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Sample records for ubiquitin-proteasome degradation pathway

  1. Kip1 degradation via the ubiquitin-proteasome pathway.

    PubMed

    Tam, S W; Theodoras, A M; Pagano, M

    1997-04-01

    The cell cycle has been the object of extensive studies for the past years. A complex network of molecular interactions has been identified. In particular, a class of cell cycle inhibitory proteins has been identified but details of the molecular mechanism of their action have yet to be resolved. These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity. The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis. Kip1 is a potent inhibitor of Cdks. In quiescent cells Kip1 accumulates without an increase in mRNA or protein synthesis. We demonstrated that cell cycle regulation of Kip1 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway. In a crude in vitro system, Kip1 is ubiquitinated and degraded in an ATP dependent manner and inhibition or depletion of the proteasome blocks Kip1 degradation. Human Ubc2 and Ubc3, the homologs of yeast Rad6 and Cdc34 gene products respectively, are specifically involved in the ubiquitination of Kip1. Compared to proliferating cells, quiescent cells contain a far lower amount of Kip1 ubiquitinating activity. These results represent the first demonstration that the ubiquitin-proteasome pathway plays a role in the regulation of a cell cycle protein in human cells, namely the Cdk inhibitor Kip1. The specific proteolysis of Kip1 may be involved in the pathway of inactivation of Cdks. PMID:9209391

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

    ERIC Educational Resources Information Center

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

    2006-01-01

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

  3. Activin-?C modulates cachexia by repressing the ubiquitin-proteasome and autophagic degradation pathways

    PubMed Central

    Marino, Francesco Elia; Risbridger, Gail; Gold, Elspeth

    2015-01-01

    Background Cancer-associated cachexia and muscle wasting are considered key determinants of cancer-related death and reduction in the quality of life of cancer patients. A crucial link has been established between activin signaling and skeletal muscle atrophy-hypertrophy. We previously showed that activin-?C, a novel activin-A antagonist, is a tumor modulator that abolishes the cancer-associated cachexia in a mouse genetic model of gonadal tumorigenesis, in which the normal balance of inhibin/activin signalling is disrupted by a targeted mutation in the Inha gene (inhibin ?-KO mouse). This study aimed to identify the molecular mechanism by which activin-?C increases survival and abolishes cancer-associated cachexia in ?-KO mice. We hypothesized that overexpression of activin-?C modulates the cachexia phenotype by antagonizing the activin signaling pathway and repressing muscle wasting via the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways. Methods Male and female ActC++, ?-KO, and ?-KO/ActC++ mice and WT littermate controls were studied. Western blot analysis for the specific E3 ubiquitin ligases, atrogin-1 and MuRF1, markers of the autophagic-lysosomal pathway, Beclin-1, p62, and LC3A/B, effectors Smad-2, Smad-3 and myostatin was performed in the gastrocnemius of age-matched mice. Histopathology of the gastrocnemius and survival analysis were also conducted in animals from the same breeding cohort. Serum levels of activin-A, inflammatory cytokines, hormonal profile, and bone density were also assessed. Results Increased levels of atrogin-1, MuRF-1, Beclin-1, p62, LC3A/B-I, Smad-2 and serum levels of activin-A were noted in the ?-KO mice. These mice developed gonadal cancers followed by severe weight loss, and reduced survival. Overexpression of activin- ?C antagonized the activin signaling cascade, attenuating the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways, and reduced serum levels of activin-A. ?-KO/ActC++ mice displayed a less aggressive cachectic phenotype, reduced tumor weight, and prolonged survival. Conclusion Our findings show for the first time a specific effect of activin-?C on muscle wasting and transcription factors involved in muscle protein degradation. The study indicates that activin-?C may be a novel therapy to abrogate cancer-associated weight loss and prolong survival.

  4. Neuropathy Target Esterase Is Degraded by the Ubiquitin-Proteasome Pathway with ARA54 as the Ubiquitin Ligase.

    PubMed

    Long, Ding-Xin; Wang, Pan; Sun, Ying-Jian; Chen, Rui; Wu, Yi-Jun

    2015-12-22

    Neuropathy target esterase (NTE) is an endoplasmic reticulum membrane-associated phospholipase B, which is essential for embryonic and nervous system development. However, the regulation of NTE at the protein level had not been thoroughly investigated. Our previous study showed that NTE was degraded not only by the macroautophagy-lysosome pathway but also by the ubiquitin-proteasome pathway. Here we further reveal that androgen receptor-associated protein 54 (ARA54) regulated the ubiquitin-proteasome degradation of NTE. We find that deletion of the regulatory domain of NTE, which possesses a putative destruction box and thus is essential for its degradation by the proteasome, prevented its degradation by the proteasome. In addition, we demonstrate that ARA54, which has a RING finger domain and E3 ligase activity, interacts directly with NTE. Overexpression of ARA54 downregulates the protein level of NTE, and knockdown of ARA54 inhibits the degradation of NTE. The mutation in the RING domain of ARA54 blocks the degradation of NTE by ARA54, which indicates that the RING domain is essential for ARA54's E3 activity. These findings suggest that ARA54 acts as the ubiquitin ligase to regulate the ubiquitin-proteasome degradation of NTE. PMID:26606397

  5. Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin–proteasome pathway

    PubMed Central

    Nie, Xiao-hu; Ou-yang, Jia; Xing, Ying; Li, Dan-yan; Dong, Xing-yu; Liu, Ru-en; Xu, Ru-xiang

    2015-01-01

    We investigated the underlying mechanism for the potent proapoptotic effect of paeoniflorin (PF) on human glioma cells in vitro, focusing on signal transducer and activator of transcription 3 (STAT3) signaling. Significant time- and dose-dependent apoptosis and inhibition of proliferation were observed in PF-treated U87 and U251 glioma cells. Expression of STAT3, its active form phosphorylated STAT3 (p-STAT3), and several downstream molecules, including HIAP, Bcl-2, cyclin D1, and Survivin, were significantly downregulated upon PF treatment. Overexpression of STAT3 induced resistance to PF, suggesting that STAT3 was a critical target of PF. Interestingly, rapid downregulation of STAT3 was consistent with its accelerated degradation, but not with its dephosphorylation or transcriptional modulation. Using specific inhibitors, we demonstrated that the prodegradation effect of PF on STAT3 was mainly through the ubiquitin–proteasome pathway rather than via lysosomal degradation. These findings indicated that PF-induced growth suppression and apoptosis in human glioma cells through the proteasome-dependent degradation of STAT3. PMID:26508835

  6. REG? regulates ER? degradation via ubiquitin–proteasome pathway in breast cancer

    SciTech Connect

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Jiang, Jun

    2015-01-02

    Highlights: • High expression of REG? is correlated with ER? status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REG? knockdown. • REG? indirectly regulates ER? protein expression. - Abstract: REG? is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REG? is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REG? was highly expressed in breast cancers, and the expression of REG? was positively correlated with breast cancer patient estrogen receptor alpha (ER?) status. Moreover, the expression of REG? was found positively associated with poor clinical features and low survival rates in ER? positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REG? knockdown. Lastly, we demonstrated that REG? indirectly regulates the degradation of ER? protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REG? expression was positively correlated with ER? status and poor clinical prognosis in ER? positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

  7. Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1? via the N-terminal Pathway*

    PubMed Central

    Trausch-Azar, Julie; Leone, Teresa C.; Kelly, Daniel P.; Schwartz, Alan L.

    2010-01-01

    PGC-1? is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1? levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1? is rapidly degraded in the nucleus (t½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t½ 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1? and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1? is cytoplasmic and stable (t½ >7 h), providing additional evidence that PGC-1? is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1? cellular degradation. In contrast, the cellular localization of NT-PCG-1? results in a longer-half-life and possible distinct temporal and potentially biological actions. PMID:20713359

  8. Rapid degradation of Bim by the ubiquitin-proteasome pathway mediates short-term ischemic tolerance in cultured neurons.

    PubMed

    Meller, Robert; Cameron, Jennifer Anastasia; Torrey, Daniel John; Clayton, Corrin Erin; Ordonez, Andrea Nicole; Henshall, David Clifford; Minami, Manabu; Schindler, Clara Kay; Saugstad, Julie Anne; Simon, Roger Pancoast

    2006-03-17

    A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke. PMID:16431916

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin–proteasome pathway

    PubMed Central

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-01-01

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin–proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein. PMID:25375378

  11. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-01-01

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein. PMID:25375378

  12. Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin–proteasomal degradation

    PubMed Central

    Miao, Ji; Xiao, Zhen; Kanamaluru, Deepthi; Min, Gyesik; Yau, Peter M.; Veenstra, Timothy D.; Ellis, Ewa; Strom, Steve; Suino-Powell, Kelly; Xu, H. Eric; Kemper, Jongsook Kim

    2009-01-01

    Small Heterodimer Partner (SHP) inhibits activities of numerous transcription factors involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin–proteasomal pathway and that bile acids or bile acid-induced intestinal fibroblast growth factor 19 (FGF19) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes. PMID:19390091

  13. The triage of damaged proteins: degradation by the ubiquitin-proteasome pathway or repair by molecular chaperones.

    PubMed

    Marques, Carla; Guo, Weimin; Pereira, Paulo; Taylor, Allen; Patterson, Cam; Evans, Paul C; Shang, Fu

    2006-04-01

    Accumulation of damaged proteins is causally related to many age-related diseases. The ubiquitin-proteasome pathway (UPP) plays a role in selective degradation of damaged proteins, whereas molecular chaperones, such as heat shock proteins, are involved in refolding denatured proteins. This work demonstrates for the first time that the UPP and molecular chaperones work in a competitive manner and that the fates of denatured proteins are determined by the relative activities of the UPP and molecular chaperones. Enhanced UPP activity suppresses the refolding of denatured proteins whereas elevated chaperone activity inhibits the degradation of denatured proteins. CHIP, a co-chaperone with E3 activity, plays a pivotal role in determining the fates of the damaged proteins. The delicate balance between UPP-mediated degradation and refolding of denatured proteins is governed by relative levels of CHIP and other molecular chaperones. Isopeptidases, the enzymes that reverse the actions of CHIP, also play an important role in determining the fate of denatured proteins. PMID:16469848

  14. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  15. 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…

  16. Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications

    PubMed Central

    Huang, Qian; Figueiredo-Pereira, Maria E.

    2010-01-01

    The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention. PMID:20131003

  17. Tripartite motif 16 suppresses breast cancer stem cell properties through regulation of Gli-1 degradation via the ubiquitin-proteasome pathway.

