochemical outcomes using different surface features. PMID:27870837

Synthetic CO.sub.2 acceptor

DOEpatents

Lancet, Michael S.; Curran, George P.

1981-08-18

A synthetic CO.sub.2 acceptor consisting essentially of at least one compound selected from the group consisting of calcium oxide and calcium carbonate supported in a refractory carrier matrix, the carrier having the general formula Ca.sub.5 (SiO.sub.4).sub.2 CO.sub.3. A method for producing the synthetic CO.sub.2 acceptor is also disclosed.

The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.

PubMed

Nakata, Shiori; Watanabe, Tadashi; Nakagawa, Koji; Takeda, Hiroshi; Ito, Akihiro; Fujimuro, Masahiro

2016-03-25

Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter

Atypical binding of the Swa2p UBA domain to ubiquitin.

PubMed

Matta-Camacho, Edna; Kozlov, Guennadi; Trempe, Jean-François; Gehring, Kalle

2009-02-20

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix alpha1 and the N-terminal part of helix alpha2 bind to Ub. Mutation of Ala148, a key residue in helix alpha1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices alpha1 and alpha3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix alpha1 of UBA domains involved in membrane protein trafficking.

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

Ubiquitin-mediated modulation of the cytoplasmic viral RNA sensor RIG-I.

PubMed

Oshiumi, Hiroyuki; Matsumoto, Misako; Seya, Tsukasa

2012-01-01

RIG-I-like receptors, including RIG-I, MDA5 and LGP2, recognize cytoplasmic viral RNA. The RIG-I protein consists of N-terminal CARDs, central RNA helicase and C-terminal domains. RIG-I activation is regulated by ubiquitination. Three ubiquitin ligases target the RIG-I protein. TRIM25 and Riplet ubiquitin ligases are positive regulators of RIG-I and deliver the K63-linked polyubiquitin moiety to RIG-I CARDs and the C-terminal domain. RNF125, another ubiquitin ligase, is a negative regulator of RIG-I and mediates K48-linked polyubiquitination of RIG-I, leading to the degradation of the RIG-I protein by proteasomes. The K63-linked polyubiquitin chains of RIG-I are removed by a deubiquitin enzyme, CYLD. Thus, CYLD is a negative regulator of RIG-I. Furthermore, TRIM25 itself is regulated by ubiquitination. HOIP and HOIL proteins are ubiquitin ligases and are also known as linear ubiquitin assembly complexes (LUBACs). The TRIM25 protein is ubiquitinated by LUBAC and then degraded by proteasomes. The splice variant of RIG-I encodes a protein that lacks the first CARD of RIG-I, and the variant RIG-I protein is not ubiquitinated by TRIM25. Therefore, ubiquitin is the key regulator of the cytoplasmic viral RNA sensor RIG-I.

The elusive structural role of ubiquitinated histones.

PubMed

Moore, Susan C; Jason, Laure; Ausió, Juan

2002-01-01

It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

BIOREMEDIATION AT WOOD-PRESERVING SITES

EPA Science Inventory

The removal of organic compounds from ground water during bioremediation at wood-preserving sites is a function of the stoichiometric demand for electron acceptors (oxygen, nitrate, and sulfate) to metabolize the organic contaminants and the supply of the electron acceptors in th...

Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haeggblom, M.M.; Rivera, M.D.; Young, L.Y.

1993-04-01

Methanogeneic conditions can promote the biodegradation of a number of halogenated aromatic compounds. This study, using sediments from freshwater and estuarine sites, is an evaluation of the anaerobic biodegradability of monochlorinated phenols and benzoic acids coupled to denitrification, sulfidogenesis, and methanogenesis. The results indicate that chlorinated phenols and benzoic acids are biodegradable under at least one set of anaerobic conditions. Metabolism depends both on the electron acceptor available and on the position of the chlorine substituent. Presence of alternative electron acceptors, nitrate, sulfate, and carbonate, can affect degradation rates and substrate specificities. Since contaminated sites usually have mixtures of wastes,more » bioremediation efforts may need to consider the activities of diverse anaerobic communities to carry out effective treatment of all components. 37 refs., 4 figs., 4 tabs.« less

Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin

NASA Astrophysics Data System (ADS)

Zanzoni, Serena; Ceccon, Alberto; Assfalg, Michael; Singh, Rajesh K.; Fushman, David; D'Onofrio, Mariapina

2015-04-01

The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with 15N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which

Role of Growth Arrest and DNA Damage–inducible α in Akt Phosphorylation and Ubiquitination after Mechanical Stress-induced Vascular Injury

PubMed Central

Mitra, Sumegha; Sammani, Saad; Wang, Ting; Boone, David L.; Meyer, Nuala J.; Dudek, Steven M.; Moreno-Vinasco, Liliana; Garcia, Joe G. N.

2011-01-01

Rationale: The stress-induced growth arrest and DNA damage–inducible α (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a−/−) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown. Objectives: We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress. Methods: VILI-challenged GADD45a−/− mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methyl-specific polymerase chain reaction assay. Measurements and Main Results: Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI-challenged GADD45a−/− mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a-silenced endothelial cells and GADD45a−/− mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a

The Ubiquitin Ligase RNF125 Targets Innate Immune Adaptor Protein TRIM14 for Ubiquitination and Degradation.

PubMed

Jia, Xue; Zhou, Hongli; Wu, Chao; Wu, Qiankun; Ma, Shichao; Wei, Congwen; Cao, Ye; Song, Jingdong; Zhong, Hui; Zhou, Zhuo; Wang, Jianwei

2017-06-15

Tripartite motif-containing 14 (TRIM14) is a mitochondrial adaptor that facilitates innate immune signaling. Upon virus infection, the expression of TRIM14 is significantly induced, which stimulates the production of type-I IFNs and proinflammatory cytokines. As excessive immune responses lead to harmful consequences, TRIM14-mediated signaling needs to be tightly balanced. In this study, we identify really interesting new gene-type zinc finger protein 125 (RNF125) as a negative regulator of TRIM14 in the innate antiviral immune response. Overexpression of RNF125 inhibits TRIM14-mediated antiviral response, whereas knockdown of RNF125 has the opposite effect. RNF125 interacts with TRIM14 and acts as an E3 ubiquitin ligase that catalyzes TRIM14 ubiquitination. RNF125 promotes K48-linked polyubiquitination of TRIM14 and mediates its degradation via the ubiquitin-proteasome pathway. Consequently, wild-type mouse embryonic fibroblasts show significantly reduced TRIM14 protein levels in late time points of viral infection, whereas TRIM14 protein is retained in RNF125-deficient mouse embryonic fibroblasts. Collectively, our data suggest that RNF125 plays a new role in innate immune response by regulating TRIM14 ubiquitination and degradation. Copyright © 2017 by The American Association of Immunologists, Inc.

Cdk5 regulates PSD-95 ubiquitination in neurons

PubMed Central

Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

2011-01-01

The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ 1

PubMed Central

Sullivan, Michael L.; Callis, Judy; Vierstra, Richard D.

1990-01-01

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 125I-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 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667769

Ubiquitination in the antiviral immune response.

PubMed

Davis, Meredith E; Gack, Michaela U

2015-05-01

Ubiquitination has long been known to regulate fundamental cellular processes through the induction of proteasomal degradation of target proteins. More recently, 'atypical' non-degradative types of polyubiquitin chains have been appreciated as important regulatory moieties by modulating the activity or subcellular localization of key signaling proteins. Intriguingly, many of these non-degradative types of ubiquitination regulate the innate sensing pathways initiated by pattern recognition receptors (PRRs), ultimately coordinating an effective antiviral immune response. Here we discuss recent advances in understanding the functional roles of degradative and atypical types of ubiquitination in innate immunity to viral infections, with a specific focus on the signaling pathways triggered by RIG-I-like receptors, Toll-like receptors, and the intracellular viral DNA sensor cGAS. Copyright © 2015 Elsevier Inc. All rights reserved.

Ubiquitination as an efficient molecular strategy employed in salmonella infection

USDA-ARS?s Scientific Manuscript database

The ubiquitin modification has various functions in the host innate immune system in response to the bacterial infection. To counteract the host immunity, Salmonella can specifically target ubiquitin pathways by its effector proteins. In this review, we describe the multiple facets of ubiquitin func...

Promoters active in interphase are bookmarked during mitosis by ubiquitination

PubMed Central

Arora, Mansi; Zhang, Jie; Heine, George F.; Ozer, Gulcin; Liu, Hui-wen; Huang, Kun; Parvin, Jeffrey D.

2012-01-01

We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis. PMID:22941662

Kinetic modeling of electron transfer reactions in photosystem I complexes of various structures with substituted quinone acceptors.

PubMed

Milanovsky, Georgy E; Petrova, Anastasia A; Cherepanov, Dmitry A; Semenov, Alexey Yu

2017-09-01

The reduction kinetics of the photo-oxidized primary electron donor P 700 in photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 were analyzed within the kinetic model, which considers electron transfer (ET) reactions between P 700 , secondary quinone acceptor A 1 , iron-sulfur clusters and external electron donor and acceptors - methylviologen (MV), 2,3-dichloro-naphthoquinone (Cl 2 NQ) and oxygen. PS I complexes containing various quinones in the A 1 -binding site (phylloquinone PhQ, plastoquinone-9 PQ and Cl 2 NQ) as well as F X -core complexes, depleted of terminal iron-sulfur F A /F B clusters, were studied. The acceleration of charge recombination in F X -core complexes by PhQ/PQ substitution indicates that backward ET from the iron-sulfur clusters involves quinone in the A 1 -binding site. The kinetic parameters of ET reactions were obtained by global fitting of the P 700 + reduction with the kinetic model. The free energy gap ΔG 0 between F X and F A /F B clusters was estimated as -130 meV. The driving force of ET from A 1 to F X was determined as -50 and -220 meV for PhQ in the A and B cofactor branches, respectively. For PQ in A 1A -site, this reaction was found to be endergonic (ΔG 0  = +75 meV). The interaction of PS I with external acceptors was quantitatively described in terms of Michaelis-Menten kinetics. The second-order rate constants of ET from F A /F B , F X and Cl 2 NQ in the A 1 -site of PS I to external acceptors were estimated. The side production of superoxide radical in the A 1 -site by oxygen reduction via the Mehler reaction might comprise ≥0.3% of the total electron flow in PS I.

Interactions of chloride and formate at the donor and the acceptor side of photosystem II.

PubMed

Jajoo, Anjana; Bharti, Sudhakar; Kawamori, Asako

2005-02-01

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S(2) state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe(2)+Q-A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.

Identification and Structure-Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors.

PubMed

Ferreira de Freitas, Renato; Harding, Rachel J; Franzoni, Ivan; Ravichandran, Mani; Mann, Mandeep K; Ouyang, Hui; Lautens, Mark; Santhakumar, Vijayaratnam; Arrowsmith, Cheryl H; Schapira, Matthieu

2018-05-24

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155, are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

ERIC Educational Resources Information Center

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…

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

PubMed

Gan, Jin; Qiao, Niu; Strahan, Roxanne; Zhu, Caixia; Liu, Lei; Verma, Subhash C; Wei, Fang; Cai, Qiliang

2016-11-01

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Acceptors in bulk and nanoscale ZnO

NASA Astrophysics Data System (ADS)

McCluskey, M. D.

2012-02-01

Zinc oxide (ZnO) is a semiconductor that emits bright UV light, with little wasted heat. This intrinsic feature makes it a promising material for energy-efficient white lighting, nano-lasers, and other optical applications. For devices to be competitive, however, it is necessary to develop reliable p-type doping. Although substitutional nitrogen has been considered as a potential p-type dopant for ZnO, theoretical and experimental work indicates that nitrogen is a deep acceptor and will not lead to p-type conductivity. This talk will highlight recent experiments on ZnO:N at low temperatures. A red/near-IR photoluminescence (PL) band is correlated with the presence of deep nitrogen acceptors. PL excitation (PLE) measurements show an absorption threshold of 2.26 eV, in good agreement with theory. Magnetic resonance experiments provide further evidence for this assignment. The results of these studies seem to rule out group-V elements as shallow acceptors in ZnO, contradicting numerous reports in the literature. If these acceptors do not work as advertised, is there a viable alternative? Optical studies on ZnO nanocrystals show some intriguing leads. At liquid-helium temperatures, a series of sharp IR absorption peaks arise from an unknown acceptor impurity. The data are consistent with a hydrogenic acceptor 0.46 eV above the valence band edge. While this binding energy is still too deep for many practical applications, it represents a significant improvement over the ˜ 1.3 eV binding energy for nitrogen acceptors. Nanocrystals present another twist. Due to their high surface-to-volume ratio, surface states are especially important. Specifically, electron-hole recombination at the surface give rises to a red luminescence band. From our PL and IR experiments, we have developed a ``unified'' model that attempts to explain acceptor and surface states in ZnO nanocrystals. This model could provide a useful framework for designing future nanoscale ZnO devices.

Chemical trends for acceptor impurities in GaN

NASA Astrophysics Data System (ADS)

Neugebauer, Jörg; Van de Walle, Chris G.

1999-03-01

We present a comprehensive investigation of acceptor impurities in GaN, based on first-principles total-energy calculations. Two main factors are identified that determine acceptor incorporation: the strength of chemical bonding between the acceptor and its neighbors (which can be assessed by comparison with existing compounds) and the atomic size match between the acceptor and the host atom for which it substitutes. None of the candidates (Li, Na, K, Be, Zn, and Ca) exhibits characteristics which surpass those of Mg in all respects. Only Be emerges as a potential alternative dopant, although it may suffer from compensation by Be interstitial donors.

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

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 asmore » 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.« less

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

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

Proteomic snapshot of the EGF-induced ubiquitin network

PubMed Central

Argenzio, Elisabetta; Bange, Tanja; Oldrini, Barbara; Bianchi, Fabrizio; Peesari, Raghunath; Mari, Sara; Di Fiore, Pier Paolo; Mann, Matthias; Polo, Simona

2011-01-01

The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses. PMID:21245847

Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus.

PubMed

Barrera-Vargas, Ana; Gómez-Martín, Diana; Carmona-Rivera, Carmelo; Merayo-Chalico, Javier; Torres-Ruiz, Jiram; Manna, Zerai; Hasni, Sarfaraz; Alcocer-Varela, Jorge; Kaplan, Mariana J

2018-06-01

To assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects. We studied 74 subjects with SLE and 77 healthy controls. Neutrophils and low-density granulocytes were isolated, and NETs were induced. Ubiquitin content was quantified in NETs by western blot analysis, ELISA and immunofluorescence microscopy, while ubiquitination of NET proteins was assessed by immunoprecipitation. Monocyte-derived macrophages from SLE and controls were isolated and stimulated with NETs or ubiquitin. Calcium flux and cytokine synthesis were measured following these stimuli. NETs contain ubiquitinated proteins, with a lower expression of polyubiquitinated proteins in subjects with SLE than in controls. Myeloperoxidase (MPO) is present in ubiquitinated form in NETs. Patients with SLE develop antiubiquitinated MPO antibodies, and titres positively correlate with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (P<0.01), and negatively correlate with complement components (P<0.01). Stimulation of monocyte-derived macrophages with NETs or with ubiquitin led to enhanced calcium flux. In addition, stimulation with NETs led to enhanced cytokine (tumour necrosis factor-α and interleukin-10) production in macrophages from patients with SLE when compared with controls, which was hampered by inhibition of NET internalisation by macrophages. This is the first study to find ubiquitinated proteins in NETs, and evidence for adaptive immune responses directed towards ubiquitinated NET proteins in SLE. The distinct differences in ubiquitin species profile in NETs compared with healthy controls may contribute to dampened anti-inflammatory responses observed in SLE. These results also support a role for extracellular ubiquitin in inflammation in SLE. © Article author(s) (or their employer(s) unless otherwise stated

Regulation of transcriptional activators by DNA-binding domain ubiquitination

PubMed Central

Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

2017-01-01

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

Template Dimerization Promotes an Acceptor Invasion-Induced Transfer Mechanism during Human Immunodeficiency Virus Type 1 Minus-Strand Synthesis

PubMed Central

Balakrishnan, Mini; Roques, Bernard P.; Fay, Philip J.; Bambara, Robert A.

2003-01-01

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism. PMID:12663778

Effective Tuning of Ketocyanine Derivatives through Acceptor Substitution.

PubMed

Poe, Ambata; Della Pelle, Andrea; Byrnes, Sean; Thayumanavan, S

2015-05-18

A series of ketocyanine derivatives possessing bis(diarylamino)fluorenyl donors and variable acceptors installed at the bridging carbon atom were synthesized to investigate how the electronic structure of the dye can be systemically tuned through stabilization of the cyanine-like character of the donor by increasing the acceptor strength. Analysis of the (1) H NMR spectra indicates that the "charge-separated" species dominates in these dyes, given that carbons possessing a positive or negative charge in the resonance structures of this state purposefully shift downfield or upfield, respectively, depending on the strength of the acceptor moiety. In DAA-Fl-PI, the acceptor strength and the gain of acceptor aromaticity indicates a predisposition of the separated state, indicated by asymmetry in the (1) H NMR spectrum, as well as uneven distribution of the HOMO on the fluorenyl donor. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular dynamics simulations of human E3 ubiquitin ligase Parkin

PubMed Central

Qiu, Shi; Zhu, Shun; Xu, Shan; Han, Yanyan; Liu, Wen; Zuo, Ji

2017-01-01

Human E3 ubiquitin protein ligase parkin (Parkin) mediates mitophagy to maintain mitochondrial homeostasis. Parkin mutations are common genetic causes of early onset familial Parkinson's disease. The molecular mechanism of Parkin activation has been widely studied with emerging evidence suggesting an essential role of the phosphorylated (phospho)-ubiquitin interaction. However, the underlying mechanism of the phospho-ubiquitin interaction remains elusive. In the present study, replica exchange molecular dynamics simulations were performed to examine the conformational dynamics of Parkin in monomer and phospho-ubiquitin-bound states. In the Parkin monomer state, high structural flexibilities were observed in the majority of regions of Parkin particularly in the loop domain between the ubiquitin-like (UBL) and really interesting new gene (RING)0 domain. Binding of phospho-ubiquitin stabilizes the RING1/RING in between RING interface but destabilizes the RING1-UBL interface. Furthermore, using steered molecular dynamics simulations of Parkin mutations, it was demonstrated that salt bridge interactions contribute significantly to the interdomain interactions between the RING1 and UBL domain. Taken together, the results of the present study revealed the conformational dynamics of human full-length Parkin in monomer and phospho-ubiquitin-bound states, providing insights into designing potential therapeutics against Parkinson's disease. PMID:28765939

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

USDA-ARS?s Scientific Manuscript database

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. T...

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

Mechanisms of electron acceptor utilization: Implications for simulating anaerobic biodegradation

USGS Publications Warehouse

Schreiber, M.E.; Carey, G.R.; Feinstein, D.T.; Bahr, J.M.

2004-01-01

Simulation of biodegradation reactions within a reactive transport framework requires information on mechanisms of terminal electron acceptor processes (TEAPs). In initial modeling efforts, TEAPs were approximated as occurring sequentially, with the highest energy-yielding electron acceptors (e.g. oxygen) consumed before those that yield less energy (e.g., sulfate). Within this framework in a steady state plume, sequential electron acceptor utilization would theoretically produce methane at an organic-rich source and Fe(II) further downgradient, resulting in a limited zone of Fe(II) and methane overlap. However, contaminant plumes often display much more extensive zones of overlapping Fe(II) and methane. The extensive overlap could be caused by several abiotic and biotic processes including vertical mixing of byproducts in long-screened monitoring wells, adsorption of Fe(II) onto aquifer solids, or microscale heterogeneity in Fe(III) concentrations. Alternatively, the overlap could be due to simultaneous utilization of terminal electron acceptors. Because biodegradation rates are controlled by TEAPs, evaluating the mechanisms of electron acceptor utilization is critical for improving prediction of contaminant mass losses due to biodegradation. Using BioRedox-MT3DMS, a three-dimensional, multi-species reactive transport code, we simulated the current configurations of a BTEX plume and TEAP zones at a petroleum- contaminated field site in Wisconsin. Simulation results suggest that BTEX mass loss due to biodegradation is greatest under oxygen-reducing conditions, with smaller but similar contributions to mass loss from biodegradation under Fe(III)-reducing, sulfate-reducing, and methanogenic conditions. Results of sensitivity calculations document that BTEX losses due to biodegradation are most sensitive to the age of the plume, while the shape of the BTEX plume is most sensitive to effective porosity and rate constants for biodegradation under Fe(III)-reducing and

Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses.

PubMed

Zhao, Yi; Long, Marcus J C; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2018-02-28

Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true k cat / K m conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

PubMed Central

Xie, Xingqiao; Li, Faxiang; Wang, Yuanyuan; Wang, Yingli; Lin, Zhijie; Cheng, Xiaofang; Liu, Jianping; Chen, Changbin; Pan, Lifeng

2015-01-01

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations. PMID:26506893

Structure of PINK1 in complex with its substrate ubiquitin

PubMed Central

Schubert, Alexander F.; Gladkova, Christina; Pardon, Els; Wagstaff, Jane L.; Freund, Stefan M.V.; Steyaert, Jan; Maslen, Sarah L.; Komander, David

2018-01-01

Autosomal recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular in the E3 ubiquitin ligase Parkin (PARK2), and in its upstream protein kinase PINK1 (PARK6). PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain on structurally protected Ser65 to trigger mitophagy. We here report a crystal structure of a nanobody stabilised complex between Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the ‘C-terminally retracted’ (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the PINK1 N-lobe binds ubiquitin via a unique insertion. The flexible Ser65-loop in the Ub-CR conformation reaches the activation segment, facilitating placement of Ser65 in a phosphate accepting position. The structure also explains how autophosphorylation in the N-lobe stabilises structurally and functionally important insertions, and reveals the molecular basis for AR-JP causing mutations, some of which disrupt ubiquitin binding. PMID:29160309

Structure of PINK1 in complex with its substrate ubiquitin.

PubMed

Schubert, Alexander F; Gladkova, Christina; Pardon, Els; Wagstaff, Jane L; Freund, Stefan M V; Steyaert, Jan; Maslen, Sarah L; Komander, David

2017-12-07

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.

Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb.

PubMed

Kitagawa, K; Hiramatsu, Y; Uchida, C; Isobe, T; Hattori, T; Oda, T; Shibata, K; Nakamura, S; Kikuchi, A; Kitagawa, M

2009-06-25

Expression of oncoprotein c-Myb oscillates during hematopoiesis and hematological malignancies. Its quantity is not only regulated through transcriptional control but also through the ubiquitin-proteasome pathway, accompanied by phosphorylation, although the mechanisms are poorly understood. In this report, we tried to identify an E3 ubiquitin ligase, which targets c-Myb for ubiquitin-dependent degradation. We found that an F-box protein, Fbw7, interacted with c-Myb, which is mutated in numerous cancers. Fbw7 facilitated ubiquitylation and degradation of c-Myb in intact cells. Moreover, depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of c-Myb in myeloid leukemia cells concomitantly, and suppressed the transcriptional level of gamma-globin, which receives transcriptional repression from c-Myb. In addition, we analysed sites required for both ubiquitylation and degradation of c-Myb. We found that Thr-572 is critical for Fbw7-mediated ubiquitylation in mouse c-Myb using site-directed mutagenesis. Fbw7 recognized the phosphorylation of Thr-572, which was mediated by glycogen synthase kinase 3 (GSK3). In consequence, the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. These observations suggest that SCF(Fbw7) ubiquitin ligase regulates phosphorylation-dependent degradation of c-Myb protein.

Shallow versus deep nature of Mg acceptors in nitride semiconductors

NASA Astrophysics Data System (ADS)

Lyons, John; Janotti, Anderson; van de Walle, Chris G.

2012-02-01

Although Mg doping is the only known method for achieving p-type conductivity in nitride semiconductors, Mg is not a perfect acceptor. Hydrogen is known to passivate the Mg acceptor, necessitating a post-growth anneal for acceptor activation. Furthermore, the acceptor ionization energy of Mg is relatively large (200 meV) in GaN, thus only a few percent of Mg acceptors are ionized at room temperature. Surprisingly, despite the importance of this impurity, open questions remain regarding the nature of the acceptor. Optical and magnetic resonance measurements on Mg-doped GaN indicate intriguing and complex behavior that depends on the growth, doping level, and thermal treatment of the samples. Motivated by these studies, we have revisited this topic by performing first-principles calculations based on a hybrid functional. We investigate the electrical and optical properties of the isolated Mg acceptor and its complexes with hydrogen in GaN, InN, and AlN. With the help of these advanced techniques we explain the deep or shallow nature of the Mg acceptor and its relation to optical signals often seen in Mg-doped GaN. We also explore the properties of the Mg acceptor in InN and AlN, allowing predictions of the behavior of the Mg dopant in ternary nitride alloys.