    PubMed

    Yao, Juntao; Xu, Tao; Tian, Tao; Fu, Xiao; Wang, Wenjuan; Li, Suoni; Shi, Tingting; Suo, Aili; Ruan, Zhiping; Guo, Hui; Yao, Yu

    2016-02-01

    Cancer stem cells (CSCs) are responsible for cancer progression and patient prognosis. Tripartite motif 16 (TRIM16) is a proteasome coactivator that regulates proteolytic activity in eukaryotic cells. Abundant evidence has shown that TRIM16 is lowly expressed in a number of human carcinomas. In a previous study, we demonstrated that TRIM16 suppressed cancer malignancy and that TRIM16 expression levels were associated with favorable prognostic parameters of patients with cancer. However, the precise role of this motif in the pathogenesis of breast cancer remains unknown. In the present study, we examined 29 human breast cancer specimens, and found that TRIM16 was lowly expressed in breast cancers; thus, TRIM16 expression is negatively correlated with metastasis in breast cancer patients. Moreover, we showed that TRIM16 suppressed CSC properties in a population of breast cancer cells. TRIM16 depletion resulted in an increased proportion of CSCs relative to breast cancer cells when several assays were used to assess CSC properties. Finally, we demonstrated that TRIM16 directly regulated the degradation of Gli?1 protein via the ubiquitin?proteasome pathway. In conclusion, we propose that inhibition of CSC properties may be one of the functions of TRIM16 as a suppressor of breast cancer progression. PMID:26718507

  18. Ligand-dependent switching of ubiquitin–proteasome pathways for estrogen receptor

    PubMed Central

    Tateishi, Yukiyo; Kawabe, Yoh-ichi; Chiba, Tomoki; Murata, Shigeo; Ichikawa, Ken; Murayama, Akiko; Tanaka, Keiji; Baba, Tadashi; Kato, Shigeaki; Yanagisawa, Junn

    2004-01-01

    Recent evidence indicates that the transactivation of estrogen receptor ? (ER?) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ER? is also ubiquitinated and degraded through a ubiquitin–proteasome pathway. To investigate this ubiquitin–proteasome pathway, we purified the ubiquitin ligase complex for unliganded ER? and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ER? and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ER?. In CHIP?/? cells, the degradation of unliganded ER? was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ER? is regulated by two independent ubiquitin–proteasome pathways, which are switched by ligand binding to ER?. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor. PMID:15538384

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  20. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

    PubMed Central

    Wang, Feng; Deng, Xing Wang

    2011-01-01

    The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth. PMID:21788985

  1. Ubiquitin-Proteasome-mediated Degradation of Keratin Intermediate Filaments in Mechanically Stimulated A549 Cells*

    PubMed Central

    Jaitovich, Ariel; Mehta, Semil; Na, Ni; Ciechanover, Aaron; Goldman, Robert D.; Ridge, Karen M.

    2008-01-01

    We previously reported that shear stress induces phosphorylation and disassembly of keratin intermediate filaments (IFs). Shear stress also induces a time- and strain-dependent degradation of keratin IFs, and the current study examines the mechanisms involved in degradation of keratin proteins in human A549 cells exposed to 0–24 h of shear stress (7.5–30 dynes/cm2). Ubiquitin was found to be covalently associated with keratin proteins immunoprecipitated from shear-stressed cells, and pretreatment with the proteasomal inhibitor MG132 prevented the degradation of the keratin IF network. Importantly, phosphorylation of K8 Ser-73 is required for the shear stress-mediated ubiquitination, disassembly, and degradation of the keratin IF network. Immunofluorescence microscopy revealed that shear stress caused the thin array of keratin fibrils observed in control cells to be reorganized into a perinuclear aggregate, known as an aggresome, and that ubiquitin was also associated with this structure. Finally, the E2 enzymes, UbcH5b, -c, and Ubc3, but not E2-25K are required for the shear stress-mediated ubiquitin-proteasomal degradation of keratin proteins. These data suggest that shear stress promotes the disassembly and degradation of the keratin IF network via phosphorylation and the ubiquitin-proteasome pathway. PMID:18617517

  2. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    ERIC Educational Resources Information Center

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  3. Ubiquitin-proteasome pathway function is required for lens cell proliferation and differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the differentia...

  4. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system.

    PubMed

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  5. TGF-? Induces Degradation of PTHrP Through Ubiquitin-Proteasome System in Hepatocellular Carcinoma

    PubMed Central

    Li, Hao; He, Guangchun; Yao, Hui; Song, Liujiang; Zeng, Liang; Peng, Xiaoning; Rosol, Thomas J.; Deng, Xiyun

    2015-01-01

    Both transforming growth factor-? (TGF-?) and parathyroid hormone-related protein (PTHrP) regulate important cellular processes, such as apoptosis in the development of hepatocellular carcinoma. However, the mechanisms of regulation of PTHrP by TGF-? are largely unknown. We hypothesized that TGF-? regulates the expression of PTHrP protein through a post-translational mechanism. Using hepatocellular carcinoma cell lines as the in vitro model, we investigated the effects of TGF-? on protein expression and post-translational processing of PTHrP. We found that TGF-? treatment led to protein degradation of PTHrP through the ubiquitin-proteasome-dependent pathway. We also provided evidence to show that Smurf2 was the E3 ligase responsible for the ubiquitination of PTHrP. Furthermore, using immunohistochemistry on human hepatocellular carcinoma specimens and a tissue array, we found that the expression of PTHrP was predominantly in the cancer cells, whereas the expression of TGF-? was present in non-neoplastic liver tissue adjacent to hepatocellular carcinoma. Our findings reveal a novel mechanism whereby TGF-? may regulate PTHrP in hepatocellular carcinogenesis and lack of TGF-? in hepatocellular carcinoma may promote cancer progression. Promotion of PTHrP degradation provides a novel target of therapeutic intervention to sensitize hepatocellular carcinoma cells to cytostatic and/or pro-apoptotic signals. PMID:26000041

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

    PubMed Central

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

    2014-01-01

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

  7. Regulation of ubiquitin proteasome pathway molecular markers in response to endurance and resistance exercise and training.

    PubMed

    Stefanetti, Renae J; Lamon, Séverine; Wallace, Marita; Vendelbo, Mikkel H; Russell, Aaron P; Vissing, Kristian

    2015-07-01

    Knowledge on the effects of divergent exercise on ostensibly protein degradation pathways may be valuable for counteracting muscle wasting and for understanding muscle remodelling. This study examined mRNA and/or protein levels of molecular markers of the ubiquitin proteasome pathway (UPP), including FBXO32 (atrogin-1), MURF-1, FBXO40, FOXO1 and FOXO3. Protein substrates of atrogin-1-including EIF3F, MYOG and MYOD1-and of MURF-1-including PKM and MHC-were also measured. Subjects completed 10 weeks of endurance training (ET) or resistance training (RT) followed by a single-bout of endurance exercise (EE) or resistance exercise (RE). Following training, atrogin-1, FBXO40, FOXO1 and FOXO3 mRNA increased independently of exercise mode, whereas MURF-1 mRNA and FOXO3 protein increased following ET only. No change in other target proteins occurred post-training. In the trained state, single-bout EE, but not RE, increased atrogin-1, MURF-1, FBXO40, FOXO1, FOXO3 mRNA and FOXO3 protein. In contrast to EE, FBXO40 mRNA and protein decreased following single-bout RE. MURF-1 and FOXO1 protein levels as well as the protein substrates of atrogin-1 and MURF-1 were unchanged following training and single-bout exercise. This study demonstrates that the intracellular signals elicited by ET and RT result in an upregulation of UPP molecular markers, with a greater increase following ET. However, in the trained state, the expression levels of UPP molecular markers are increased following single-bout EE, but are less responsive to single-bout RE. This suggests that adaptations following endurance exercise training are more reliant on protein UPP degradation processes than adaptations following resistance exercise training. PMID:25104573

  8. Limiting the power of p53 through the ubiquitin proteasome pathway

    PubMed Central

    Pant, Vinod

    2014-01-01

    The ubiquitin proteasome pathway is critical in restraining the activities of the p53 tumor suppressor. Numerous E3 and E4 ligases regulate p53 levels. Additionally, deubquitinating enzymes that modify p53 directly or indirectly also impact p53 function. When alterations of these proteins result in increased p53 activity, cells arrest in the cell cycle, senesce, or apoptose. On the other hand, alterations that result in decreased p53 levels yield tumor-prone phenotypes. This review focuses on the physiological relevance of these important regulators of p53 and their therapeutic implications. PMID:25128494

  9. Enzymatic Blockade of the Ubiquitin-Proteasome Pathway

    E-print Network

    Ernst, Robert

    Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ...

  10. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

    PubMed

    Pagano, M; Tam, S W; Theodoras, A M; Beer-Romero, P; Del Sal, G; Chau, V; Yew, P R; Draetta, G F; Rolfe, M

    1995-08-01

    The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases. PMID:7624798

  11. Oxidative stress, NF-?B and the ubiquitin proteasomal pathway in the pathology of calpainopathy.

    PubMed

    Rajakumar, Dhanarajan; Alexander, Mathew; Oommen, Anna

    2013-10-01

    The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-?B and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-?B signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-?B/IKK ? signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-?B signaling could be considered for treatment of LGMD 2A. PMID:23846623

  12. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    SciTech Connect

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O.; Kang, Seok-Seong

    2011-01-14

    Research highlights: {yields} Distinct inclusion bodies are developed by inhibition of UPP and ALP. {yields} The inclusion bodies differ in morphology, localization and formation process. {yields} The inclusion bodies are distinguishable by the localization of TSC2. {yields} Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  13. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

    PubMed Central

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation. PMID:26678046

  14. Activation of the Ubiquitin Proteasome Pathway by Silk Fibroin Modified Chitosan Nanoparticles in Hepatic Cancer Cells

    PubMed Central

    Yang, Ming-Hui; Chung, Tze-Wen; Lu, Yi-Shan; Chen, Yi-Ling; Tsai, Wan-Chi; Jong, Shiang-Bin; Yuan, Shyng-Shiou; Liao, Pao-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

    2015-01-01

    Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 ?g/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. PMID:25588218

  15. Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

    PubMed Central

    Choy, Milly M.; Sessions, October M.; Gubler, Duane J.; Ooi, Eng Eong

    2015-01-01

    Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector. PMID:26566123

  16. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    PubMed Central

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro

    2014-01-01

    The ubiquitin–proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  17. Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

    PubMed

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-09-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  18. Vitamin D deficiency-induced muscle wasting occurs through the ubiquitin proteasome pathway and is partially corrected by calcium in male rats.

    PubMed

    Bhat, Mehrajuddin; Kalam, Ramesh; Qadri, Syed Syh; Madabushi, Seshacharyulu; Ismail, Ayesha

    2013-11-01

    Vitamin D deficiency leads to muscle wasting in both animals and humans. A vitamin D-deficient rat model was created using Sprague Dawley male rats. We studied the involvement of the ubiquitin proteasome and other proteolytic pathways in vitamin D deficiency-induced muscle atrophy. To delineate the effect of hypocalcemia that accompanies D deficiency, a group of deficient rats was supplemented with high calcium alone. Total protein degradation in muscle was assessed by release of tyrosine; proteasomal, lysosomal, and calpain enzyme activities were studied using specific substrates by fluorometry, and E2 enzyme expression was assessed by Western blot analysis. Muscle histology was done by myosin ATPase staining method, whereas 3-methylhistidine in the urine was estimated using HPLC. Muscle gene expression was measured by semiquantitative RT-PCR. Total protein degradation in muscle and the level of 3-methylhistidine in urine were increased in the deficient group compared with the control group. Proteasomal enzyme activities, expression of the E2 ubiquitin conjugating enzyme, and ubiquitin conjugates were increased in the deficient group compared with controls. On the other hand, lysosomal and calpain activities were not altered. Type II fiber area, a marker for muscle atrophy, was decreased in the deficient muscle compared with control muscle. Muscle atrophy marker genes and proteasomal subunit genes were up-regulated, whereas myogenic genes were down-regulated in D-deficient muscle. From the results it appears that the ubiquitin proteasome pathway is the major pathway involved in vitamin D deficiency-induced muscle protein degradation and that calcium supplementation alone in the absence of vitamin D partially corrects the changes. PMID:23928374

  19. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    SciTech Connect

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  20. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

    PubMed

    Shibata, Norihito; Ohoka, Nobumichi; Sugaki, Yusuke; Onodera, Chiaki; Inoue, Mizuho; Sakuraba, Yoshiyuki; Takakura, Daisuke; Hashii, Noritaka; Kawasaki, Nana; Gondo, Yoichi; Naito, Mikihiko

    2015-11-20

    During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders. PMID:26442586

  1. Neuroinflammation and J2 prostaglandins: linking impairment of the ubiquitin-proteasome pathway and mitochondria to neurodegeneration

    PubMed Central

    Figueiredo-Pereira, Maria E.; Rockwell, Patricia; Schmidt-Glenewinkel, Thomas; Serrano, Peter

    2015-01-01

    The immune response of the CNS is a defense mechanism activated upon injury to initiate repair mechanisms while chronic over-activation of the CNS immune system (termed neuroinflammation) may exacerbate injury. The latter is implicated in a variety of neurological and neurodegenerative disorders such as Alzheimer and Parkinson diseases, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, HIV dementia, and prion diseases. Cyclooxygenases (COX-1 and COX-2), which are key enzymes in the conversion of arachidonic acid into bioactive prostanoids, play a central role in the inflammatory cascade. J2 prostaglandins are endogenous toxic products of cyclooxygenases, and because their levels are significantly increased upon brain injury, they are actively involved in neuronal dysfunction induced by pro-inflammatory stimuli. In this review, we highlight the mechanisms by which J2 prostaglandins (1) exert their actions, (2) potentially contribute to the transition from acute to chronic inflammation and to the spreading of neuropathology, (3) disturb the ubiquitin-proteasome pathway and mitochondrial function, and (4) contribute to neurodegenerative disorders such as Alzheimer and Parkinson diseases, and amyotrophic lateral sclerosis, as well as stroke, traumatic brain injury (TBI), and demyelination in Krabbe disease. We conclude by discussing the therapeutic potential of targeting the J2 prostaglandin pathway to prevent/delay neurodegeneration associated with neuroinflammation. In this context, we suggest a shift from the traditional view that cyclooxygenases are the most appropriate targets to treat neuroinflammation, to the notion that J2 prostaglandin pathways and other neurotoxic prostaglandins downstream from cyclooxygenases, would offer significant benefits as more effective therapeutic targets to treat chronic neurodegenerative diseases, while minimizing adverse side effects. PMID:25628533

  2. Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system.

    PubMed

    Wang, Xiaoju; Fan, Huadong; Ying, Zheng; Li, Bin; Wang, Hongfeng; Wang, Guanghui

    2010-01-18

    TAR DNA-binding protein-43 (TDP-43) is a nuclear protein functioning in the regulation of transcription and mRNA splicing. TDP-43 is accumulated in ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) diseased brains. However, the pathways involved in the clearance of TDP-43 and its pathogenic form (TDP-25), a truncated form of TDP-43, are still not elucidated. In this study, we demonstrated that the protein levels of TDP-43 and TDP-25 were increased in cells treated with a proteasome inhibitor, MG132, or an autophagy inhibitor, 3-MA, whereas, they were decreased in cells treated with an enhancer of autophagy, trehalose. Furthermore, more protein level changes of TDP-25 than TDP-43 were observed in cells treated with above inhibitors or enhancer. Thus, our data suggest that TDP-43 and TDP-25 are degraded by both proteasome and autophagy with TDP-25 being more regulated. PMID:19944744

  3. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  4. Role of ubiquitin-proteasome in protein quality control and signaling: implication in the pathogenesis of eye diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin–proteasome pathway (UPP) plays important roles in many cellular functions, such as protein quality control, cell cycle control, and signal transduction. The selective degradation of aberrant proteins by the UPP is essential for the timely removal of potential cytotoxic damaged or other...

  5. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

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

  6. Stuck between a ROS and a hard place: Analysis of the ubiquitin proteasome pathway in selenocysteine treated Brassica napus reveals different toxicities during selenium assimilation.

    PubMed

    Dimkovikj, Aleksandar; Fisher, Brian; Hutchison, Kim; Van Hoewyk, Doug

    2015-06-01

    During the selenium assimilation pathway, inorganic selenate and selenite are reduced to form selenocysteine (Sec). Tolerance to selenium in plants has long been attributable to minimizing the replacement of cysteine with selenocysteine, which can result in nonspecific selenoproteins that are potentially misfolded. Despite this widely accepted assumption, there is no evidence in higher plants demonstrating that selenocysteine induces toxicity by resulting in malformed proteins. In this study, we use Brassica napus to analyze the ubiquitin-proteasome pathway, which is capable of removing misfolded proteins. Sec rapidly increased proteasome activity and levels of ubiquitinated proteins, strongly indicating that selenocysteine induces protein misfolding. Proteasome inhibition increased the amount of selenium in protein in Sec-treated plants. Collectively, these data provide a mechanism that accounts for Sec toxicity. Additionally, Sec did not cause oxidative stress as judged by examining levels of superoxide using fluorescent microscopy. Therefore, the cellular response to Sec is different compared to selenite, which was recently shown to increase antioxidant metabolism in response to elevated mitochondrial superoxide that ultimately impaired proteasome activity. Therefore, plants must contend with two divergent modes of cytotoxicity during selenium assimilation. Selenite can result in oxidative stress, but increased flux of selenite reduction can yield Sec that in turn can cause protein misfolding. PMID:25974369

  7. Intracellular Dynamics of the Ubiquitin-Proteasome-System

    PubMed Central

    Chowdhury, Maisha; Enenkel, Cordula

    2015-01-01

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

  8. Inhibition of the ubiquitin-proteasome pathway does not protect against ventilator-induced accelerated proteolysis or atrophy in the diaphragm

    PubMed Central

    Smuder, Ashley J.; Nelson, W. Bradley; Hudson, Matthew B.; Kavazis, Andreas N.; Powers, Scott K.

    2014-01-01

    Background Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. However, prolonged MV results in ventilator-induced diaphragm dysfunction (VIDD), a condition characterized by both diaphragm fiber atrophy and contractile dysfunction. Previous work has shown calpain, caspase-3 and the ubiquitin-proteasome pathway (UPP) are all activated in the diaphragm during prolonged MV. However, while it is established that both calpain and caspase-3 are important contributors to VIDD, the role that the UPP plays in VIDD remains unknown. These experiments tested the hypothesis that inhibition of the UPP will protect the diaphragm against VIDD. Methods We tested this prediction in an established animal model of MV using a highly specific UPP inhibitor, epoxomicin, to prevent MV-induced activation of the proteasome in the diaphragm (n = 8/group). Results Our results reveal that inhibition of the UPP did not prevent ventilator-induced diaphragm muscle fiber atrophy and contractile dysfunction during 12 hours of MV. Also, inhibition of the UPP does not impact MV-induced increases in calpain and caspase-3 activity in the diaphragm. Finally, administration of the proteasome inhibitor did not protect against the MV-induced increases in the expression of the E3 ligases, MuRF1 and atrogin-1/MaFbx. Conclusions Collectively, these results indicate that proteasome activation does not play a required role in VIDD during the first 12 hours of MV. PMID:24681580

  9. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

    NASA Technical Reports Server (NTRS)

    Baracos, V. E.; DeVivo, C.; Hoyle, D. H.; Goldberg, A. L.

    1995-01-01

    Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

  10. The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway

    PubMed Central

    Õunap, Kadri; Leetsi, Lilian; Matsoo, Maarja; Kurg, Reet

    2015-01-01

    The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells. PMID:26214185

  11. Differential ubiquitin-proteasome and autophagy signaling following rotator cuff tears and suprascapular nerve injury.

    PubMed

    Joshi, Sunil K; Kim, Hubert T; Feeley, Brian T; Liu, Xuhui

    2014-01-01

    Previous studies have evaluated role of Akt/mTOR signaling in rotator cuff muscle atrophy and determined that there was differential in signaling following tendon transection (TT) and suprascapular nerve (SSN) denervation (DN), suggesting that atrophy following TT and DN was modulated by different protein degradation pathways. In this study, two muscle proteolytic systems that have been shown to be potent regulators of muscle atrophy in other injury models, the ubiquitin-proteasome pathway and autophagy, were evaluated following TT and DN. In addition to examining protein degradation, this study assessed protein synthesis rate following these two surgical models to understand how the balance between protein degradation and synthesis results in atrophy following rotator cuff injury. In contrast to the traditional theory that protein synthesis is decreased during muscle atrophy, this study suggests that protein synthesis is up-regulated in rotator cuff muscle atrophy following both surgical models. While the ubiquitin-proteasome pathway was a major contributor to the atrophy seen following DN, autophagy was a major contributor following TT. The findings of this study suggest that protein degradation is the primary factor contributing to atrophy following rotator cuff injury. However, different proteolytic pathways are activated if SSN injury is involved. PMID:24018537

  12. Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

    PubMed Central

    Ma, Yi-ming; Han, Wei; Li, Jia; Hu, Li-hong; Zhou, Yu-bo

    2015-01-01

    Aim: To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms. Methods: Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye. Results: Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 ?mol/L. Treatment of the cells with physalin B (2.5–10 ?mol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis. Conclusion: Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro. PMID:25832431

  13. Targeting the ubiquitin–proteasome system for cancer therapy

    PubMed Central

    Shen, Min; Schmitt, Sara; Buac, Daniela; Dou, Q Ping

    2013-01-01

    Introduction The ubiquitin–proteasome system (UPS) degrades 80 – 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway. Areas covered This review provides a broad overview of the UPS and its role in supporting cancer development and progression, especially in aspects of p53 inactivation, p27 turnover and NF-?B activation. Also, efforts toward the development of small molecule inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed. Expert opinion Whereas some of the targets in the UPS, such as the 20S pro-teasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest. PMID:23822887

  14. Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system.

    PubMed Central

    Singer, Thorsten; Haefner, Stefan; Hoffmann, Michael; Fischer, Michael; Ilyina, Julia; Hilt, Wolfgang

    2003-01-01

    Using a synthetic lethality screen we found that the Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system. Yeast cells harboring sit4 mutations and an impaired proteasome (due to pre1-1 pre4-1 mutations) exhibited defective growth on minimal medium. Nearly identical synthetic effects were found when sit4 mutations were combined with defects of the Rad6/Ubc2- and Cdc34/Ubc3-dependent ubiquitination pathways. Under synthetic lethal conditions, sit4 pre or sit4 ubc mutants formed strongly enlarged unbudded cells with a DNA content of 1N, indicating a defect in the maintenance of cell integrity during starvation-induced G(1) arrest. Sit4-related synthetic effects could be cured by high osmotic pressure or by the addition of certain amino acids to the growth medium. These results suggest a concerted function of the Sit4 phosphatase and the ubiquitin-proteasome system in osmoregulation and in the sensing of nutrients. Further analysis showed that Sit4 is not a target of proteasome-dependent protein degradation. We could also show that Sit4 does not contribute to regulation of proteasome activity. These data suggest that both Sit4 phosphatase and the proteasome act on a common target protein. PMID:12930741

  15. The ubiquitin-proteasomal system is critical for multiple myeloma: implications in drug discovery

    PubMed Central

    Cao, Biyin; Mao, Xinliang

    2011-01-01

    Bortezomib is a specific inhibitor of proteasomes, the most important protease complexes in protein degradation. Bortezomib can induce apoptosis of a variety of cancer cells, including leukemia, lymphoma, multiple myeloma, breast cancers, prostate cancers, lung cancers, and so on. However, extensive studies and overall evaluation suggested that multiple myeloma is the most sensitive and the best responsive disease which was later approved by Food and Drug Administration for bortezomib treatment. Because proteasomes are an essential component in the ubiquitin-proteasomal protein degradation pathway, the discovery of bortezomib implicates that the UPS is critical for myeloma pathophysiology. The UPS also contains ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3) and deubiquitinases (Dubs). In this review, we examined and analyzed the recent advancements of the UPS components in multiple myeloma and its implications in drug discovery for myeloma treatment. PMID:22432065