Spectral, thermal and kinetic studies of charge-transfer complexes formed between the highly effective antibiotic drug metronidazole and two types of acceptors: σ- and π-acceptors

NASA Astrophysics Data System (ADS)

Refat, Moamen S.; Saad, Hosam A.; Adam, Abdel Majid A.

2015-04-01

Understanding the interaction between drugs and small inorganic or organic molecules is critical in being able to interpret the drug-receptor interactions and acting mechanism of these drugs. A combined solution and solid state study was performed to describe the complexation chemistry of drug metronidazole (MZ) which has a broad-spectrum antibacterial activity with two types of acceptors. The acceptors include, σ-acceptor (i.e., iodine) and π-acceptors (i.e., dichlorodicyanobenzoquinone (DDQ), chloranil (CHL) and picric acid (PA)). The molecular structure, spectroscopic characteristics, the binding modes as well as the thermal stability were deduced from IR, UV-vis, 1H NMR and thermal studies. The binding ratio of complexation (MZ: acceptor) was determined to be 1:2 for the iodine acceptor and 1:1 for the DDQ, CHL or PA acceptor, according to the CHN elemental analyses and spectrophotometric titrations. It has been found that the complexation with CHL and PA acceptors increases the values of enthalpy and entropy, while the complexation with DDQ and iodine acceptors decreases the values of these parameters compared with the free MZ donor.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Spectral, thermal, XRD and SEM studies of charge-transfer complexation of hexamethylenediamine and three types of acceptors: π-, σ- and vacant orbital acceptors that include quinol, picric acid, bromine, iodine, SnCl4 and ZnCl2 acceptors

NASA Astrophysics Data System (ADS)

Adam, Abdel Majid A.; Refat, Moamen S.; Saad, Hosam A.

2013-11-01

In this work, structural, thermal, morphological and pharmacological characterization was performed on the interactions between a hexamethylenediamine (HMDA) donor and three types of acceptors to understand the complexation behavior of diamines. The three types of acceptors include π-acceptors (i.e., quinol (QL) and picric acid (PA)), σ-acceptors (i.e., bromine and iodine) and vacant orbital acceptors (i.e., tin(IV) tetrachloride (SnCl4) and zinc chloride (ZnCl2)). The characterization of the obtained CT complexes was performed using elemental analysis, infrared (IR), Raman, 1H NMR and electronic absorption spectroscopy, powder X-ray diffraction (XRD) and thermogravimetric (TG) analysis. Their morphologies were studied using scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX). The biological activities of the obtained CT complexes were tested for their antibacterial activities. The complex containing the QL acceptor exhibited a remarkable electronic spectrum with a strong, broad absorption band, which had an observed λmax that was at a much longer wavelength than those of the free reactants. In addition, this complex exhibited strong antimicrobial activities against various bacterial and fungal strains compared to standard drugs. The complexes containing the PA, iodine, Sn(IV) and Zn(II) acceptors exhibited good thermal stability up to 240, 330, 275 and 295 °C, respectively. The complexes containing bromine, Sn(IV) and Zn(II) acceptors exhibited good crystallinity. In addition to its good crystallinity properties, the complex containing the bromine acceptor exhibits a remarkable morphology feature.

The ubiquitin-proteasome system in spongiform degenerative disorders

PubMed Central

Whatley, Brandi R.; Li, Lian; Chin, Lih-Shen

2008-01-01

Summary Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer's disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan's spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin-proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin-protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders. PMID:18790052

PINK1 autophosphorylation is required for ubiquitin recognition.

PubMed

Rasool, Shafqat; Soya, Naoto; Truong, Luc; Croteau, Nathalie; Lukacs, Gergely L; Trempe, Jean-François

2018-04-01

Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin. © 2018 The Authors.

APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

PubMed

Dussart, Sylvie; Douaisi, Marc; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne

2005-01-21

APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

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.

TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

NASA Technical Reports Server (NTRS)

Li, Yi-Ping; Lecker, Stewart H.; Chen, Yuling; Waddell, Ian D.; Goldberg, Alfred L.; Reid, Michael B.

2003-01-01

In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

Contrasting performance of donor-acceptor copolymer pairs in ternary blend solar cells and two-acceptor copolymers in binary blend solar cells.

PubMed

Khlyabich, Petr P; Rudenko, Andrey E; Burkhart, Beate; Thompson, Barry C

2015-02-04

Here two contrasting approaches to polymer-fullerene solar cells are compared. In the first approach, two distinct semi-random donor-acceptor copolymers are blended with phenyl-C61-butyric acid methyl ester (PC61BM) to form ternary blend solar cells. The two poly(3-hexylthiophene)-based polymers contain either the acceptor thienopyrroledione (TPD) or diketopyrrolopyrrole (DPP). In the second approach, semi-random donor-acceptor copolymers containing both TPD and DPP acceptors in the same polymer backbone, termed two-acceptor polymers, are blended with PC61BM to give binary blend solar cells. The two approaches result in bulk heterojunction solar cells that have the same molecular active-layer components but differ in the manner in which these molecular components are mixed, either by physical mixing (ternary blend) or chemical "mixing" in the two-acceptor (binary blend) case. Optical properties and photon-to-electron conversion efficiencies of the binary and ternary blends were found to have similar features and were described as a linear combination of the individual components. At the same time, significant differences were observed in the open-circuit voltage (Voc) behaviors of binary and ternary blend solar cells. While in case of two-acceptor polymers, the Voc was found to be in the range of 0.495-0.552 V, ternary blend solar cells showed behavior inherent to organic alloy formation, displaying an intermediate, composition-dependent and tunable Voc in the range from 0.582 to 0.684 V, significantly exceeding the values achieved in the two-acceptor containing binary blend solar cells. Despite the differences between the physical and chemical mixing approaches, both pathways provided solar cells with similar power conversion efficiencies, highlighting the advantages of both pathways toward highly efficient organic solar cells.

Energy bands and acceptor binding energies of GaN

NASA Astrophysics Data System (ADS)

Xia, Jian-Bai; Cheah, K. W.; Wang, Xiao-Liang; Sun, Dian-Zhao; Kong, Mei-Ying

1999-04-01

The energy bands of zinc-blende and wurtzite GaN are calculated with the empirical pseudopotential method, and the pseudopotential parameters for Ga and N atoms are given. The calculated energy bands are in agreement with those obtained by the ab initio method. The effective-mass theory for the semiconductors of wurtzite structure is established, and the effective-mass parameters of GaN for both structures are given. The binding energies of acceptor states are calculated by solving strictly the effective-mass equations. The binding energies of donor and acceptor are 24 and 142 meV for the zinc-blende structure, 20 and 131, and 97 meV for the wurtzite structure, respectively, which are consistent with recent experimental results. It is proposed that there are two kinds of acceptor in wurtzite GaN. One kind is the general acceptor such as C, which substitutes N, which satisfies the effective-mass theory. The other kind of acceptor includes Mg, Zn, Cd, etc., the binding energy of these acceptors is deviated from that given by the effective-mass theory. In this report, wurtzite GaN is grown by the molecular-beam epitaxy method, and the photoluminescence spectra were measured. Three main peaks are assigned to the donor-acceptor transitions from two kinds of acceptors. Some of the transitions were identified as coming from the cubic phase of GaN, which appears randomly within the predominantly hexagonal material.

Ubiquitin-dependent and independent roles of SUMO in proteostasis.

PubMed

Liebelt, Frauke; Vertegaal, Alfred C O

2016-08-01

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. Copyright © 2016 the American Physiological Society.

Immune defects caused by mutations in the ubiquitin system.

PubMed

Etzioni, Amos; Ciechanover, Aaron; Pikarsky, Eli

2017-03-01

The importance of the ubiquitin system in health and disease has been widely recognized in recent decades, with better understanding of the various components of the system and their function. Ubiquitination, which is essential to almost all biological processes in eukaryotes, was also found to play an important role in innate and adaptive immune responses. Thus it is not surprising that mutations in genes coding for components of the ubiquitin system cause immune dysregulation. The first defect in the system was described 30 years ago and is due to mutations in the nuclear factor κB (NF-κB) essential modulator, a key regulator of the NF-κB pathway. With use of novel sequencing techniques, many additional mutations in different genes involved in ubiquitination and related to immune system function were identified. This can be clearly illustrated in mutations in the different activation pathways of NF-κB, which result in aberrations in production of various proinflammatory cytokines. The inherited diseases typically manifest with immunodeficiency, autoimmunity, or autoinflammation. In this perspective we provide a short description of the ubiquitin system, with specific emphasis given to its role in the immune system. The various immunodeficiency conditions identified thus far in association with defective ubiquitination are discussed in more detail. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

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

2016-01-01

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

Dual nature of acceptors in GaN and ZnO: The curious case of the shallow MgGa deep state

NASA Astrophysics Data System (ADS)

Lany, Stephan; Zunger, Alex

2010-04-01

Employing a Koopmans corrected density functional method, we find that the metal-site acceptors Mg, Be, and Zn in GaN and Li in ZnO bind holes in deep levels that are largely localized at single anion ligand atoms. In addition to this deep ground state (DGS), we observe an effective-masslike delocalized state that can exist as a short lived shallow transient state (STS). The Mg dopant in GaN represents the unique case where the ionization energy of the localized deep level exceeds only slightly that of the shallow effective-mass acceptor, which explains why Mg works so exceptionally well as an acceptor dopant.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

PubMed

Huseinovic, Angelina; van Leeuwen, Jolanda S; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P E; Kooter, Jan M; Vos, J Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Organic solar cells based on non-fullerene acceptors

NASA Astrophysics Data System (ADS)

Hou, Jianhui; Inganäs, Olle; Friend, Richard H.; Gao, Feng

2018-02-01

Organic solar cells (OSCs) have been dominated by donor:acceptor blends based on fullerene acceptors for over two decades. This situation has changed recently, with non-fullerene (NF) OSCs developing very quickly. The power conversion efficiencies of NF OSCs have now reached a value of over 13%, which is higher than the best fullerene-based OSCs. NF acceptors show great tunability in absorption spectra and electron energy levels, providing a wide range of new opportunities. The coexistence of low voltage losses and high current generation indicates that new regimes of device physics and photophysics are reached in these systems. This Review highlights these opportunities made possible by NF acceptors, and also discuss the challenges facing the development of NF OSCs for practical applications.

Effects of exogenous ubiquitin in a polytrauma model with blunt chest trauma

PubMed Central

Baker, Todd A.; Romero, Jacqueline; Bach, Harold H.; Strom, Joel A.; Gamelli, Richard L.; Majetschak, Matthias

2013-01-01

Objective To determine whether treatment with the CXC chemokine receptor (CXCR) 4 agonist ubiquitin results in beneficial effects in a polytrauma model consisting of bilateral femur fractures plus blunt chest trauma (Injury Severity Score 18-25). Design Treatment study. Setting Research Laboratory. Subjects Seventeen Yorkshire pigs. Interventions Intravenous (i.v.) injection of 1.5 mg/kg ubiquitin or albumin (=control) at 60 min after polytrauma. Measurements and Main Results Anesthetized, mechanically ventilated pigs underwent polytrauma, followed by a simulated 60 min shock phase. At the end of the shock phase ubiquitin or albumin were administered and animals were resuscitated to a mean arterial blood pressure of 70 mmHg until t = 420 min. After i.v. ubiquitin, ubiquitin plasma concentrations increased sixteen-fold to 2870 ± 1015 ng/mL at t = 90 min and decreased with t1/2 = 60 min. Endogenous plasma ubiquitin increased two-fold in the albumin group with peak levels of 359 ± 210 ng/mL. Plasma levels of the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α were unchanged in both groups. Ubiquitin treatment reduced arterial lactate levels and prevented a continuous decrease in arterial oxygenation, which occurred in the albumin group during resuscitation. Wet weight to dry weight ratios of the lung contralateral from the injury, heart, spleen and jejunum were lower with ubiquitin. With ubiquitin treatment, tissue levels of IL-8, IL-10, TNFα and SDF-1α were reduced in the injured lung and of IL-8 in the contralateral lung, respectively. Conclusions Administration of exogenous ubiquitin modulates the local inflammatory response, improves resuscitation, reduces fluid shifts into tissues and preserves arterial oxygenation after blunt polytrauma with lung injury. This study further supports the notion that ubiquitin is a promising protein therapeutic and implies CXCR4 as a drug target after polytrauma. PMID:22622399

Ubiquitin Chains Modified by the Bacterial Ligase SdeA Are Protected from Deubiquitinase Hydrolysis.