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

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2012-01-01

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

  17. Ubiquitin-proteasome system and mitochondria - reciprocity.

    PubMed

    Livnat-Levanon, Nurit; Glickman, Michael H

    2011-02-01

    Recently, sporadic links have been published between mitochondria - membrane-confined organelles - and the cytosolic ubiquitin-proteasome system (UPS) for removal of cellular proteins. For example, Fzo1, a mitochondrial outer membrane mitofusin was shown to be ubiquitinated by a ubiquitin ligase, Cdc53(MDM30), and degraded by the proteasome. Two additional ubiquitin ligases, MITOL/MARCH-V and MULAN, as well as a deubiquitinating enzyme, Ubp16/USP30, are embedded in mitochondrial outer membranes and participate in mitochondrial dynamics. Defects in mitochondrial morphology or respiration capacity are also reported for mutations in other UPS components such as the Ub ligases Parkin and Rsp5 as well as in proteasome subunits. These examples are likely to reflect a pervasive involvement of UPS in recycling of mitochondria-associated proteins. The flux of imported proteins and the proximity to oxidative phosphorylation results in abundant damaged or misfolded proteins that generate the need for a responsive quality control system. Within the mitochondrial matrix there is a self-contained ATP-dependent system for protein turnover. However at the outer membrane, the UPS may play a corresponding role in recycling either membrane-embedded or imported proteins. In a parallel process, ubiquitination also partakes in selection of damaged mitochondria to the lysozome/vacuole via autophagy. In the reverse direction, components of the UPS are sensitive to cellular REDOX potential, and as such are affected by reactive oxygen species (ROS) generated as a byproduct of mitochondrial respiration. This review will try to address the regulation of mitochondrial morphology and metabolic function by UPS, as well as the reciprocal relationship between aberrant ROS produced by mitochondria and ubiquitination or proteasome activity. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough! PMID:20674813

  18. The Impact of Paeoniflorin on ?-Synuclein Degradation Pathway

    PubMed Central

    2015-01-01

    Paeoniflorin (PF) is the major active ingredient in the traditional Chinese medicine Radix. It plays a neuroprotective role by regulating autophagy and the ubiquitin-proteasome degradation pathway. In this study, we found PF significantly reduced cell damage caused by MPP+, returning cells to normal state. Cell viability significantly improved after 24 h exposure to RAPA and PF in the MPP+ group (all P < 0.01). CAT and SOD activities were significantly decreased after PF and RAPA treatment, compared with MPP+ (P < 0.001). In addition, MPP+ activated both LC3-II and E1; RAPA increased LC3-II but inhibited E1. PF significantly upregulated both LC3-II (autophagy) and E1 (ubiquitin-proteasome pathway) expression (P < 0.001), promoted degradation of ?-synuclein, and reduced cell damage. We show MPP+ enhanced immunofluorescence signal of intracellular ?-synuclein and LC3. Fluorescence intensity of ?-synuclein decreased after PF treatment. In conclusion, these data show PF reversed the decline of proteasome activity caused by MPP+ and significantly upregulated both autophagy and ubiquitin-proteasome pathways, promoted the degradation of ?-synuclein, and reduced cell damage. These findings suggest PF is a potential therapeutic medicine for neurodegenerative diseases. PMID:26693241

  19. Failure of ubiquitin proteasome system: risk for neurodegenerative diseases.

    PubMed

    Zheng, Chen; Geetha, Thangiah; Babu, Jeganathan Ramesh

    2014-01-01

    The ubiquitin proteasome system (UPS) is the primary proteolytic quality control system in cells and has an essential function in the nervous system. UPS dysfunction has been linked to neurodegenerative conditions, including Alzheimer's, Parkinson's and Huntington's diseases. The pathology of neurodegenerative diseases is characterized by the abnormal accumulation of insoluble protein aggregates or inclusion bodies within neurons. The failure or dysregulation of the UPS prevents the degradation of misfolded/aberrant proteins, leading to deficient synaptic function that eventually affects the nervous system. In this review, we discuss the UPS and its physiological roles in the nervous system, its influence on neuronal function, and how UPS dysfunction contributes to the development of neurodegenerative diseases. PMID:25413678

  20. The Role of the Ubiquitin Proteasome System in Ischemia and Ischemic Tolerance

    PubMed Central

    Meller, Robert

    2010-01-01

    Ubiquitin modification targets a protein for rapid degradation by the proteasome. However, poly-ubiquitination of proteins can result in multiple functions depending on the topology of the ubiquitin chain. Therefore ubiquitin signaling offers a more complex and versatile biology compared to many other post translational modifications. One area of potential for the application of this knowledge is the field of ischemia-induced brain damage, as occurs following a stroke. The ubiquitin proteasome system may exert a dual role on neuronal outcome following ischemia. Harmful ischemia results in an overload of the ubiquitin proteasome system, and blocking the proteasome reduces brain infarction following ischemia. However, the rapid and selective degradation of proteins following brief ischemia results in endogenous protection against ischemia. Therefore further understanding of the molecular signaling mechanisms which regulate the ubiquitin proteasome system may reveal novel therapeutic targets to reduce brain damage when ischemia is predicted, or to reduce the activation of the cell death mechanisms and the inflammatory response following stroke. The aim of this review is to discuss some of the recent advances in the understanding of protein ubiquitination and its implications for novel stroke therapies. PMID:19181875

  1. Ubiquitin, Proteasomes and Proteolytic Mechanisms Activated by Kidney Disease

    PubMed Central

    Rajan, Vik; Mitch, William E.

    2008-01-01

    Summary The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle. The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function. PMID:18723090

  2. Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.

    PubMed

    Melvin, Adam T; Woss, Gregery S; Park, Jessica H; Waters, Marcey L; Allbritton, Nancy L

    2013-09-01

    The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington's disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples. PMID:23686610

  3. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites

    PubMed Central

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target. PMID:26090380

  4. Inhibition of Proteasome Activity Strongly Affects Kiwifruit Pollen Germination. Involvement of the Ubiquitin/Proteasome Pathway as a Major Regulator1

    PubMed Central

    Speranza, Anna; Scoccianti, Valeria; Crinelli, Rita; Calzoni, Gian Lorenzo; Magnani, Mauro

    2001-01-01

    The 26S proteasome is a multicatalytic complex that acts as primary protease of the ubiquitin-mediated proteolytic pathway in eukaryotes. We provide here the first evidence that the proteasome plays a key role in regulating pollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germinating pollen of kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev) C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed fluorometrically, accompanied the progression of germination. Specific inhibitors of proteasome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin ?-lactone, and epoxomicin significantly decreased tube growth or altered tube morphology. High-molecular mass, ubiquitinated proteins accumulated in MG-132- and ?-lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were also able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succinyl-l-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine proteases, was investigated. Some reduction in tube growth rate was observed, but only at 80 ?m E-64, and no abnormal tubes were produced. Furthermore, no inhibition of tube growth was observed when another inhibitor of cysteine proteases, leupeptin, or inhibitors of serine and aspartic proteases (phenylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate that protein turnover during tube organization and elongation in kiwifruit pollen is important, and our results also implicate the ubiquitin/26S proteasome as the major proteolytic pathway involved. PMID:11457965

  5. The ubiquitin proteasome system postsynaptically regulates glutamatergic synaptic function

    PubMed Central

    Haas, Kevin F.; Miller, Stephanie L. H.; Friedman, David B.; Broadie, Kendal

    2007-01-01

    The ubiquitin proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome ?2 and ?6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors. PMID:17363264

  6. The Ubiquitin-Proteasome System as a Prospective Molecular Target for Cancer Treatment and Prevention

    PubMed Central

    Chen, Di; Dou, Q. Ping

    2012-01-01

    Proteasomes are large multicatalytic proteinase complexes located in the cytosol and the nucleus of eukaryotic cells. The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins and therefore plays an essential regulatory role in critical cellular processes including cell cycle progression, proliferation, differentiation, angiogenesis and apoptosis. Besides involving in normal cellular functions and homeostasis, the alteration of proteasomal activity contributes to the pathological states of several clinical disorders including inflammation, neurodegeneration and cancer. It has been reported that human cancer cells possess elevated level of proteasome activity and are more sensitive to proteasome inhibitors than normal cells, indicating that the inhibition of the ubiquitin-proteasome system could be used as a novel approach for cancer therapy. In this review we summarize several specific aspects of research for the proteasome complex, including the structure and catalytic activities of the proteasome, properties and mechanisms of action of various proteasome inhibitors, and finally the clinical development of proteasome inhibitors as novel anticancer agents. PMID:20491623

  7. Regulation of Mitochondrial Genome Inheritance by Autophagy and Ubiquitin-Proteasome System: Implications for Health, Fitness, and Fertility

    PubMed Central

    Ballard, John William Oman; Yi, Young-Joo

    2014-01-01

    Mitochondria, the energy-generating organelles, play a role in numerous cellular functions including adenosine triphosphate (ATP) production, cellular homeostasis, and apoptosis. Maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is universally observed in humans and most animals. In general, high levels of mitochondrial heteroplasmy might contribute to a detrimental effect on fitness and disease resistance. Therefore, a disposal of the sperm-derived mitochondria inside fertilized oocytes assures normal preimplantation embryo development. Here we summarize the current research and knowledge concerning the role of autophagic pathway and ubiquitin-proteasome-dependent proteolysis in sperm mitophagy in mammals, including humans. Current data indicate that sperm mitophagy inside the fertilized oocyte could occur along multiple degradation routes converging on autophagic clearance of paternal mitochondria. The influence of assisted reproductive therapies (ART) such as intracytoplasmic sperm injection (ICSI), mitochondrial replacement (MR), and assisted fertilization of oocytes from patients of advanced reproductive age on mitochondrial function, inheritance, and fitness and for the development and health of ART babies will be of particular interest to clinical audiences. Altogether, the study of sperm mitophagy after fertilization has implications in the timing of evolution and developmental and reproductive biology and in human health, fitness, and management of mitochondrial disease. PMID:25028670

  8. Sent to Destroy: The Ubiquitin Proteasome System Regulates Cell Signaling and Protein Quality Control in Cardiovascular Development and Disease

    PubMed Central

    Willis, Monte S.; Townley-Tilson, W.H. Davin; Kang, Eunice Y.; Homeister, Jonathon W.; Patterson, Cam

    2010-01-01

    The ubiquitin proteasome system (UPS) plays a crucial role in biological processes integral to the development of the cardiovascular system and cardiovascular diseases. The UPS prototypically recognizes specific protein substrates and places polyubiquitin chains on them for subsequent destruction by the proteasome. This system is in place to degrade not only misfolded and damaged proteins, but is essential also in regulating a host of cell signaling pathways involved in proliferation, adaptation to stress, regulation of cell size, and cell death. During the development of the cardiovascular system, the UPS regulates cell signaling by modifying transcription factors, receptors, and structural proteins. Later, in the event of cardiovascular diseases as diverse as atherosclerosis, cardiac hypertrophy, and ischemia reperfusion injury, ubiquitin ligases and the proteasome are implicated in protecting and exacerbating clinical outcomes. However, when misfolded and damaged proteins are ubiquitinated by the UPS, their destruction by the proteasome is not always possible due to their aggregated confirmations. Recent studies have discovered how these ubiquitinated misfolded proteins can be destroyed by alternative “specific” mechanisms. The cytosolic receptors p62, NBR, and HDAC6 recognize aggregated ubiquitinated proteins and target them for autophagy in the process of “selective autophagy”. Even the ubiquitination of multiple proteins within whole organelles that drive the more general macro-autophagy may be due, in part, to similar ubiquitin-driven mechanisms. In summary, the cross-talk between the UPS and autophagy highlight the pivotal and diverse roles the UPS plays in maintaining protein quality control and regulating cardiovascular development and disease. PMID:20167943

  9. Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

    PubMed Central

    Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

    2015-01-01

    Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-?, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-? induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-? via a NF-?B dependent pathway in C2C12 myotubes, while inhibition of NF-?B activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems. PMID:26448817

  10. Regulation of mitochondrial genome inheritance by autophagy and ubiquitin-proteasome system: implications for health, fitness, and fertility.

    PubMed

    Song, Won-Hee; Ballard, John William Oman; Yi, Young-Joo; Sutovsky, Peter

    2014-01-01

    Mitochondria, the energy-generating organelles, play a role in numerous cellular functions including adenosine triphosphate (ATP) production, cellular homeostasis, and apoptosis. Maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is universally observed in humans and most animals. In general, high levels of mitochondrial heteroplasmy might contribute to a detrimental effect on fitness and disease resistance. Therefore, a disposal of the sperm-derived mitochondria inside fertilized oocytes assures normal preimplantation embryo development. Here we summarize the current research and knowledge concerning the role of autophagic pathway and ubiquitin-proteasome-dependent proteolysis in sperm mitophagy in mammals, including humans. Current data indicate that sperm mitophagy inside the fertilized oocyte could occur along multiple degradation routes converging on autophagic clearance of paternal mitochondria. The influence of assisted reproductive therapies (ART) such as intracytoplasmic sperm injection (ICSI), mitochondrial replacement (MR), and assisted fertilization of oocytes from patients of advanced reproductive age on mitochondrial function, inheritance, and fitness and for the development and health of ART babies will be of particular interest to clinical audiences. Altogether, the study of sperm mitophagy after fertilization has implications in the timing of evolution and developmental and reproductive biology and in human health, fitness, and management of mitochondrial disease. PMID:25028670

  11. Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein.

    PubMed

    Greussing, Ruth; Unterluggauer, Hermann; Koziel, Rafal; Maier, Andrea B; Jansen-Dürr, Pidder

    2012-01-01

    Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins (1, 2). Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response (3), cell cycle regulation and cellular differentiation (4) or in immune system response (5). The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases (4, 6). Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging (7, 8, 9, 10). Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable (11)) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry. PMID:23169445

  12. Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise

    PubMed Central

    Stefanetti, Renae J.; Zacharewicz, Evelyn; Della Gatta, Paul; Garnham, Andrew; Russell, Aaron P.; Lamon, Séverine

    2014-01-01

    Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP). Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE). We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60–75 years) vs. 10 younger (18–30 years) healthy male subjects at basal as well as 2 h after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signaling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown (MPB) measurements following RE in older vs. younger subjects. PMID:24550841

  13. Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise.

    PubMed

    Stefanetti, Renae J; Zacharewicz, Evelyn; Della Gatta, Paul; Garnham, Andrew; Russell, Aaron P; Lamon, Séverine

    2014-01-01

    Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP). Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE). We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60-75 years) vs. 10 younger (18-30 years) healthy male subjects at basal as well as 2 h after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signaling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown (MPB) measurements following RE in older vs. younger subjects. PMID:24550841

  14. Coordinated Regulation of Nuclear Receptor CAR by CCRP/DNAJC7, HSP70 and the Ubiquitin-Proteasome System

    PubMed Central

    Timsit, Yoav E.; Negishi, Masahiko

    2014-01-01

    The constitutive active/androstane receptor (CAR) plays an important role as a coordinate transcription factor in the regulation of various hepatic metabolic pathways for chemicals such as drugs, glucose, fatty acids, bilirubin, and bile acids. Currently, it is known that in its inactive state, CAR is retained in the cytoplasm in a protein complex with HSP90 and the tetratricopeptide repeat protein cytosoplasmic CAR retention protein (CCRP). Upon activation by phenobarbital (PB) or the PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), CAR translocates into the nucleus. We have identified two new components to the cytoplasmic regulation of CAR: ubiquitin-dependent degradation of CCRP and protein-protein interaction with HSP70. Treatment with the proteasome inhibitor MG132 (5 µM) causes CAR to accumulate in the cytoplasm of transfected HepG2 cells. In the presence of MG132, TCPOBOP increases CCRP ubiquitination in HepG2 cells co-expressing CAR, while CAR ubiquitination was not detected. MG132 treatment of HepG2 also attenuated of TCPOBOP-induced CAR transcriptional activation on reporter constructs which contain CAR-binding DNA elements derived from the human CYP2B6 gene. The elevation of cytoplasmic CAR protein with MG132 correlated with an increase of HSP70, and to a lesser extent HSP60. Both CCRP and CAR were found to interact with endogenous HSP70 in HepG2 cells by immunoprecipitation analysis. Induction of HSP70 levels by heat shock also increased cytoplasmic CAR levels, similar to the effect of MG132. Lastly, heat shock attenuated TCPOBOP-induced CAR transcriptional activation, also similar to the effect of MG132. Collectively, these data suggest that ubiquitin-proteasomal regulation of CCRP and HSP70 are important contributors to the regulation of cytoplasmic CAR levels, and hence the ability of CAR to respond to PB or PB-like inducers. PMID:24789201

  15. An Extract of Artemisia dracunculus L. Inhibits Ubiquitin-Proteasome Activity and Preserves Skeletal Muscle Mass in a Murine Model of Diabetes

    PubMed Central

    Kirk-Ballard, Heather; Wang, Zhong Q.; Acharya, Priyanka; Zhang, Xian H.; Yu, Yongmei; Kilroy, Gail; Ribnicky, David; Cefalu, William T.; Floyd, Z. Elizabeth

    2013-01-01

    Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011) improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-Ay murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-Ay murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes. PMID:23437325

  16. Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact

    PubMed Central

    Rajan, Vik R.

    2007-01-01

    Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin–proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions. PMID:17987322

  17. The ubiquitin proteasome system in glia and its role in neurodegenerative diseases

    PubMed Central

    Jansen, Anne H. P.; Reits, Eric A. J.; Hol, Elly M.

    2014-01-01

    The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal function and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS function in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell function might provide essential information in unraveling mechanisms of neurodegenerative diseases. PMID:25152710

  18. The regulation of glucose on milk fat synthesis is mediated by the ubiquitin-proteasome system in bovine mammary epithelial cells.

    PubMed

    Liu, Lily; Jiang, Li; Ding, Xiang-dong; Liu, Jian-feng; Zhang, Qin

    2015-09-11

    Glucose as one of the nutrition factors plays a vital role in the regulation of milk fat synthesis. Ubiquitin-proteasome system (UPS) is a vital proteolytic pathway in all eukaryotic cells through timely marking, recognizing and degrading the poly-ubiquitinated protein substrates. Previous studies indicated that UPS plays a considerable role in controlling the triglyceride (TG) synthesis. Therefore, the aim of this study is to confirm the link between high-glucose and UPS and its regulation mechanism on milk fat synthesis in BMEC (bovine mammary epithelial cells). We incubated BMEC with normal (17.5 mm/L) and high-glucose (25 mm/L) with and without proteasome inhibitor epoxomicin and found that, compared with the control (normal glucose and without proteasome inhibitor), both high-glucose concentration and proteasome inhibitor epoxomicin could increase the accumulation of TG and poly-ubiquitinated proteins, and reduce significantly three proteasome activities (chymotrypsin-like, caspase-like, and trypsin-like). In addition, high-glucose concentration combined with proteasome inhibitor further enhanced the increase of the poly-ubiquitinated protein level and the decrease of proteasome activities. Our results suggest that the regulation of high-glucose on milk fat synthesis is mediated by UPS in BMEC, and high-glucose exposure could lead to a hypersensitization of BMEC to UPS inhibition which in turn results in increased milk fat synthesis. PMID:26231798

  19. The ubiquitin proteasome system – Implications for cell cycle control and the targeted treatment of cancer

    PubMed Central

    Bassermann, Florian; Eichner, Ruth; Pagano, Michele

    2013-01-01

    Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin-proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (Anaphase Promoting Complex or Cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/CCdc20) and establishes a stable G1 phase (APC/CCdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. PMID:23466868

  20. The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution

    PubMed Central

    Dantuma, Nico P.; Bott, Laura C.

    2014-01-01

    The ubiquitin-proteasome system (UPS) has been implicated in neurodegenerative diseases based on the presence of deposits consisting of ubiquitylated proteins in affected neurons. It has been postulated that aggregation-prone proteins associated with these disorders, such as ?-synuclein, ?-amyloid peptide, and polyglutamine proteins, compromise UPS function, and delay the degradation of other proteasome substrates. Many of these substrates play important regulatory roles in signaling, cell cycle progression, or apoptosis, and their inadvertent stabilization due to an overloaded and improperly functioning UPS may thus be responsible for cellular demise in neurodegeneration. Over the past decade, numerous studies have addressed the UPS dysfunction hypothesis using various model systems and techniques that differ in their readout and sensitivity. While an inhibitory effect of some disease proteins on the UPS has been demonstrated, increasing evidence attests that the UPS remains operative in many disease models, which opens new possibilities for treatment. In this review, we will discuss the paradigm shift that repositioned the UPS from being a prime suspect in the pathophysiology of neurodegeneration to an attractive therapeutic target that can be harnessed to accelerate the clearance of disease-linked proteins. PMID:25132814

  1. The ubiquitin-proteasome system and autophagy are defective in the taurine-deficient heart.

    PubMed

    Jong, Chian Ju; Ito, Takashi; Schaffer, Stephen W

    2015-12-01

    Taurine depletion leads to impaired mitochondrial function, as characterized by reduced ATP production and elevated superoxide generation. These defects can fundamentally alter cardiomyocyte function and if left unchanged can result in cell death. To protect against these stresses, cardiomyocytes possess quality control processes, such as the ubiquitin-proteasome system (UPS) and autophagy, which can rejuvenate cells through the degradation of damaged proteins and organelles. Hence, the present study tested the hypothesis that reactive oxygen species generated by damaged mitochondria initiates UPS and autophagy in the taurine-deficient heart. Using transgenic mice lacking the taurine transporter (TauTKO) as a model of taurine deficiency, it was shown that the levels of ubiquitinated protein were elevated, an effect associated with a decrease in ATP-dependent 26S ?5 proteasome activity. Treating the TauTKO mouse with the mitochondria-specific antioxidant, mitoTEMPO, largely abolished the increase in ubiquitinated protein content. The TauTKO heart was also associated with impaired autophagy, characterized by an increase in the initiator, Beclin-1, and autophagosome content, but a defect in the generation of active autophagolysosomes. Although mitoTEMPO treatment only restores the oxidative balance within the mitochondria, it appeared to completely disrupt the crosstalk between the damaged mitochondria and the quality control processes. Thus, mitochondrial oxidative stress is the main trigger initiating the quality control systems in the taurine-deficient heart. We conclude that the activation of the UPS and autophagy is another fundamental function of mitochondria. PMID:26193770

  2. Atrophy, hypertrophy, and hypoxemia induce transcriptional regulators of the ubiquitin proteasome system in the rat heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In skeletal muscle, transcript levels of proteins regulating the ubiquitin proteasome system (UPS) increase with atrophy and decrease with hypertrophy. Whether the same is true for heart muscle is not known. We set out to characterize the transcriptional profile of regulators of the UPS during atrop...