PubMed

Puvar, Kedar; Zhou, Yiyang; Qiu, Jiazhang; Luo, Zhao-Qing; Wirth, Mary J; Das, Chittaranjan

2017-09-12

The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD + -dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA's modulatory functions during Legionella infection.

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

PubMed

Vriend, Jerry; Liu, Wenjun; Reiter, Russel J

2017-01-01

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. 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. 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. 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 responsive, in vitro, to treatment with

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

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 have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65

Ubiquitin and Parkinson's disease through the looking glass of genetics.

PubMed

Walden, Helen; Muqit, Miratul M K

2017-04-13

Biochemical alterations found in the brains of Parkinson's disease (PD) patients indicate that cellular stress is a major driver of dopaminergic neuronal loss. Oxidative stress, mitochondrial dysfunction, and ER stress lead to impairment of the homeostatic regulation of protein quality control pathways with a consequent increase in protein misfolding and aggregation and failure of the protein degradation machinery. Ubiquitin signalling plays a central role in protein quality control; however, prior to genetic advances, the detailed mechanisms of how impairment in the ubiquitin system was linked to PD remained mysterious. The discovery of mutations in the α-synuclein gene, which encodes the main protein misfolded in PD aggregates, together with mutations in genes encoding ubiquitin regulatory molecules, including PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, has provided an opportunity to dissect out the molecular basis of ubiquitin signalling disruption in PD, and this knowledge will be critical for developing novel therapeutic strategies in PD that target the ubiquitin system. © 2017 The Author(s).

Regulation of the Hippo signaling pathway by ubiquitin modification.

PubMed

Kim, Youngeun; Jho, Eek-Hoon

2018-03-01

The Hippo signaling pathway plays an essential role in adult tissue homeostasis and organ size control. Abnormal regulation of Hippo signaling can be a cause for multiple types of human cancers. Since the awareness of the importance of the Hippo signaling in a wide range of biological fields has been continually grown, it is also understood that a thorough and well-rounded comprehension of the precise dynamics could provide fundamental insights for therapeutic applications. Several components in the Hippo signaling pathway are known to be targeted for proteasomal degradation via ubiquitination by E3 ligases. β-TrCP is a well-known E3 ligase of YAP/TAZ, which leads to the reduction of YAP/TAZ levels. The Hippo signaling pathway can also be inhibited by the E3 ligases (such as ITCH) which target LATS1/2 for degradation. Regulation via ubiquitination involves not only complex network of E3 ligases but also deubiquitinating enzymes (DUBs), which remove ubiquitin from its targets. Interestingly, non-degradative ubiquitin modifications are also known to play important roles in the regulation of Hippo signaling. Although there has been much advanced progress in the investigation of ubiquitin modifications acting as regulators of the Hippo signaling pathway, research done to date still remains inadequate due to the sheer complexity and diversity of the subject. Herein, we review and discuss recent developments that implicate ubiquitin-mediated regulatory mechanisms at multiple steps of the Hippo signaling pathway. [BMB Reports 2018; 51(3): 143-150].

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

PubMed Central

Huseinovic, Angelina; van Leeuwen, Jolanda S.; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P. E.; Kooter, Jan M.; Vos, J. Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity. PMID:28291796

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism.

PubMed

Lavie, Julie; De Belvalet, Harmony; Sonon, Sessinou; Ion, Ana Madalina; Dumon, Elodie; Melser, Su; Lacombe, Didier; Dupuy, Jean-William; Lalou, Claude; Bénard, Giovanni

2018-06-05

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles

NASA Technical Reports Server (NTRS)

Riley, Danny A.; Bain, James L. W.; Haas, Arthur L.; Ellis, Stanley

1988-01-01

Solid-phase immunochemical methods were employed to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscles. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

Identification and expression of the protein ubiquitination system in Giardia intestinalis.

PubMed

Gallego, Eva; Alvarado, Magda; Wasserman, Moises

2007-06-01

Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite's differentiation processes, whereas variation in transcription was observed in the other genes under study.

An Overview of Electron Acceptors in Microbial Fuel Cells

PubMed Central

Ucar, Deniz; Zhang, Yifeng; Angelidaki, Irini

2017-01-01

Microbial fuel cells (MFC) have recently received increasing attention due to their promising potential in sustainable wastewater treatment and contaminant removal. In general, contaminants can be removed either as an electron donor via microbial catalyzed oxidization at the anode or removed at the cathode as electron acceptors through reduction. Some contaminants can also function as electron mediators at the anode or cathode. While previous studies have done a thorough assessment of electron donors, cathodic electron acceptors and mediators have not been as well described. Oxygen is widely used as an electron acceptor due to its high oxidation potential and ready availability. Recent studies, however, have begun to assess the use of different electron acceptors because of the (1) diversity of redox potential, (2) needs of alternative and more efficient cathode reaction, and (3) expanding of MFC based technologies in different areas. The aim of this review was to evaluate the performance and applicability of various electron acceptors and mediators used in MFCs. This review also evaluated the corresponding performance, advantages and disadvantages, and future potential applications of select electron acceptors (e.g., nitrate, iron, copper, perchlorate) and mediators. PMID:28469607

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.

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

A study of acceptors and non-acceptors of family planning methods among three tribal communities.

PubMed

Mutharayappa, R

1995-03-01

Primary data were collected from 399 currently married women of the Marati, Malekudiya, and Koraga tribes in the Dakshina Kannada district of Karnataka State in this study of the implementation of family planning programs in tribal areas. The Marati, Malekudiya, and Koraga tribes are three different endogamous tribal populations living in similar ecological conditions. Higher levels of literacy and a high rate of acceptance of family planning methods, however, have been observed among these tribes compared to the rest of the tribal population in the state. 46.4% of currently married women aged 15-49 years in the tribes were acceptors of family planning methods, having a mean 3.7 children. The majority of acceptors opted for tubectomy and vasectomy. The adoption of spacing methods is less common among tribal people. Most acceptors received their operations through government health facilities. They were motivated mainly by female health workers and received both cash and other incentives to accept family planning. The main reason for non-acceptance of family planning among non-acceptors was the desire to conceive and bear more children. The data indicate that most of the tribal households are nuclear families with household size more or less similar to that of the general population. They have a higher literacy rate than the rest of the tribal population in the state, with literacy levels between males and females and between the three tribes being quite different; the school enrollment ratio is relatively higher for both boys and girls.

Regulation of human MutYH DNA glycosylase by the E3 ubiquitin ligase mule.

PubMed

Dorn, Julia; Ferrari, Elena; Imhof, Ralph; Ziegler, Nathalie; Hübscher, Ulrich

2014-03-07

Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.

Towards building artificial light harvesting complexes: enhanced singlet-singlet energy transfer between donor and acceptor pairs bound to albumins.

PubMed

Kumar, Challa V; Duff, Michael R

2008-12-01

Specific donor and acceptor pairs have been assembled in bovine serum albumin (BSA), at neutral pH and room temperature, and these dye-protein complexes indicated efficient donor to acceptor singlet-singlet energy transfer. For example, pyrene-1-butyric acid served as the donor and Coumarin 540A served as the acceptor. Both the donor and the acceptor bind to BSA with affinity constants in excess of 2x10(5) M(-1), as measured in absorption and circular dichroism (CD) spectral titrations. Simultaneous binding of both the donor and the acceptor chromophores was supported by CD spectra and one chromophore did not displace the other from the protein host, even when limited concentrations of the host were used. For example, a 1:1:1 complex between the donor, acceptor and the host can be readily formed, and spectral data clearly show that the binding sites are mutually exclusive. The ternary complexes (two different ligands bound to the same protein molecule) provided opportunities to examine singlet-singlet energy transfer between the protein-bound chromophores. Donor emission was quenched by the addition of the acceptor, in the presence of limited amounts of BSA, while no energy transfer was observed in the absence of the protein host, under the same conditions. The excitation spectra of the donor-acceptor-host complexes clearly show the sensitization of acceptor emission by the donor. Protein denaturation, as induced by the addition of urea or increasing the temperature to 360 K, inhibited energy transfer, which indicate that protein structure plays an important role. Sensitization also proceeded at low temperature (77 K) and diffusion of the donor or the acceptor is not required for energy transfer. Stern-Volmer quenching plots show that the quenching constant is (3.1+/-0.2)x10(4) M(-1), at low acceptor concentrations (<35 microM). Other albumins such as human and porcine proteins also served as good hosts for the above experiments. For the first time, non

Method for producing and regenerating a synthetic CO.sub.2 acceptor

DOEpatents

Lancet, Michael S [Pittsburgh, PA; Curran, George P [Pittsburgh, PA; Gorin, Everett [San Rafael, CA

1982-01-01

A method for producing a synthetic CO.sub.2 acceptor by feeding a mixture of finely divided silica and at least one finely divided calcium compound selected from the group consisting of calcium oxide and calcium carbonate to a fluidized bed; operating the fluidized bed at suitable conditions to produce pellets of synthetic CO.sub.2 acceptor and recovering the pellets of synthetic CO.sub.2 acceptor from the fluidized bed. Optionally, spent synthetic CO.sub.2 acceptor can be charged to the fluidized bed to produce regenerated pellets of synthetic CO.sub.2 acceptor.

Assessment of a New Type of Coin Acceptor

DOT National Transportation Integrated Search

1983-04-01

An assessment of the Mars Money Systems Model CD 540-1 coin acceptor associated with farecard vendors was conducted at the Port Authority Transit Corp. as part of an appraisal of automatic fare collection (AFC) equipment. The Mars acceptor consistent...

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

FANCL ubiquitinates β-catenin and enhances its nuclear function

PubMed Central

Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.

2012-01-01

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss. PMID:22653977

FANCL ubiquitinates β-catenin and enhances its nuclear function.

PubMed

Dao, Kim-Hien T; Rotelli, Michael D; Petersen, Curtis L; Kaech, Stefanie; Nelson, Whitney D; Yates, Jane E; Hanlon Newell, Amy E; Olson, Susan B; Druker, Brian J; Bagby, Grover C

2012-07-12

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes.

PubMed

Wang, Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-beta activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-beta enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-beta type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Solution structure of lysine-free (K0) ubiquitin

PubMed Central

Huang, Tao; Li, Jess; Byrd, R Andrew

2014-01-01

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub 15N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations. PMID:24591328

Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress.

PubMed

Biswas, Siddhartha; Willis, Leslie G; Fang, Minggang; Nie, Yingchao; Theilmann, David A

2018-02-01

During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV. IMPORTANCE Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the

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.

Alternansucrase acceptor reactions with D-tagatose and L-glucose.

PubMed

Côté, Gregory L; Dunlap, Christopher A; Appell, Michael; Momany, Frank A

2005-02-07

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are D-tagatose and L-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide alpha-d-glucopyranosyl-(1-->1)-beta-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from L-glucose, but the trisaccharide alpha-D-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-(1-->4)-l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, alpha-D-glucopyranosyl-(1-->4)-L-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of alpha-D-glucopyranosyl-(1-->4)-L-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.

Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.

PubMed

Peisley, Alys; Wu, Bin; Xu, Hui; Chen, Zhijian J; Hur, Sun

2014-05-01

Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.

Method for producing and regenerating a synthetic CO[sub 2] acceptor

DOEpatents

Lancet, M. S.; Curran, G. P.; Gorin, E.

1982-05-18

A method is described for producing a synthetic CO[sub 2] acceptor by feeding a mixture of finely divided silica and at least one finely divided calcium compound selected from the group consisting of calcium oxide and calcium carbonate to a fluidized bed; operating the fluidized bed at suitable conditions to produce pellets of synthetic CO[sub 2] acceptor and recovering the pellets of synthetic CO[sub 2] acceptor from the fluidized bed. Optionally, spent synthetic CO[sub 2] acceptor can be charged to the fluidized bed to produce regenerated pellets of synthetic CO[sub 2] acceptor. 1 fig.