  3. Regulation of TRAIL-Receptor Expression by the Ubiquitin-Proteasome System

    PubMed Central

    Sarhan, Dhifaf; D’Arcy, Padraig; Lundqvist, Andreas

    2014-01-01

    The tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor (TRAIL-R) family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC), caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS) has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB) activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients. PMID:25318057

  4. Mitochondrial and Ubiquitin Proteasome System Dysfunction in Ageing and Disease: Two Sides of the Same Coin?

    PubMed Central

    Ross, Jaime M.; Olson, Lars; Coppotelli, Giuseppe

    2015-01-01

    Mitochondrial dysfunction and impairment of the ubiquitin proteasome system have been described as two hallmarks of the ageing process. Additionally, both systems have been implicated in the etiopathogenesis of many age-related diseases, particularly neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Interestingly, these two systems are closely interconnected, with the ubiquitin proteasome system maintaining mitochondrial homeostasis by regulating organelle dynamics, the proteome, and mitophagy, and mitochondrial dysfunction impairing cellular protein homeostasis by oxidative damage. Here, we review the current literature and argue that the interplay of the two systems should be considered in order to better understand the cellular dysfunction observed in ageing and age-related diseases. Such an approach may provide valuable insights into molecular mechanisms underlying the ageing process, and further discovery of treatments to counteract ageing and its associated diseases. Furthermore, we provide a hypothetical model for the heterogeneity described among individuals during ageing. PMID:26287188

  5. Dysfunction of the ubiquitin-proteasome system in atherosclerotic cardiovascular disease

    PubMed Central

    Wang, Feilong; Lerman, Amir; Herrmann, Joerg

    2015-01-01

    The ubiquitin-proteasome system (UPS) is an integral part of the protein metabolism and protein quality control in eukaryotic cells. It is involved in a number of biological processes of significance for vascular biology and pathology such as oxidative stress, inflammation, foam cell formation, and apoptosis. This review summarizes both indirect and direct lines of evidence for a role of the UPS in atherosclerosis from the initiation to the progression and complication stage and concludes with a future perspective. PMID:26064796

  6. Breast cancer cells: Modulation by melatonin and the ubiquitin-proteasome system - A review.

    PubMed

    Vriend, Jerry; Reiter, Russel J

    2015-12-01

    Melatonin inhibits human breast cancer cells stimulated with estrogen. This antiproliferative action depends on the presence of the estrogen receptor alpha (ER?) in the human MCF-7 cell line and is strictly dose-dependent. Since researchers concerned with melatonin and breast cancer have not considered the relevance of the ubiquitin-proteasome system to this research in this review we do so. The fact that the first breast cancer susceptibility gene to be identified, Brca1, functions as a ubiquitin ligase indicates that the ubiquitin-proteasome system has a role in regulating susceptibility to breast cancer. While mutations of this gene increase the incidence of breast cancer, the wild type gene suppresses estrogen-dependent transcriptional events relying on the estrogen receptor ER?. Three other ubiquitin ligases, SCF(Skp2), E6AP and APC, interact directly with ER? at the ERE and AP-1 promoters of ER? target genes. Melatonin, like proteasome inhibitors, decreases estrogen-induced gene transcription. Indeed, it has been reported that melatonin specifically inhibits estrogen-induced transcription mediated by ER? at the ERE and AP1 gene promoters. Herein, we present a model in which the inhibitory action of melatonin on MCF-7 cells is mediated, directly or indirectly, by the ubiquitin-proteasome system. In this model ER?, apoptotic proteins, and cell cycle proteins, all influenced by melatonin, are substrates of key ubiquitin ligases including SCF(Skp2), E6AP, and SCF(B-TrCP). Since dysfunction of the ubiquitin-proteasome system is a risk factor for breast cancer, this model provides a context in which to test the clinical potential, and limitations, of melatonin and proteasome inhibitors. PMID:26363225

  7. The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

    PubMed Central

    Finley, Daniel; Ulrich, Helle D.; Sommer, Thomas; Kaiser, Peter

    2012-01-01

    Protein modifications provide cells with exquisite temporal and spatial control of protein function. Ubiquitin is among the most important modifiers, serving both to target hundreds of proteins for rapid degradation by the proteasome, and as a dynamic signaling agent that regulates the function of covalently bound proteins. The diverse effects of ubiquitylation reflect the assembly of structurally distinct ubiquitin chains on target proteins. The resulting ubiquitin code is interpreted by an extensive family of ubiquitin receptors. Here we review the components of this regulatory network and its effects throughout the cell. PMID:23028185

  8. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    PubMed Central

    Sommers, Joshua A.; Suhasini, Avvaru N.; Brosh, Robert M.

    2015-01-01

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. PMID:25906194

  9. Mechanisms Stimulating Muscle Wasting in Chronic Kidney Disease: The Roles of the Ubiquitin-Proteasome System and Myostatin

    PubMed Central

    Thomas, Sandhya S.; Mitch, William E.

    2013-01-01

    Catabolic conditions including chronic kidney disease (CKD), cancer, and diabetes cause muscle atrophy. The loss of muscle mass worsens the burden of disease because it is associated with increased morbidity and mortality. To avoid these problems or to develop treatment strategies, the mechanisms leading to muscle wasting must be identified. Specific mechanisms uncovered in CKD generally occur in other catabolic conditions. These include stimulation of protein degradation in muscle arising from activation of caspase-3 and the ubiquitin-proteasome system (UPS). These proteases act in a coordinated fashion with caspase-3 initially cleaving the complex structure of proteins in muscle yielding fragments that are substrates which are degraded by the UPS. Fortunately, the UPS exhibits remarkable specificity for proteins to be degraded because it is the major intracellular proteolytic system. Without a high level of specificity cellular functions would be disrupted. The specificity is accomplished by complex reactions that depend on recognition of a protein substrate by specific E3 ubiquitin ligases. In muscle, the specific ligases are Atrogin-1 and MuRF1 and their expression has characteristics of a biomarker of accelerated muscle proteolysis. Specific complications of CKD (metabolic acidosis, insulin resistance, inflammation, and angiotensin II) activate caspase-3 and the UPS through mechanisms that include glucocorticoids and impaired insulin or IGF-1 signaling. Mediators activate myostatin which functions as a negative growth factor in muscle. In models of cancer or CKD, strategies that block myostatin prevent muscle wasting suggesting that therapies which block myostatin could prevent muscle wasting in catabolic conditions. PMID:23292175

  10. Metabolomic Quantitative Trait Loci (mQTL) Mapping Implicates the Ubiquitin Proteasome System in Cardiovascular Disease Pathogenesis

    PubMed Central

    Kraus, William E.; Muoio, Deborah M.; Stevens, Robert; Craig, Damian; Bain, James R.; Grass, Elizabeth; Haynes, Carol; Kwee, Lydia; Qin, Xuejun; Slentz, Dorothy H.; Krupp, Deidre; Muehlbauer, Michael; Hauser, Elizabeth R.; Gregory, Simon G.; Newgard, Christopher B.; Shah, Svati H.

    2015-01-01

    Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6–2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk. PMID:26540294

  11. Metabolomic Quantitative Trait Loci (mQTL) Mapping Implicates the Ubiquitin Proteasome System in Cardiovascular Disease Pathogenesis.

    PubMed

    Kraus, William E; Muoio, Deborah M; Stevens, Robert; Craig, Damian; Bain, James R; Grass, Elizabeth; Haynes, Carol; Kwee, Lydia; Qin, Xuejun; Slentz, Dorothy H; Krupp, Deidre; Muehlbauer, Michael; Hauser, Elizabeth R; Gregory, Simon G; Newgard, Christopher B; Shah, Svati H

    2015-11-01

    Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6-2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk. PMID:26540294

  12. Elimination of C/EBP? through the ubiquitin-proteasome system promotes the development of liver cancer in mice

    PubMed Central

    Wang, Guo-Li; Shi, Xiurong; Haefliger, Simon; Jin, Jingling; Major, Angela; Iakova, Polina; Finegold, Milton; Timchenko, Nikolai A.

    2010-01-01

    Despite significant advancements in our understanding of cancer development, the molecular mechanisms that underlie the formation of liver cancer remain largely unknown. C/EBP? is a transcription factor that regulates liver quiescence. Phosphorylation of C/EBP? at serine 193 (S193-ph) is upregulated in older mice and is thought to contribute to age-associated liver dysfunction. Because development of liver tumors is associated with increasing age, we investigated the role of S193-ph in the development of liver cancer using knockin mice expressing a phospho-mimetic aspartic acid residue in place of serine at position 193 (S193D) of C/EBP?. The S193D isoform of C/EBP? was able to completely inhibit liver proliferation in vivo after partial hepatectomy. However, treatment of these mice with diethylnitrosamine/phenobarbital (DEN/PB), which induces formation of liver cancer, actually resulted in earlier development of liver tumors. DEN/PB treatment was associated with specific degradation of both the S193-ph and S193D isoforms of C/EBP? through activation of the ubiquitin-proteasome system (UPS). The mechanism of UPS-mediated elimination of C/EBP? during carcinogenesis involved elevated levels of gankyrin, a protein that was found to interact with the S193-ph isoform of C/EBP? and target it for UPS-mediated degradation. This study identifies a molecular mechanism that supports the development of liver cancer in older mice and potential therapeutic targets for the prevention of liver cancer. PMID:20516642

  13. Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of ?-synuclein in PC12 cells

    PubMed Central

    WANG, RUNQING; ZHAO, JIE; ZHANG, JIEWEN; LIU, WEI; ZHAO, MEIYING; LI, JIANGTAO; LV, JUAN; LI, YANAN

    2015-01-01

    The aim of this study was to investigate the effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of ?-synuclein, and to analyze its role in the pathogenesis of Parkinson's disease (PD). PC12 cells subjected to nerve growth factor-induced differentiation were used as the cell model to study the dopaminergic neurons, and the lysosomal and proteasomal inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64) and, respectively, were used exclusively and in combination to treat the PC12 cells. The viability and metabolic state of the cells was assessed using the MTT assay; flow cytometry was used to measure the rate of cell apoptosis; and the double immunofluorescence method was applied to observe the formation of thioflavin S- and ?-synuclein protein-positive aggregates and inclusion bodies in the PC12 cells. In addition, the Hoechst 33258 staining method was used to observe the apoptosis of the ?-synuclein protein and thioflavin-S double-labeled cells. Following the administration of the lysosomal and proteasomal pathway inhibitors, the cell viability decreased in a concentration-dependent manner and the cell apoptosis rate increased. The proportion of PC12 cells with ?-synuclein protein-positive aggregates and inclusion bodies in the E64 group was 7.94%, compared with 20.33 and 36.77% in the lactacystin and combination treatment groups, respectively. Statistical analysis indicated that the number of inclusion body-positive cells in the treatment groups was significantly higher than that in the control group (3.78%) (P<0.05). Apoptosis was evident in the double-positive cells with ?-synuclein protein-positive inclusion bodies (17.29±1.54%). In conclusion, lysosomal and proteasomal dysfunction may play an important role in the pathogenesis of PD through the induction of abnormal ?-synuclein protein aggregation in dopaminergic neurons. PMID:26136940

  14. A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation | Office of Cancer Genomics

    Cancer.gov

    Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood.

  15. A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation

    Cancer.gov

    Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood.

  16. Functions of plastid protein import and the ubiquitin-proteasome system in plastid development.

    PubMed

    Ling, Qihua; Jarvis, Paul

    2015-09-01

    Plastids, such as chloroplasts, are widely distributed endosymbiotic organelles in plants and algae. Apart from their well-known functions in photosynthesis, they have roles in processes as diverse as signal sensing, fruit ripening, and seed development. As most plastid proteins are produced in the cytosol, plastids have developed dedicated translocon machineries for protein import, comprising the TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes. Multiple lines of evidence reveal that protein import via the TOC complex is actively regulated, based on the specific interplay between distinct receptor isoforms and diverse client proteins. In this review, we summarize recent advances in our understanding of protein import regulation, particularly in relation to control by the ubiquitin-proteasome system (UPS), and how such regulation changes plastid development. The diversity of plastid import receptors (and of corresponding preprotein substrates) has a determining role in plastid differentiation and interconversion. The controllable turnover of TOC components by the UPS influences the developmental fate of plastids, which is fundamentally linked to plant development. Understanding the mechanisms by which plastid protein import is controlled is critical to the development of breakthrough approaches to increase the yield, quality and stress tolerance of important crop plants, which are highly dependent on plastid development. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25762164

  17. Ubiquitin-proteasome system components are up-regulated during intestinal regeneration

    PubMed Central

    Pasten, Consuelo; Ortiz-Pineda, Pablo A.; E.García-Arrarás, José

    2011-01-01

    The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence suggests that the UPS plays a regulatory role in regeneration processes. Here we explore the possibility that the UPS is involved during intestinal regeneration of the sea cucumber Holothuria glaberrima. These organisms can regenerate most of their digestive tract following a process of evisceration. Initially, we identified components of H. glaberrima UPS, including sequences for Rpn10, ?3 and ubiquitin-RPL40. Predicted proteins from the mRNA sequences showed high degree of conservation that ranged from 60% (Rpn10) to 98% (Ub-RPL40). Microarrays and RT-PCR experiments showed that these genes were up-regulated during intestinal regeneration. In addition, we demonstrated expression of alpha 20S proteasome subunits and ubiquitinated proteins during intestinal regeneration and detected them in the epithelium and connective tissue of the regenerating intestine. Finally, the intestinal regeneration was altered in animals treated with MG132, a proteasome inhibitor. These findings support our contention that proteasomes are playing an important role during intestinal regeneration. PMID:21913312

  18. Polypharmacology of small molecules targeting the ubiquitin–proteasome and ubiquitin-like systems

    PubMed Central

    Amelio, Ivano; Landré, Vivien; Knight, Richard A.; Lisitsa, Andrey; Melino, Gerry; Antonov, Alexey V.