Newly translated proteins are substrates for ubiquitin, ISG15, and FAT10.

PubMed

Spinnenhirn, Valentina; Bitzer, Annegret; Aichem, Annette; Groettrup, Marcus

2017-01-01

The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins. © 2016 Federation of European Biochemical Societies.

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

Mapping the Relationship between Glycosyl Acceptor Reactivity and Glycosylation Stereoselectivity.

PubMed

van der Vorm, Stefan; van Hengst, Jacob M A; Bakker, Marloes; Overkleeft, Herman S; van der Marel, Gijsbert A; Codée, Jeroen D C

2018-03-30

The reactivity of both coupling partners-the glycosyl donor and acceptor-is decisive for the outcome of a glycosylation reaction, in terms of both yield and stereoselectivity. Where the reactivity of glycosyl donors is well understood and can be controlled through manipulation of the functional/protecting-group pattern, the reactivity of glycosyl acceptor alcohols is poorly understood. We here present an operationally simple system to gauge glycosyl acceptor reactivity, which employs two conformationally locked donors with stereoselectivity that critically depends on the reactivity of the nucleophile. A wide array of acceptors was screened and their structure-reactivity/stereoselectivity relationships established. By systematically varying the protecting groups, the reactivity of glycosyl acceptors can be adjusted to attain stereoselective cis-glucosylations. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity.

PubMed

Yao, Fan; Zhou, Zhicheng; Kim, Jongchan; Hang, Qinglei; Xiao, Zhenna; Ton, Baochau N; Chang, Liang; Liu, Na; Zeng, Liyong; Wang, Wenqi; Wang, Yumeng; Zhang, Peijing; Hu, Xiaoyu; Su, Xiaohua; Liang, Han; Sun, Yutong; Ma, Li

2018-06-11

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCF SKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

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

Defining donor and acceptor strength in conjugated copolymers

NASA Astrophysics Data System (ADS)

Hedström, Svante; Wang, Ergang; Persson, Petter

2017-03-01

The progress in efficiency of organic photovoltaic devices is largely driven by the development of new donor-acceptor (D-A) copolymers. The number of possible D-A combinations escalates rapidly with the ever-increasing number of donor and acceptor units, and the design process often involves a trial-and-error approach. We here present a computationally efficient methodology for the prediction of optical and electronic properties of D-A copolymers based on density functional theory calculations of donor- and acceptor-only homopolymers. Ten donors and eight acceptors are studied, as well as all of their 80 D-A copolymer combinations, showing absorption energies of 1.3-2.3 eV, and absorption strengths varying by up to a factor of 2.5. Focus lies on exhibited trends in frontier orbital energies, optical band gaps, and absorption intensities, as well as their relation to the molecular structure. Based on the results, we define the concept of donor and acceptor strength, and calculate this quantity for all investigated units. The light-harvesting capabilities of the 80 D-A copolymers were also assessed. This gives a valuable theoretical guideline to the design of D-A copolymers with the potential to reduce the synthesis efforts in the development of new polymers.

Nanographenes as electron-deficient cores of donor-acceptor systems.

PubMed

Liu, Yu-Min; Hou, Hao; Zhou, Yan-Zhen; Zhao, Xin-Jing; Tang, Chun; Tan, Yuan-Zhi; Müllen, Klaus

2018-05-15

Conjugation of nanographenes (NGs) with electro-active molecules can establish donor-acceptor π-systems in which the former generally serve as the electron-donating moieties due to their electronic-rich nature. In contrast, here we report a series of reversed donor-acceptor structures are obtained by C-N coupling of electron-deficient perchlorinated NGs with electron-rich anilines. Selective amination at the vertexes of the NGs is unambiguously shown through X-ray crystallography. By varying the donating ability of the anilino groups, the optical and assembly properties of donor-acceptor NGs can be finely modulated. The electron-deficient concave core of the resulting conjugates can host electron-rich guest molecules by intermolecular donor-acceptor interactions and gives rise to charge-transfer supramolecular architectures.

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

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

PubMed

Manczyk, Noah; Yates, Bradley P; Veggiani, Gianluca; Ernst, Andreas; Sicheri, Frank; Sidhu, Sachdev S

2017-05-01

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type. © 2017 The Protein Society.

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.

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-{beta} activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-{beta} enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-{beta} type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smadmore » complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.« less

Quantitative regulation of histone variant H2A.Z during cell cycle by ubiquitin proteasome system and SUMO-targeted ubiquitin ligases.

PubMed

Takahashi, Daisuke; Orihara, Yuki; Kitagawa, Saho; Kusakabe, Masayuki; Shintani, Takahiro; Oma, Yukako; Harata, Masahiko

2017-08-01

Quantitative control of histones and histone variants during cell cycle is relevant to their epigenetic functions. We found that the level of yeast histone variant H2A.Z in the G2/M-phase is actively kept low by the ubiquitin proteasome system and SUMO-targeted ubiquitin ligases. Overexpression of H2A.Z induced defects in mitotic progression, suggesting functional importance of this quantitative control.

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 ...

Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover

PubMed Central

Swaney, Danielle L; Rodríguez-Mias, Ricard A; Villén, Judit

2015-01-01

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology. PMID:26142280

Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells.

PubMed

Eckerdt, Frank; Yuan, Juping; Saxena, Krishna; Martin, Bernd; Kappel, Sven; Lindenau, Christine; Kramer, Andrea; Naumann, Steffen; Daum, Sebastian; Fischer, Gunter; Dikic, Ivan; Kaufmann, Manfred; Strebhardt, Klaus

2005-11-04

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

Recognition of Poly-Ubiquitins by the Proteasome through Protein Refolding Guided by Electrostatic and Hydrophobic Interactions.

PubMed

Zhang, Yi; Vuković, Lela; Rudack, Till; Han, Wei; Schulten, Klaus

2016-08-25

Specificity of protein degradation by cellular proteasomes comes from tetra-ubiquitin recognition. We carry out molecular dynamics simulations to characterize how the ubiquitin receptor Rpn10 recognizes in the 26S proteasome K48-linked tetra-ubiquitin. In the binding pose, ubiquitin and Rpn10 interact primarily through hydrophobic patches. However, K48-linked tetra-ubiquitin mostly assumes a closed form in solution prior to binding, in which its hydrophobic patches are not exposed to solvent. Likewise, the hydrophobic ubiquitin interacting motifs (UIMs) of Rpn10 are mostly protected prior to binding. As a result, ubiquitin recognition in the proteasome requires refolding of both K48-linked tetra-ubiquitin and Rpn10. Simulations suggest that conserved complementary electrostatic patterns of Rpn10 and ubiquitins guide protein association (stage 1 in the recognition process), which induces refolding (stage 2), and then facilitates formation of hydrophobic contacts (stage 3). The simulations also explain why Rpn10 has a higher affinity for K48-linked tetra-ubiquitin than for mono-ubiquitin and K48-linked di- and tri-ubiquitins. Simulation results expand on the current view that the flexible arm of Rpn10 acts as an extended fragment of α-helices and flexible coils in the recognition process.

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

PubMed

Wang, Chien-Hsiang; Huang, Yi-Chun; Chen, Pei-Yi; Cheng, Ying-Ju; Kao, Hsiu-Hua; Pi, Haiwei; Chien, Cheng-Ting

2017-05-10

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

PubMed

Rodrigues, Elizabeth M; Scudder, Samantha L; Goo, Marisa S; Patrick, Gentry N

2016-02-03

Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. Copyright © 2016 the authors 0270-6474/16/361590-06$15.00/0.

Impact of Thermal Annealing on Organic Photovoltaic Cells Using Regioisomeric Donor-Acceptor-Acceptor Molecules.

PubMed

Zhang, Tao; Han, Han; Zou, Yunlong; Lee, Ying-Chi; Oshima, Hiroya; Wong, Ken-Tsung; Holmes, Russell J

2017-08-02

We report a promising set of donor-acceptor-acceptor (D-A-A) electron-donor materials based on coplanar thieno[3,2-b]/[2,3-b]indole, benzo[c][1,2,5]thiadiazole, and dicyanovinylene, which are found to show broadband absorption with high extinction coefficients. The role of the regioisomeric electron-donating thienoindole moiety on the physical and structural properties is examined. Bulk heterojunction (BHJ) organic photovoltaic cells (OPVs) based on the thieno[2,3-b]indole-based electron donor NTU-2, using C 70 as an electron acceptor, show a champion power conversion efficiency of 5.2% under AM 1.5G solar simulated illumination. This efficiency is limited by a low fill factor (FF), as has previously been the case in D-A-A systems. In order to identify the origin of the limited FF, further insight into donor layer charge-transport behavior is realized by examining planar heterojunction OPVs, with emphasis on the evolution of film morphology with thermal annealing. Compared to as-deposited OPVs that exhibit insufficient donor crystallinity, crystalline OPVs based on annealed thin films show an increase in the short-circuit current density, FF, and power conversion efficiency. These results suggest that that the crystallization of D-A-A molecules might not be realized spontaneously at room temperature and that further processing is needed to realize efficient charge transport in these materials.

Halocarbons as hydrogen bond acceptors: a spectroscopic study of haloethylbenzenes (PhCH2CH2X, X = F, Cl, Br) and their hydrate clusters.

PubMed

Robertson, Patrick A; Villani, Luigi; Dissanayake, Uresha L M; Duncan, Luke F; Abbott, Belinda M; Wilson, David J D; Robertson, Evan G

2018-03-28

The electronic spectra of 2-bromoethylbenzene and its chloro and fluoro analogues have been recorded by resonant two-photon ionisation (R2PI) spectroscopy. Anti and gauche conformers have been assigned by rotational band contour analysis and IR-UV ion depletion spectroscopy in the CH region. Hydrate clusters of the anti conformers have also been observed, allowing the role of halocarbons as hydrogen bond acceptors to be examined in this context. The donor OH stretch of water bound to chlorine is red-shifted by 36 cm -1 , or 39 cm -1 in the case of bromine. Although classed as weak H-bond acceptors, halocarbons are favourable acceptor sites compared to π systems. Fluorine stands out as the weakest H-bond acceptor amongst the halogens. Chlorine and bromine are also weak H-bond acceptors, but allow for more geometric lability, facilitating complimentary secondary interactions within the host molecule. Ab initio and DFT quantum chemical calculations, both harmonic and anharmonic, aid the structural assignments and analysis.

Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

PubMed Central

Liu, Chao; Liu, Weixiao; Ye, Yihong; Li, Wei

2017-01-01

Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome. PMID:28165462

Activity‐Based Probes for HECT E3 Ubiquitin Ligases

PubMed Central

Byrne, Robert; Mund, Thomas

2017-01-01

Abstract Activity‐based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine‐based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin‐ABPs (Ub‐VME and Ub‐PA) and a novel set of E2–Ub‐ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin‐ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub‐ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub‐PA and His‐UBE2D2–Ub‐ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases. PMID:28425671

Satisfied IUD acceptors as family planning motivators in Sri Lanka.

PubMed

Fisher, A A; de Silva, V

1986-01-01

In this study, government midwives were teamed with currently satisfied IUD acceptors to strengthen field motivational and recruitment efforts. The objective was to increase the number of new IUD acceptors. In the experimental study areas, a total of 3,019 new IUD acceptors were recruited. Time series regression analysis revealed a significant difference between the experimental and comparison areas that was over and above what might be expected on the basis of the past history of differences between these two areas. These and other findings suggest that teaming currently satisfied acceptors with government field-workers can have a substantial impact on the recruitment of new family planning users.

Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

PubMed

Zhao, Huzi; Zhang, Lina; Zhang, Yongchen; Zhao, Lei; Wan, Qing; Wang, Bei; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-05-30

The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

Influence of acceptor on charge mobility in stacked π-conjugated polymers

NASA Astrophysics Data System (ADS)

Sun, Shih-Jye; Menšík, Miroslav; Toman, Petr; Gagliardi, Alessio; Král, Karel

2018-02-01

We present a quantum molecular model to calculate mobility of π-stacked P3HT polymer layers with electron acceptor dopants coupled next to side groups in random position with respect to the linear chain. The hole density, the acceptor LUMO energy and the hybridization transfer integral between the acceptor and polymer were found to be very critical factors to the final hole mobility. For a dopant LUMO energy close and high above the top of the polymer valence band we have found a significant mobility increase with the hole concentration and with the dopant LUMO energy approaching the top of the polymer valence band. Higher mobility was achieved for small values of hybridization transfer integral between polymer and the acceptor, corresponding to the case of weakly bound acceptor. Strong couplings between the polymer and the acceptor with Coulomb repulsion interactions induced from the electron localizations was found to suppress the hole mobility.

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

PubMed Central

Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

2015-01-01

ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

E3 ubiquitin ligases: key regulators of hormone signaling in plants.

PubMed

Kelley, Dior

2018-03-07

Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. While E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Characteristics of ovulation method acceptors: a cross-cultural assessment.

PubMed

Klaus, H; Labbok, M; Barker, D

1988-01-01

Five programs of instruction in the ovulation method (OM) in diverse geographic and cultural settings are described, and characteristics of approximately 200 consecutive OM acceptors in each program are examined. Major findings include: the religious background and family size of acceptors are variable, as is the level of previous contraceptive use. Acceptors are drawn from a wide range of socioeconomic and religious backgrounds; however, family planning intention was similarly distributed in all five countries. In sum, the ovulation method is accepted by persons from a variety of backgrounds within and between cultural setting.

TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic ALS are low frequency and widely distributed in the lower motor neuron columns independent of disease spread

PubMed Central

Bodansky, Aaron; Kim, Jae Mun ‘Hugo’; Tempest, Lynne; Velagapudi, Amit; Libby, Ryan; Ravits, John

2010-01-01

Abstract Ubiqitinated and TDP-43 immunoreactive cytoplasmic aggregates are hallmark features of ALS molecular pathology. Since clinically most ALS begins focally and advances contiguously, it is important to characterize their distribution. Our objective was to determine the extent and distribution of TDP-43 immunoreactive aggregates in the lower motor neuron columns as a function of disease onset, and to correlate ubiquitinated with TDP-43 aggregates in the lumbar region. We examined TDP-43 cytoplasmic aggregates at four separate neuraxis levels – hypoglossal nucleus and cervical, thoracic, and lumbar anterior horns – in five controls and 20 sporadic ALS nervous systems from patients whose disease began in various sites, i.e. five bulbar, five arm, five trunk, and five leg onsets. We correlated ubiquitinated to TDP-43 aggregates on adjacent histological sections for the lumbar regions. We found that TDP-43 cytoplasmic aggregates are seen in about 8% of motor neurons but there is marked variability between nervous systems, ranging from 0.4% to 20.6%. The aggregates are uniformly distributed within individual nervous systems. There is no obvious correlation between site of disease onset and rate of spread. Almost all ubiquitinated aggregates correlate to TDP-43 aggregates. Thus, TDP-43 immunoreactive cytoplasmic aggregates have a low overall average frequency that does not correlate with either disease course or clinical spread and is the prime ubiquitinated protein. PMID:20225928

BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus*

PubMed Central

Kang, Eugene L.; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-01-01

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules. PMID:23109336

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer

PubMed Central

Nguyen, Thanh Hung

2018-01-01

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system. PMID:29673168

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer.

PubMed

Nguyen, Thanh Hung; Kugler, Jan-Michael

2018-04-17

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system.

Constructing and decoding unconventional ubiquitin chains.

PubMed

Behrends, Christian; Harper, J Wade

2011-05-01

One of the most notable discoveries in the ubiquitin system during the past decade is the extensive use of diverse chain linkages to control signaling networks. Although the utility of Lys48- and Lys63-linked chains in protein turnover and molecular assembly, respectively, are well known, we are only beginning to understand how unconventional chain linkages are formed on target proteins and how such linkages are decoded by specific binding proteins. In this review, we summarize recent efforts to elucidate the machinery and mechanisms controlling assembly of Lys11-linked and linear (or Met1-linked) ubiquitin chains, and describe current models for how these chain types function in immune signaling and cell-cycle control.

UbSRD: The Ubiquitin Structural Relational Database.

PubMed

Harrison, Joseph S; Jacobs, Tim M; Houlihan, Kevin; Van Doorslaer, Koenraad; Kuhlman, Brian

2016-02-22

The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

PubMed

Kumar, Ish; Pratt, R F

2005-08-02

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

Skp2 regulates androgen receptor through ubiquitin-mediated degradation independent of Akt/mTOR pathways in prostate cancer.

PubMed

Li, Bo; Lu, Wenfu; Yang, Qing; Yu, Xiuping; Matusik, Robert J; Chen, Zhenbang

2014-04-01

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling. © 2013 Wiley Periodicals, Inc.

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

PubMed

Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

A20 restricts ubiquitination of pro-interleukin-1β protein complexes and suppresses NLRP3 inflammasome activity

PubMed Central

Duong, Bao H.; Onizawa, Michio; Oses-Prieto, Juan A.; Advincula, Rommel; Burlingame, Alma; Malynn, Barbara A.; Ma, Averil

2015-01-01

SUMMARY Inappropriate inflammasome activation contributes to multiple human diseases, but the mechanisms by which inflammasomes are suppressed are poorly understood. The NFκB inhibitor A20 is a ubiquitin-modifying enzyme that may prevent human inflammatory diseases and lymphomas. Here, we report that A20-deficient macrophages, unlike normal cells, exhibit spontaneous NLRP3 inflammasome activity to LPS alone. The kinase RIPK3, but not the adaptor MyD88, is required for this response. In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 recruitment to the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1 and caspase-8 in a complex that is modified with K63-linked and unanchored polyubiquitin. In A20-deficient macrophages, this pro-IL-1β-associated ubiquitination is markedly increased in a RIPK3-dependent manner. Mass spectrometric and mutational analyses reveal that K133 of pro-IL-1β is a physiological ubiquitination site that supports processing. Our study reveals a novel mechanism by which A20 prevents inflammatory diseases. PMID:25607459

Ubiquitin-Fused and/or Multiple Early Genes from Cottontail Rabbit Papillomavirus as DNA Vaccines

PubMed Central

Leachman, Sancy A.; Shylankevich, Mark; Slade, Martin D.; Levine, Dana; K. Sundaram, Ranjini; Xiao, Wei; Bryan, Marianne; Zelterman, Daniel; Tiegelaar, Robert E.; Brandsma, Janet L.

2002-01-01

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection. PMID:12097575

Fragment charge difference method for estimating donor-acceptor electronic coupling: Application to DNA π-stacks

NASA Astrophysics Data System (ADS)

Voityuk, Alexander A.; Rösch, Notker

2002-09-01

The purpose of this communication is two-fold. We introduce the fragment charge difference (FCD) method to estimate the electron transfer matrix element HDA between a donor D and an acceptor A, and we apply this method to several aspects of hole transfer electronic couplings in π-stacks of DNA, including systems with several donor-acceptor sites. Within the two-state model, our scheme can be simplified to recover a convenient estimate of the electron transfer matrix element HDA=(1-Δq2)1/2(E2-E1)/2 based on the vertical excitation energy E2-E1 and the charge difference Δq between donor and acceptor. For systems with strong charge separation, Δq≳0.95, one should resort to the FCD method. As favorable feature, we demonstrate the stability of the FCD approach for systems which require an approach beyond the two-state model. On the basis of ab initio calculations of various DNA related systems, we compared three approaches for estimating the electronic coupling: the minimum splitting method, the generalized Mulliken-Hush (GMH) scheme, and the FCD approach. We studied the sensitivity of FCD and GMH couplings to the donor-acceptor energy gap and found both schemes to be quite robust; they are applicable also in cases where donor and acceptor states are off resonance. In the application to π-stacks of DNA, we demonstrated for the Watson-Crick pair dimer [(GC),(GC)] how structural changes considerably affect the coupling strength of electron hole transfer. For models of three Watson-Crick pairs, we showed that the two-state model significantly overestimates the hole transfer coupling whereas simultaneous treatment of several states leads to satisfactory results.

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

Bioinformatic investigation of the role of ubiquitins in cucumber flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena; Osipowski, Paweł; Wojcieszek, Michał; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2016-09-01

Three cDNA clones were used to screen cucumber genome in order to find genes and proteins. Functional annotation reveals that they are correlated with ubiquitination pathways. Various bioinformatics tools were used to screen and check protein sequences features such as: the presence of specific domains, transmembrane regions, cleavage site and cellular placement. The computational analysis for promotor region shows many binding sites for transcription factors, which could regulate the expression of genes. In order to check gene expression levels in developing flower buds of monoecious (B10) and gynoecious (2gg) cucumber lines, the real - time PCR technique was applied. The expression was checked for the whole buds and only for the 3rd and 4th whorls of bud when generative organ are form which were obtained by Laser Capture Microdissection (LCM) technique.

Characteristics of contraceptive acceptors in an urban Nigerian setting.

PubMed

Ayangade, O

1984-02-01

Intensive efforts in promoting family planning concepts and contraceptive delivery in the Third World over the past two- and one-half decades have yielded only token dividends . This has occurred in Nigeria, despite the favorable government attitude. A study of the characteristics of current contraceptive acceptors showed an overwhelming percentage of acceptors are uneducated , married and from the lower socioeconomic class, a striking departure from usual expectations. Most acceptors prefer oral contraceptives. The status of acceptance of modern contraception by the educated population is still undetermined. Contraceptives appeared to be used primarily by women aged 30 and older in our population.

USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin

PubMed Central

Durcan, Thomas M; Tang, Matthew Y; Pérusse, Joëlle R; Dashti, Eman A; Aguileta, Miguel A; McLelland, Gian-Luca; Gros, Priti; Shaler, Thomas A; Faubert, Denis; Coulombe, Benoit; Fon, Edward A

2014-01-01

Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. PMID:25216678

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

2013-11-01

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

Prohibitin-mediated mitochondrial ubiquitination during spermiogenesis in Chinese mitten crab Eriocheir sinensis

PubMed Central

Hou, Cong-Cong; Wei, Chao-Guang; Lu, Cheng-Peng; Gao, Xin-Ming; Yang, Wan-Xi; Zhu, Jun-Quan

2017-01-01

The sperm of Eriocheir sinensis has a cup-shaped nucleus that contains several mitochondria embedded at the opening of the cup. The acrosome vesicle also contains derivants of mitochondria. The mitochondria distribution pattern involves a decrease in the number and changes in the structure and transportation of these organelles. The decreased number of sperm mitochondria is achieved through autophagy or the ubiquitination pathway. Prohibitin (PHB), the mitochondria inner membrane protein, is an evolutionarily highly conserved protein, is closely associated with spermatogenesis and sperm quality control and is also a potential substrate of ubiquitination. However, whether PHB protein mediates the ubiquitination pathway of sperm mitochondria in crustacean animals remains poorly understood. In the present study, we revealed that PHB, a substrate of ubiquitin, participates in the ubiquitination and degradation of mitochondria during spermiogenesis in E. sinensis. To confirm this finding, we used shRNA interference to reduce PHB expression and an overexpression technique to increase PHB expression in vitro. The interference experiment showed that the reduced PHB expression directly affected the polyubiquitination level and mitochondria status, whereas PHB overexpression markedly increased the polyubiquitination level. In vitro experiments also showed that PHB and its ubiquitination decide the fate of mitochondria. PMID:29228727

BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72.

PubMed

Schönbühler, Bianca; Schmitt, Verena; Huesmann, Heike; Kern, Andreas; Gamerdinger, Martin; Behl, Christian

2016-12-30

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

PubMed Central

Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

2015-01-01

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

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

PubMed Central

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

2014-01-01

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. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, 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 wild-type. 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. PMID:24361330

Anaerobic methanotrophy in tidal wetland: Effects of electron acceptors

NASA Astrophysics Data System (ADS)

Lin, Li-Hung; Yu, Zih-Huei; Wang, Pei-Ling

2016-04-01

Wetlands have been considered to represent the largest natural source of methane emission, contributing substantially to intensify greenhouse effect. Despite in situ methanogenesis fueled by organic degradation, methanotrophy also plays a vital role in controlling the exact quantity of methane release across the air-sediment interface. As wetlands constantly experience various disturbances of anthropogenic activities, biological burrowing, tidal inundation, and plant development, rapid elemental turnover would enable various electron acceptors available for anaerobic methanotrophy. The effects of electron acceptors on stimulating anaerobic methanotrophy and the population compositions involved in carbon transformation in wetland sediments are poorly explored. In this study, sediments recovered from tidally influenced, mangrove covered wetland in northern Taiwan were incubated under the static conditions to investigate whether anaerobic methanotrophy could be stimulated by the presence of individual electron acceptors. Our results demonstrated that anaerobic methanotrophy was clearly stimulated in incubations amended with no electron acceptor, sulfate, or Fe-oxyhydroxide. No apparent methane consumption was observed in incubations with nitrate, citrate, fumarate or Mn-oxides. Anaerobic methanotrophy in incubations with no exogenous electron acceptor appears to proceed at the greatest rates, being sequentially followed by incubations with sulfate and Fe-oxyhydroxide. The presence of basal salt solution stimulated methane oxidation by a factor of 2 to 3. In addition to the direct impact of electron acceptor and basal salts, incubations with sediments retrieved from low tide period yielded a lower rate of methane oxidation than from high tide period. Overall, this study demonstrates that anaerobic methanotrophy in wetland sediments could proceed under various treatments of electron acceptors. Low sulfate content is not a critical factor in inhibiting methane

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

Fluorescent and colorimetric molecular recognition probe for hydrogen bond acceptors.