    2015-01-01

    Targeting the ubiquitin–proteasome system (UPS) and ubiquitin-like signalling systems (UBL) has been considered a promising therapeutic strategy to treat cancer, neurodegenerative and immunological disorders. There have been multiple efforts recently to identify novel compounds that efficiently modulate the activities of different disease-specific components of the UPS-UBL. However, it is evident that polypharmacology (the ability to affect multiple independent protein targets) is a basic property of small molecules and even highly potent molecules would have a number of “off target” effects. Here we have explored publicly available high-throughput screening data covering a wide spectrum of currently accepted drug targets in order to understand polypharmacology of small molecules targeting different components of the UPS-UBL. We have demonstrated that molecules targeting a given UPS-UBL protein also have high odds to target a given off target spectrum. Moreover, the off target spectrum differs significantly between different components of UPS-UBL. This information can be utilized further in drug discovery efforts, to improve drug efficiency and to reduce the risk of potential side effects of the prospective drugs designed to target specific UPS-UBL components. PMID:25991664

  19. Cereblon is recruited to aggresome and shows cytoprotective effect against ubiquitin-proteasome system dysfunction.

    PubMed

    Sawamura, Naoya; Wakabayashi, Satoru; Matsumoto, Kodai; Yamada, Haruka; Asahi, Toru

    2015-09-01

    Cereblon (CRBN) is encoded by a candidate gene for autosomal recessive nonsyndromic intellectual disability (ID). The nonsense mutation, R419X, causes deletion of 24 amino acids at the C-terminus of CRBN, leading to mild ID. Although abnormal CRBN function may be associated with ID disease onset, its cellular mechanism is still unclear. Here, we examine the role of CRBN in aggresome formation and cytoprotection. In the presence of a proteasome inhibitor, exogenous CRBN formed perinuclear inclusions and co-localized with aggresome markers. Endogenous CRBN also formed perinuclear inclusions under the same condition. Treatment with a microtubule destabilizer or an inhibitor of the E3 ubiquitin ligase activity of CRBN blocked formation of CRBN inclusions. Biochemical analysis showed CRBN containing inclusions were high-molecular weight, ubiquitin-positive. CRBN overexpression in cultured cells suppressed cell death induced by proteasome inhibitor. Furthermore, knockdown of endogenous CRBN in cultured cells increased cell death induced by proteasome inhibitor, compared with control cells. Our results show CRBN is recruited to aggresome and has functional roles in cytoprotection against ubiquitin-proteasome system impaired condition. PMID:26188093

  20. Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension

    PubMed Central

    Kasahara, Kousuke; Kawakami, Yoshitaka; Kiyono, Tohru; Yonemura, Shigenobu; Kawamura, Yoshifumi; Era, Saho; Matsuzaki, Fumio; Goshima, Naoki; Inagaki, Masaki

    2014-01-01

    Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis. PMID:25270598

  1. The PML-nuclear inclusion of human supraoptic neurons: a new compartment with SUMO-1- and ubiquitin-proteasome-associated domains.

    PubMed

    Villagra, Nuria T; Navascues, Joaquin; Casafont, Iñigo; Val-Bernal, J Fernando; Lafarga, Miguel; Berciano, Maria T

    2006-01-01

    It is well known that the cell nucleus is organized in structural and functional compartments involved in transcription, RNA processing and protein modifications such as conjugation with SUMO-1 and proteolysis. Promyelocytic leukaemia (PML) bodies are dynamic nuclear structures that concentrate PML protein, SUMO-1 and several sumoylated and non-sumoylated protein regulators of nuclear functions. PML bodies and their associated CBP has been involved in neuronal survival. By light and electron microscopy immunocytochemistry and in situ hybridization we reported the presence, in non-pathological conditions, of a large PML-nuclear inclusion (PML-NI) in human supraoptic neurons. This inclusion appears as a single nuclear structure composed of a capsule enriched in PML, SUMO-1 and CBP proteins and a central lattice of filaments immunoreactive for class III beta-tubulin, ubiquitinated proteins and proteasomes. Furthermore, the PML-NI concentrates the SUMO-conjugating enzyme E2 (UBC9). The PML-NI may be considered a nuclear factory involved in sumoylation and proteolysis via ubiquitin-proteasome system, two nuclear pathways engaged in the control of the nucleoplasmic concentration of active transcriptional regulators. Interestingly, the structural and molecular organization of the PML-NI is related to the Marinesco bodies, age-associated ubiquitinated intranuclear inclusions, and to the intranuclear rodlets enriched in class III beta-tubulin, which are nuclear structures markedly decreased in Alzheimer's disease. PMID:16125395

  2. 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

  3. The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

    PubMed Central

    Amaya, Moushimi; Keck, Forrest; Lindquist, Michael; Voss, Kelsey; Scavone, Lauren; Kehn-Hall, Kylene; Roberts, Brian; Bailey, Charles; Schmaljohn, Connie; Narayanan, Aarthi

    2015-01-01

    Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections. PMID:25927990

  4. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  5. Amino-terminal arginylation as a degradation signal for selective autophagy

    PubMed Central

    Cha-Molstad, Hyunjoo; Kwon, Yong Tae; Kim, Bo Yeon

    2015-01-01

    The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins. [BMB Reports 2015; 48(9): 487-488] PMID:26303972

  6. Degradation of Postsynaptic Scaffold GKAP and Regulation of Dendritic Spine Morphology by the TRIM[subscript 3] Ubiquitin Ligase in Rat Hippocampal Neurons

    E-print Network

    Sheng, Morgan Hwa-Tze

    Changes in neuronal activity modify the structure of dendritic spines and alter the function and protein composition of synapses. Regulated degradation of postsynaptic density (PSD) proteins by the ubiquitin-proteasome ...

  7. FBG1 Is the Final Arbitrator of A1AT-Z Degradation

    PubMed Central

    Wen, John H.; Wen, Hsiang; Gibson-Corley, Katherine N.; Glenn, Kevin A.

    2015-01-01

    Alpha-1 antitrypsin deficiency is the leading cause of childhood liver failure and one of the most common lethal genetic diseases. The disease-causing mutant A1AT-Z fails to fold correctly and accumulates in the endoplasmic reticulum (ER) of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma in a subset of patients. Furthermore, A1AT-Z sequestration in hepatocytes leads to a reduction in A1AT secretion into the serum, causing panacinar emphysema in adults. The purpose of this work was to elucidate the details by which A1AT-Z is degraded in hepatic cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT-Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT-Z through both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half-life of A1AT-Z and knocking down FBG1 in a hepatic cell line, and in mice results in an increase in ATAT. Finally, we show that FBG1 degrades A1AT-Z through a Beclin1-dependent arm of autophagy. In our model, FBG1 acts as a safety ubiquitin ligase, whose function is to re-ubiquitinate ER proteins that have previously undergone de-ubiquitination to ensure they are degraded. PMID:26295339

  8. High intensity training induces alteration of the ubiquitin-proteasome system gene expression profile and structural changes in the ovaries.

    PubMed

    Xiong, Ruohong; Ren, Xue; Wang, Guiming; Chen, Guoqing; Yang, Huijun; Yuan, Qiongjia; Zhou, Hongying

    2012-05-01

    The female reproductive system is highly sensitive to physiological stress. However, the mechanism(s) involved in dysfunction of the ovaries induced by high intensity exercise training remain unknown. In the present study, we established a rat model of high intensity exercise training, and investigated the morphological and functional changes in ovaries. Furthermore, we profiled the differential gene expression in the ovaries between high intensity exercise training rats and control rats by restriction fragment differential display PCR. Differentially expressed genes were validated by real-time PCR. The results showed that the weight and microstructure of the ovaries were significantly altered in the exercise training group rats. In addition, the levels of estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone were significantly decreased in the exercise training group rats compared with the control group rats. Immunohistochemistry revealed weak expression of estradiol, estrogen receptor and progesterone receptor in ovarian tissues from the high intensity exercise training group compared with the control group. Furthermore, the gene expression profiles of the exercise training and control group rats were analyzed, and differentially expressed genes, such as those involved in the ubiquitin-proteasome system, were identified. Our results indicate that high intensity exercise training can suppress the cellular function of the ovary glands. The mechanism may involve a series of alterations in the expression of genes particularly associated with the ubiquitin-proteasome system. PMID:22366872

  9. Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

    E-print Network

    Bogyo, Matthew

    . Ploegh*¶ , and Rickard Glas* *Department of Pathology, Harvard Medical School, 200 Longwood Avenue) was identified as one protease responsible for this activity (19). TPPII is a large cytosolic oligopeptidase

  10. cAMP STIMULATES THE UBIQUITIN/PROTEASOME PATHWAY IN RAT SPINAL CORD NEURONS

    PubMed Central

    Myeku, Natura; Wang, Hu; Figueiredo-Pereira, Maria E.

    2012-01-01

    Proteasome impairment and accumulation of ubiquitinated proteins are implicated in neurodegeneration associated with different forms of spinal cord injury. We show herein that elevating cAMP in rat spinal cord neurons increases 26S proteasome activity in a protein kinase A-dependent manner. Treating spinal cord neurons with dibutyryl-cAMP (db-cAMP) also raised the levels of various components of the UPP including proteasome subunits Rpt6 and ?5, polyubiquitin shuttling factor p62/sequestosome1, E3 ligase CHIP, AAA-ATPase p97 and the ubiquitin gene ubB. Finally, db-cAMP reduced the accumulation of ubiquitinated proteins, proteasome inhibition, and neurotoxicity triggered by the endogenous product of inflammation prostaglandin J2. We propose that optimizing the effects of cAMP/PKA-signaling on the UPP could offer an effective therapeutic approach to prevent UPP-related proteotoxicity in spinal cord neurons. PMID:22982149

  11. Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy*

    PubMed Central

    McGorum, Bruce C.; Pirie, R. Scott; Eaton, Samantha L.; Keen, John A.; Cumyn, Elizabeth M.; Arnott, Danielle M.; Chen, Wenzhang; Lamont, Douglas J.; Graham, Laura C.; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M.

    2015-01-01

    Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ?2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. PMID:26364976

  12. Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

    PubMed

    McGorum, Bruce C; Pirie, R Scott; Eaton, Samantha L; Keen, John A; Cumyn, Elizabeth M; Arnott, Danielle M; Chen, Wenzhang; Lamont, Douglas J; Graham, Laura C; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M

    2015-11-01

    Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ?2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. PMID:26364976

  13. An essential postsynaptic role for the ubiquitin proteasome system in slow homeostatic synaptic plasticity in cultured hippocampal neurons.