PubMed

Pike, Sarah J; Hunter, Christopher A

2017-11-22

The association constants for formation of 1 : 1 complexes between a H-bond donor, 1-naphthol, and a diverse range of charged and neutral H-bond acceptors have been measured using UV/vis absorption and fluorescence emission titrations. The performance of 1-naphthol as a dual colorimetric and fluorescent molecular recognition probe for determining the H-bond acceptor (HBA) parameters of charged and neutral solutes has been investigated in three solvents. The data were employed to establish self-consistent H-bond acceptor parameters (β) for benzoate, azide, chloride, thiocyanate anions, a series of phosphine oxides, phosphate ester, sulfoxide and a tertiary amide. The results demonstrate both the transferability of H-bond parameters between different solvents and the utility of the naphthol-based dual molecular recognition probe to exploit orthogonal spectroscopic techniques to determine the HBA properties of neutral and charged solutes. The benzoate anion is the strongest HBA studied with a β parameter of 15.4, and the neutral tertiary amide is the weakest H-bond acceptor investigated with a β parameter of 8.5. The H-bond acceptor strength of the azide anion is higher than that of chloride (12.8 and 12.2 respectively), and the thiocyanate anion has a β value of 10.8 and thus is a significantly weaker H-bond acceptor than both the azide and chloride anions.

Pichia anomala DBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity▿

PubMed Central

De Ingeniis, Jessica; Raffaelli, Nadia; Ciani, Maurizio; Mannazzu, Ilaria

2009-01-01

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with β-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity. PMID:19114528

Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

PubMed Central

2012-01-01

Objective To investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6 (TRAF6),-and ubiquitin in gastric cancer patients. Methods Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed. Results TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82% (60/102) muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated with TNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression. Conclusions We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia. PMID:23013936

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

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements*

PubMed Central

Smit, Judith J.; van Dijk, Willem J.; El Atmioui, Dris; Merkx, Remco; Ovaa, Huib; Sixma, Titia K.

2013-01-01

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin. PMID:24030825

The Impact of Acceptor-Acceptor Homocoupling on the Optoelectronic Properties and Photovoltaic Performance of PDTSQxff Low Bandgap Polymers.

PubMed

Pirotte, Geert; Kesters, Jurgen; Cardeynaels, Tom; Verstappen, Pieter; D'Haen, Jan; Lutsen, Laurence; Champagne, Benoît; Vanderzande, Dirk; Maes, Wouter

2018-04-22

Push-pull-type conjugated polymers applied in organic electronics do not always contain a perfect alternation of donor and acceptor building blocks. Misscouplings can occur, which have a noticeable effect on the device performance. In this work, the influence of homocoupling on the optoelectronic properties and photovoltaic performance of PDTSQx ff polymers is investigated, with a specific focus on the quinoxaline acceptor moieties. A homocoupled biquinoxaline segment is intentionally inserted in specific ratios during the polymerization. These homocoupled units cause a gradually blue-shifted absorption, while the highest occupied molecular orbital energy levels decrease only significantly upon the presence of 75-100% of homocouplings. Density functional theory calculations show that the homocoupled acceptor unit generates a twist in the polymer backbone, which leads to a decreased conjugation length and a reduced aggregation tendency. The virtually defect-free PDTSQx ff affords a solar cell efficiency of 5.4%, which only decreases substantially upon incorporating a homocoupling degree over 50%. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spatial structure of single and interacting Mn acceptors in GaAs

NASA Astrophysics Data System (ADS)

Koenraad, Paul

2005-03-01

Ferromagnetic semiconductors such as Ga1-xMnxAs are receiving a lot of attention at the moment because of their application in spintronic devices. However, despite intense study of deep acceptors in III-V semiconductors such as MnGa, little information has been obtained on their electronic properties at the atomic scale. Yet the spatial shape of the Mn acceptor state will influence the hole-mediated Mn-Mn coupling and thus all of the magnetic properties of ferromagnetic semiconductors such as Ga1-xMnxAs. This study presents an experimental and theoretical description of the spatial symmetry of the Mn acceptor wave-function in GaAs. We present measurements of the spatial mapping of the anisotropic wavefunction of a hole localized at a Mn acceptor. To achieve this, we have used the STM tip not only to image the Mn acceptor but also to manipulate its charge state A^0/A^- at room temperature. Within an envelope function effective mass model (EFM) the anisotropy in the acceptor wave-function can be traced to the influence of the cubic symmetry of the GaAs crystal which selects specific d-states that mix into the ground state due to the spin-orbit interaction in the valence band. Comparison with calculations based on a tight-binding model (TBM) for the Mn acceptor structure supports this conclusion. Using the same experimental and theoretical approach we furthermore explored the interaction between Mn acceptors directly by analyzing close Mn-Mn pairs, which were separated by less than 2 nm. We will discuss some implications of these results for Mn delta-doped layers grown on differently oriented growth surfaces.

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor-Mediated Activation of Akt Signaling

PubMed Central

Yang, Wei-Lei; Jin, Guoxiang; Li, Chien-Feng; Jeong, Yun Seong; Moten, Asad; Xu, Dazhi; Feng, Zizhen; Chen, Wei; Cai, Zhen; Darnay, Bryant; Gu, Wei; Lin, Hui-Kuan

2013-01-01

K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor-mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor-induced Akt ubiquitination have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. Here, we showed that CYLD was a DUB for Akt and suppressed growth factor-mediated Akt ubiquitination and activation. CYLD directly removed ubiquitin moieties on Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake and growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in its membrane recruitment and activation, and further identifies CYLD as a molecular switch for these processes. PMID:23300340

Inhibition of the ubiquitin-proteasome system by natural products for cancer therapy.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2010-08-01

The ubiquitin-proteasome system plays a critical role in selective protein degradation and regulates almost all cellular events such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein quality control, development, and neuronal function. The recent approval of bortezomib, a synthetic proteasome inhibitor, for the treatment of relapsed multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and ubiquitinating and deubiquitinating enzymes as well as the delivery system. To date, various synthetic and natural products have been reported to inhibit the components of the ubiquitin-proteasome system. Here, we review natural products targeting the ubiquitin-proteasome system as well as synthetic compounds with potent inhibitory effects. Georg Thieme Verlag KG Stuttgart-New York.

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

Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

PubMed

Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

2016-07-15

The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. © 2016 Höckner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Next-generation organic photovoltaics based on non-fullerene acceptors

NASA Astrophysics Data System (ADS)

Cheng, Pei; Li, Gang; Zhan, Xiaowei; Yang, Yang

2018-03-01

Over the past three years, a particularly exciting and active area of research within the field of organic photovoltaics has been the use of non-fullerene acceptors (NFAs). Compared with fullerene acceptors, NFAs possess significant advantages including tunability of bandgaps, energy levels, planarity and crystallinity. To date, NFA solar cells have not only achieved impressive power conversion efficiencies of 13-14%, but have also shown excellent stability compared with traditional fullerene acceptor solar cells. This Review highlights recent progress on single-junction and tandem NFA solar cells and research directions to achieve even higher efficiencies of 15-20% using NFA-based organic photovoltaics are also proposed.

Hidden targets of ubiquitin proteasome system: To prevent diabetic nephropathy.

PubMed

Goru, Santosh Kumar; Kadakol, Almesh; Gaikwad, Anil Bhanudas

2017-06-01

Diabetic nephropathy (DN) is the major cause of end stage renal failure. Although, several therapeutic targets have emerged to prevent the progression of DN, the number of people with DN still continues to rise worldwide, suggesting an urgent need of novel targets to prevent DN completely. Currently, the role of ubiquitin proteasome system (UPS) has been highlighted in the pathogenesis and progression of various diseases like obesity, insulin resistance, atherosclerosis, cancers, neurodegerative disorders and including secondary complications of diabetes. UPS mainly involves in protein homeostatis through ubiquitination (post translational modification) and proteasomal degradation of various proteins. Ubiquitination, not only involves in proteasomal degradation, but also directs the substrate proteins to participate in multitude of cell signalling pathways. However, very little is known about ubiquitination and UPS in the progression of DN. This review mainly focuses on UPS and its components including E2 conjugating enzymes, E3 ligases and deubiquitinases (DUBs) in the development of DN and thus may help us to find novel therapeutic targets with in UPS to prevent DN completely in future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction

Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells

DOE PAGES

Yu M. Zhong; Nam, Chang -Yong; Trinh, M. Tuan; ...

2015-09-18

Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealedmore » both electron and hole transfer processes at the donor–acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. As a result, this study describes a new motif for designing highly efficient acceptors for organic solar cells.« less

Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells.

PubMed

Zhong, Yu; Trinh, M Tuan; Chen, Rongsheng; Purdum, Geoffrey E; Khlyabich, Petr P; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y; Black, Charles T; Steigerwald, Michael L; Loo, Yueh-Lin; Ng, Fay; Zhu, X-Y; Nuckolls, Colin

2015-09-18

Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor-acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells.

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

PubMed

González-López, Lorena; Carballar-Lejarazú, Rebeca; Arrevillaga Boni, Gerardo; Cortés-Martínez, Leticia; Cázares-Raga, Febe Elena; Trujillo-Ocampo, Abel; Rodríguez, Mario H; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

2017-01-01

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Investigating charge generation in polymer:non-fullerene acceptor bulk heterojunction films

DOE PAGES

Stoltzfus, Dani M.; Larson, Bryon W.; Zarrabi, Nasim; ...

2018-01-31

Non-fullerene acceptors are now capable of being used in high efficiency bulk heterojunction (BHJ) donor-acceptor organic solar cells. Acceptors comprising single or multiple linked chromophores have been used. We have developed a new non-fullerene molecular acceptor as well as two non-polymeric macromolecular materials that contain four equivalents of a similar chromophore, but can adopt different spatial arrangements of the chromophores. We compare the effect of having single and multiple chromophores within a macromolecule on the charge generation processes in P3HT:non-fullerene acceptor BHJ films using Transient Absorption Spectroscopy (TAS) and Time Resolved Microwave Conductivity (TRMC) measurements. It was found from themore » TAS measurements that at low weight percent (5 wt%) the single chromophore formed more polarons than the acceptors in which chromophores were linked, due to it having a more even distribution within the film. At higher concentrations (50 wt%) the trend was reversed due to the single chromophore forming crystalline domains, which reduced the interface area with the P3HT donor. The TRMC measurements showed that more mobile carriers were formed in the macromolecular acceptors when used at low concentrations in the blend and, independent of concentration, mobile carriers had a longer lifetime when compared to films containing the molecular material, which we ascribe to the charges being able to sample more than one chromophore and thus reduce recombination events.« less

Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

PubMed

Wirtz, Hanna; Schäfer, Sarah; Hoberg, Claudius; Reid, Korey M; Leitner, David M; Havenith, Martina

2018-06-04

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

Emerging Role of Ubiquitination in Antiviral RIG-I Signaling

PubMed Central

Maelfait, Jonathan

2012-01-01

Summary: Detection of viruses by the innate immune system involves the action of specialized pattern recognition receptors. Intracellular RIG-I receptors sense the presence of viral nucleic acids in infected cells and trigger signaling pathways that lead to the production of proinflammatory and antiviral proteins. Over the past few years, posttranslational modification of RIG-I and downstream signaling proteins by different types of ubiquitination has been found to be a key event in the regulation of RIG-I-induced NF-κB and interferon regulatory factor 3 (IRF3) activation. Multiple ubiquitin ligases, deubiquitinases, and ubiquitin binding scaffold proteins contribute to both positive and negative regulation of the RIG-I-induced antiviral immune response. A better understanding of the function and activity of these proteins might eventually lead to the development of novel therapeutic approaches for management of viral diseases. PMID:22390971

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

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

PubMed Central

Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi

2003-01-01

Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP. PMID:12634850

Proteasomal Ubiquitin Receptor RPN-10 Controls Sex Determination in Caenorhabditis elegans

PubMed Central

Shimada, Masumi; Kanematsu, Kenji; Tanaka, Keiji; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-binding RPN-10 protein serves as a ubiquitin receptor that delivers client proteins to the 26S proteasome. Although ubiquitin recognition is an essential step for proteasomal destruction, deletion of the rpn-10 gene in yeast does not influence viability, indicating redundancy of the substrate delivery pathway. However, their specificity and biological relevance in higher eukaryotes is still enigmatic. We report herein that knockdown of the rpn-10 gene, but not any other proteasome subunit genes, sexually transforms hermaphrodites to females by eliminating hermaphrodite spermatogenesis in Caenorhabditis elegans. The feminization phenotype induced by deletion of the rpn-10 gene was rescued by knockdown of tra-2, one of sexual fate decision genes promoting female development, and its downstream target tra-1, indicating that the TRA-2–mediated sex determination pathway is crucial for the Δrpn-10–induced sterile phenotype. Intriguingly, we found that co-knockdown of rpn-10 and functionally related ubiquitin ligase ufd-2 overcomes the germline-musculinizing effect of fem-3(gf). Furthermore, TRA-2 proteins accumulated in rpn-10-defective worms. Our results show that the RPN-10–mediated ubiquitin pathway is indispensable for control of the TRA-2–mediated sex-determining pathway. PMID:17050737

Structural basis of ubiquitin modification by the Legionella effector SdeA.

PubMed

Dong, Yanan; Mu, Yajuan; Xie, Yongchao; Zhang, Yupeng; Han, Youyou; Zhou, Yu; Wang, Wenhe; Liu, Zihe; Wu, Mei; Wang, Hao; Pan, Man; Xu, Ning; Xu, Cong-Qiao; Yang, Maojun; Fan, Shilong; Deng, Haiteng; Tan, Tianwei; Liu, Xiaoyun; Liu, Lei; Li, Jun; Wang, Jiawei; Fang, Xianyang; Feng, Yue

2018-05-01

Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

PubMed Central

Xu, Yingqi; Plechanovová, Anna; Simpson, Peter; Marchant, Jan; Leidecker, Orsolya; Kraatz, Sebastian; Hay, Ronald T.; Matthews, Steve J.

2014-01-01

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer. PMID:24969970

SUMOylation Regulates the Homologous to E6-AP Carboxyl Terminus (HECT) Ubiquitin Ligase Rsp5p*

PubMed Central

Novoselova, Tatiana Vladislavovna; Rose, Ruth-Sarah; Marks, Helen Margaret; Sullivan, James Andrew

2013-01-01

The post-translational modifiers ubiquitin and small ubiquitin-related modifier (SUMO) regulate numerous critical signaling pathways and are key to controlling the cellular fate of proteins in eukaryotes. The attachment of ubiquitin and SUMO involves distinct, but related, machinery. However, it is now apparent that many substrates can be modified by both ubiquitin and SUMO and that some regulatory interaction takes place between the respective attachment machinery. Here, we demonstrate that the Saccharomyces cerevisiae ubiquitin ligase Rsp5p, a member of the highly conserved Nedd4 family of ubiquitin ligases, is SUMOylated in vivo. We further show that Rsp5p SUMOylation is mediated by the SUMO ligases Siz1p and Siz2p, members of the conserved family of PIAS SUMO ligases that are, in turn, substrates for Rsp5p-mediated ubiquitylation. Our experiments show that SUMOylated Rsp5p has reduced ubiquitin ligase activity, and similarly, ubiquitylated Siz1p demonstrates reduced SUMO ligase activity leading to respective changes in both ubiquitin-mediated sorting of the manganese transporter Smf1p and polySUMO chain formation. This reciprocal regulation of these highly conserved ligases represents an exciting and previously unidentified system of cross talk between the ubiquitin and SUMO systems. PMID:23443663

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

Three holes bound to a double acceptor - Be(+) in germanium

NASA Technical Reports Server (NTRS)

Haller, E. E.; Mcmurray, R. E., Jr.; Falicov, L. M.; Haegel, N. M.; Hansen, W. L.

1983-01-01

A double acceptor binding three holes has been observed for the first time with photoconductive far-infrared spectroscopy in beryllium-doped germanium single crystals. This new center, Be(+), has a hole binding energy of about 5 meV and is only present when free holes are generated by ionization of either neutral shallow acceptors or neutral Be double acceptors. The Be(+) center thermally ionizes above 4 K. It disappears at a uniaxial stress higher than about a billion dyn/sq cm parallel to (111) as a result of the lifting of the valence-band degeneracy.

Molecular piracy: manipulation of the ubiquitin system by Kaposi's sarcoma-associated herpesvirus.

PubMed

Fujimuro, Masahiro; Hayward, S Diane; Yokosawa, Hideyoshi

2007-01-01

Ubiquitination, one of several post-translational protein modifications, plays a key role in the regulation of cellular events, including protein degradation, signal transduction, endocytosis, protein trafficking, apoptosis and immune responses. Ubiquitin attachment at the lysine residue of cellular factors acts as a signal for endocytosis and rapid degradation by the 26S proteasome. It has recently been observed that viruses, especially oncogenic herpesviruses, utilise molecular piracy by encoding their own proteins to interfere with regulation of cell signalling. Kaposi's sarcoma- associated herpesvirus (KSHV) manipulates the ubiquitin system to facilitate cell proliferation, anti-apoptosis and evasion from immunity. In this review, we will describe the strategies used by KSHV at distinct stages of the viral life-cycle to control the ubiquitin system and promote oncogenesis and viral persistence. (c) 2007 John Wiley & Sons, Ltd.

Improving Photoconductance of Fluorinated Donors with Fluorinated Acceptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garner, Logan E.; Larson, Bryon; Oosterhout, Stefan

2016-11-21

This work investigates the influence of fluorination of both donor and acceptor materials on the generation of free charge carriers in small molecule donor/fullerene acceptor BHJ OPV active layers. A fluorinated and non-fluorinated small molecule analogue were synthesized and their optoelectronic properties characterized. The intrinsic photoconductance of blends of these small molecule donors was investigated using time-resolved microwave conductivity. Blends of the two donor molecules with a traditional non-fluorinated fullerene (PC70BM) as well as a fluorinated fullerene (C60(CF3)2-1) were investigated using 5% and 50% fullerene loading. We demonstrate for the first time that photoconductance in a 50:50 donor:acceptor BHJ blendmore » using a fluorinated fullerene can actually be improved relative to a traditional non-fluorinated fullerene by fluorinating the donor molecule as well.« less

Acceptor binding energies in GaN and AlN

NASA Astrophysics Data System (ADS)

Mireles, Francisco; Ulloa, Sergio E.

1998-08-01

We employ effective-mass theory for degenerate hole bands to calculate the acceptor binding energies for Be, Mg, Zn, Ca, C, and Si substitutional acceptors in GaN and AlN. The calculations are performed through the 6×6 Rashba-Sheka-Pikus and the Luttinger-Kohn matrix Hamiltonians for wurtzite (WZ) and zinc-blende (ZB) crystal phases, respectively. An analytic representation for the acceptor pseudopotential is used to introduce the specific nature of the impurity atoms. The energy shift due to polaron effects is also considered in this approach. The ionization energy estimates are in very good agreement with those reported experimentally in WZ GaN. The binding energies for ZB GaN acceptors are all predicted to be shallower than the corresponding impurities in the WZ phase. The binding-energy dependence upon the crystal-field splitting in WZ GaN is analyzed. Ionization levels in AlN are found to have similar ``shallow'' values to those in GaN, but with some important differences which depend on the band structure parametrizations, especially the value of the crystal-field splitting used.

Regulation of Synaptic Structure by the Ubiquitin C-terminal Hydrolase UCH-L1

PubMed Central

Cartier, Anna E.; Djakovic, Stevan N.; Salehi, Afshin; Wilson, Scott M.; Masliah, Eliezer; Patrick, Gentry N.

2009-01-01

UCH-L1 is a de-ubiquitinating 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 have found that UCH-L1 activity is rapidly up-regulated 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 pre 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. PMID:19535597

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA domain Ubiquitination

PubMed Central

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K.; Keith, Amy L.; Razani, Babak; Weihl, Conrad C.

2017-01-01

Summary p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar to that seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT expressing cells increases ubiquitinated inclusion formation, p62’s association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62 associated disease. PMID:28380357

Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region.

PubMed

Yu, Houqing; Kago, Grace; Yellman, Christopher M; Matouschek, Andreas

2016-07-15

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin-like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin-like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin-like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin-like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation. © 2016 The Authors.

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

PubMed

O'Connor, Hazel F; Huibregtse, Jon M

2017-09-01

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Mori, Chie; Osakada, Hiroko; Kobayashi, Shouhei; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-03-01

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) Promotes Hippocampus-Dependent Memory via Its Deubiquitinating Effect on TrkB.

PubMed

Guo, Yun-Yun; Lu, Yi; Zheng, Yuan; Chen, Xiao-Rong; Dong, Jun-Lu; Yuan, Rong-Rong; Huang, Shu-Hong; Yu, Hui; Wang, Yue; Chen, Zhe-Yu; Su, Bo

2017-06-21

Multiple studies have established that brain-derived neurotrophic factor (BDNF) plays a critical role in the regulation of synaptic plasticity via its receptor, TrkB. In addition to being phosphorylated, TrkB has also been demonstrated to be ubiquitinated. However, the mechanisms of TrkB ubiquitination and its biological functions remain poorly understood. In this study, we demonstrate that ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes contextual fear conditioning learning and memory via the regulation of ubiquitination of TrkB. We provide evidence that UCH-L1 can deubiquitinate TrkB directly. K460 in the juxtamembane domain of TrkB is the primary ubiquitination site and is regulated by UCH-L1. By using a peptide that competitively inhibits the association between UCH-L1 and TrkB, we show that the blockade of UCH-L1-regulated TrkB deubiquitination leads to increased BDNF-induced TrkB internalization and consequently directs the internalized TrkB to the degradation pathway, resulting in increased degradation of surface TrkB and attenuation of TrkB activation and its downstream signaling pathways. Moreover, injection of the peptide into the DG region of mice impairs hippocampus-dependent memory. Together, our results suggest that the ubiquitination of TrkB is a mechanism that controls its downstream signaling pathways via the regulation of its endocytosis and postendocytic trafficking and that UCH-L1 mediates the deubiquitination of TrkB and could be a potential target for the modulation of hippocampus-dependent memory. SIGNIFICANCE STATEMENT Ubiquitin C-terminal hydrolase L1 (UCH-L1) has been demonstrated to play important roles in the regulation of synaptic plasticity and learning and memory. TrkB, the receptor for brain-derived neurotrophic factor, has also been shown to be a potent regulator of synaptic plasticity. In this study, we demonstrate that UCH-L1 functions as a deubiquitinase for TrkB. The blockage of UCH-L1-regulated deubiquitination of Trk

The role of spartin and its novel ubiquitin binding region in DALIS occurrence

PubMed Central

Karlsson, Amelia B.; Washington, Jacqueline; Dimitrova, Valentina; Hooper, Christopher; Shekhtman, Alexander; Bakowska, Joanna C.

2014-01-01

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains—a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively—have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63–linked ubiquitin chains but does not bind Lys-48–linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR. PMID:24523286

2012 Gordon Research Conference, Electron donor-acceptor interactions, August 5-10 2012

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCusker, James

The upcoming incarnation of the Gordon Research Conference on Electron Donor Acceptor Interactions will feature sessions on classic topics including proton-coupled electron transfer, dye-sensitized solar cells, and biological electron transfer, as well as emerging areas such as quantum coherence effects in donor-acceptor interactions, spintronics, and the application of donor-acceptor interactions in chemical synthesis.

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.

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

PubMed Central

Orian, Amir; Schwartz, Alan L.; Israël, Alain; Whiteside, Simon; Kahana, Chaim; Ciechanover, Aaron

1999-01-01

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-κB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif. PMID:10207090

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*

PubMed Central

Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki

2013-01-01

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

PubMed Central

He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.