    PubMed

    Jakawich, S K; Neely, R M; Djakovic, S N; Patrick, G N; Sutton, M A

    2010-12-29

    Chronic increases or decreases in neuronal activity initiates compensatory changes in synaptic strength that emerge slowly over a 12-24 h period, but the mechanisms underlying this slow homeostatic response remain poorly understood. Here, we show an essential role for the ubiquitin proteasome system (UPS) in slow homeostatic plasticity induced by chronic changes in network activity. In cultured hippocampal neurons, UPS inhibitors drive a slow increase in miniature excitatory postsynaptic current (mEPSC) amplitude and synaptic AMPA receptor subunit GluA1 and GluA2 expression that both mirrors and occludes the changes produced by chronic suppression of network activity with tetrodotoxin (TTX). These non-additive effects were similarly observed under conditions of chronic hyperactivation of network activity with bicuculline--the increase in mEPSC amplitude and GluA1/2 expression with chronic UPS inhibition persists during network hyperactivation, which scales synaptic strength and AMPA receptor expression in the opposite direction when UPS activity is intact. Finally, cell-autonomous UPS inhibition (via expression of the ubiquitin chain elongation mutant, UbK48R) enhances mEPSC amplitude in a manner that mimics and occludes changes in network activity, demonstrating a postsynaptic role for the UPS in slow homeostatic plasticity. Taken together, our results suggest that the UPS acts as an integration point for translating sustained changes in network activity into appropriate incremental compensatory changes at synapses. PMID:20888892

  14. Oxidative stress regulates the ubiquitin-proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy.

    PubMed

    Launay, Nathalie; Ruiz, Montserrat; Fourcade, Stéphane; Schlüter, Agatha; Guilera, Cristina; Ferrer, Isidre; Knecht, Erwin; Pujol, Aurora

    2013-03-01

    Oxidative damage is a pivotal aetiopathogenic factor in X-linked adrenoleukodystrophy. This is a neurometabolic disease characterized by the accumulation of very-long-chain fatty acids owing to the loss of function of the peroxisomal transporter Abcd1. Here, we used the X-linked adrenoleukodystrophy mouse model and patient's fibroblasts to detect malfunctioning of the ubiquitin-proteasome system resulting from the accumulation of oxidatively modified proteins, some involved in bioenergetic metabolism. Furthermore, the immunoproteasome machinery appears upregulated in response to oxidative stress, in the absence of overt inflammation. i-Proteasomes are recruited to mitochondria when fibroblasts are exposed to an excess of very-long-chain fatty acids in response to oxidative stress. Antioxidant treatment regulates proteasome expression, prevents i-proteasome induction and translocation of i-proteasomes to mitochondria. Our findings support a key role of i-proteasomes in quality control in mitochondria during oxidative damage in X-linked adrenoleukodystrophy, and perhaps in other neurodegenerative conditions with similar pathogeneses. PMID:23436506

  15. Lipotoxic Stress Induces Pancreatic ?-Cell Apoptosis through Modulation of Bcl-2 Proteins by the Ubiquitin-Proteasome System

    PubMed Central

    Litwak, Sara A.; Wali, Jibran A.; Pappas, Evan G.; Saadi, Hamdi; Stanley, William J.; Varanasi, L. Chitra; Kay, Thomas W. H.; Thomas, Helen E.; Gurzov, Esteban N.

    2015-01-01

    Pancreatic ?-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of ?-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in ?-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected ?-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent ?-cell demise in type 2 diabetes. PMID:26064977

  16. Ubiquitination Regulates PSD-95 Degradation and AMPA Receptor Surface Expression

    PubMed Central

    Colledge, Marcie; Snyder, Eric M.; Crozier, Robert A.; Soderling, Jacquelyn A.; Jin, Yetao; Langeberg, Lorene K.; Lu, Hua; Bear, Mark F.; Scott, John D.

    2014-01-01

    Summary PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity. PMID:14642282

  17. Response of the Ubiquitin-Proteasome System to Memory Retrieval After Extended-Access Cocaine or Saline Self-Administration.

    PubMed

    Werner, Craig T; Milovanovic, Mike; Christian, Daniel T; Loweth, Jessica A; Wolf, Marina E

    2015-12-01

    The ubiquitin-proteasome system (UPS) has been implicated in the retrieval-induced destabilization of cocaine- and fear-related memories in Pavlovian paradigms. However, nothing is known about its role in memory retrieval after self-administration of cocaine, an operant paradigm, or how the length of withdrawal from cocaine may influence retrieval mechanisms. Here, we examined UPS activity after an extended-access cocaine self-administration regimen that leads to withdrawal-dependent incubation of cue-induced cocaine craving. Controls self-administered saline. In initial experiments, memory retrieval was elicited via a cue-induced seeking/retrieval test on withdrawal day (WD) 50-60, when craving has incubated. We found that retrieval of cocaine- and saline-associated memories produced similar increases in polyubiquitinated proteins in the nucleus accumbens (NAc), compared with rats that did not undergo a seeking/retrieval test. Measures of proteasome catalytic activity confirmed similar activation of the UPS after retrieval of saline and cocaine memories. However, in a subsequent experiment in which testing was conducted on WD1, proteasome activity in the NAc was greater after retrieval of cocaine memory than saline memory. Analysis of other brain regions confirmed that effects of cocaine memory retrieval on proteasome activity, relative to saline memory retrieval, depend on withdrawal time. These results, combined with prior studies, suggest that the relationship between UPS activity and memory retrieval depends on training paradigm, brain region, and time elapsed between training and retrieval. The observation that mechanisms underlying cocaine memory retrieval change depending on the age of the memory has implications for development of memory destabilization therapies for cue-induced relapse in cocaine addicts. PMID:26044907

  18. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  19. Prion promoted phosphorylation of heterologous amyloid is coupled with ubiquitin-proteasome system inhibition and toxicity

    PubMed Central

    Yang, Zi; Stone, David E.; Liebman, Susan W.

    2014-01-01

    Summary Many neurodegenerative diseases are associated with conversion of a soluble protein into amyloid deposits, but how this is connected to toxicity remains largely unknown. Here, we explore mechanisms of amyloid associated toxicity using yeast. [PIN+], the prion form of the Q/N-rich Rnq1 protein, was known to enhance aggregation of heterologous proteins, including the overexpressed Q/N-rich amyloid forming domain of Pin4 (Pin4C), and Pin4C aggregates were known to attract chaperones, including Sis1. Here we show that in [PIN+] but not [pin?] cells, overexpression of Pin4C is deadly and linked to hyperphosphorylation of aggregated Pin4C. Furthermore, Pin4C aggregation, hyperphosphorylation and toxicity are simultaneously reversed by Sis1 overexpression. Toxicity may result from proteasome overload because hyperphosphorylated Pin4C aggregation is associated with reduced degradation of a ubiquitin-protein degradation reporter. Finally, hyperphosphorylation of endogenous full-length Pin4 was also facilitated by [PIN+], revealing that a prion can regulate posttranslational modification of another protein. PMID:25039275

  20. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    SciTech Connect

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-10-16

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic {alpha}- and {beta}-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

  1. Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53.

    PubMed

    Zhang, Shengliang; Zhou, Lanlan; Hong, Bo; van den Heuvel, A Pieter J; Prabhu, Varun V; Warfel, Noel A; Kline, Christina Leah B; Dicker, David T; Kopelovich, Levy; El-Deiry, Wafik S

    2015-09-15

    The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling. Cancer Res; 75(18); 3842-52. ©2015 AACR. PMID:26294215

  2. S-Nitrosation and Ubiquitin-Proteasome System Interplay in Neuromuscular Disorders

    PubMed Central

    Rizza, Salvatore; Montagna, Costanza; Di Giacomo, Giuseppina; Cirotti, Claudia; Filomeni, Giuseppe

    2014-01-01

    Protein S-nitrosation is deemed as a prototype of posttranslational modifications governing cell signaling. It takes place on specific cysteine residues that covalently incorporate a nitric oxide (NO) moiety to form S-nitrosothiol derivatives and depends on the ratio between NO produced by NO synthases and nitrosothiol removal catalyzed by denitrosating enzymes. A large number of cysteine-containing proteins are found to undergo S-nitrosation and, among them, the enzymes catalyzing ubiquitination, mainly the class of ubiquitin E3 ligases and the 20S component of the proteasome, have been reported to be redox modulated in their activity. In this review we will outline the processes regulating S-nitrosation and try to debate whether and how it affects protein ubiquitination and degradation via the proteasome. In particular, since muscle and neuronal health largely depends on the balance between protein synthesis and breakdown, here we will discuss the impact of S-nitrosation in the efficiency of protein quality control system, providing lines of evidence and speculating about its involvement in the onset and maintenance of neuromuscular dysfunctions. PMID:24627685

  3. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    SciTech Connect

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  4. The role of the ubiquitin proteasome system in cerebellar development and medulloblastoma.

    PubMed

    Vriend, Jerry; Ghavami, Saeid; Marzban, Hassan

    2015-01-01

    Cerebellar granule cells precursors are derived from the upper rhombic lip and migrate tangentially independent of glia along the subpial stream pathway to form the external germinal zone. Postnatally, granule cells migrate from the external germinal zone radially through the Purkinje cell layer, guided by Bergmann glia fibers, to the internal granular cell layer.Medulloblastomas (MBs) are the most common malignant childhood brain tumor. Many of these tumors develop from precursor cells of the embryonic rhombic lips. Four main groups of MB are recognized. The WNT group of MBs arise primarily from the lower rhombic lip and embryonic brainstem. The SHH group of MBs originate from cerebellar granule cell precursors in the external germinal zone of the embryonic cerebellum. The cellular origins of type 3 and type 4 MBs are not clear.Several ubiquitin ligases are revealed to be significant factors in development of the cerebellum as well as in the initiation and maintenance of MBs. Proteasome dysfunction at a critical stage of development may be a major factor in determining whether progenitor cells which are destined to become granule cells differentiate normally or become MB cells. We propose the hypothesis that proteasomal activity is essential to regulate the critical transition between proliferating granule cells and differentiated granule cells and that proteasome dysfunction may lead to MB. Proteasome dysfunction could also account for various mutations in MBs resulting from deficiencies in DNA checkpoint and repair mechanisms prior to development of MBs.Data showing a role for the ubiquitin ligases ?-TrCP, FBW7, Huwe1, and SKP2 in MBs suggest the possibility of a classification of MBs based on the expression (over expression or under expression) of specific ubiquitin ligases which function as oncogenes, tumor suppressors or cell cycle regulators. PMID:26475605

  5. L166P MUTANT DJ-1, CAUSATIVE FOR RECESSIVE PARKINSON'S DISEASE IS DEGRADED THROUGH THE UBIQUITIN-PROTEASOME SYSTEM

    EPA Science Inventory

    Abstract

    Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, early-onset Parkinson's disease. Whilst one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point ...

  6. Tyrosine Hydroxylase Is Short-Term Regulated by the Ubiquitin-Proteasome System in PC12 Cells and Hypothalamic and Brainstem Neurons from Spontaneously Hypertensive Rats: Possible Implications in Hypertension

    PubMed Central

    Carbajosa, Nadia A. Longo; Corradi, Gerardo; Verrilli, María A. Lopez; Guil, María J.; Vatta, Marcelo S.; Gironacci, Mariela M.

    2015-01-01

    Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension. PMID:25710381

  7. Akt activation increases cellular cholesterol by promoting the proteasomal degradation of Niemann-Pick C1.

    PubMed

    Du, Ximing; Zhang, Yuxi; Jo, Sae Rom; Liu, Xiaoyun; Qi, Yanfei; Osborne, Brenna; Byrne, Frances L; Smith, Greg C; Turner, Nigel; Hoehn, Kyle L; Brown, Andrew J; Yang, Hongyuan

    2015-10-15

    Null mutations of the Niemann-Pick type C1 (NPC1) gene cause NPC disease, a lysosomal storage disorder characterized by cholesterol accumulation in late endosomes (LE) and lysosomes (Ly). Nascent or mutated NPC1 is degraded through the ubiquitin-proteasome pathway, but how NPC1 degradation is regulated remains currently unknown. In the present study, we demonstrated a link between NPC1 degradation and the Akt (protein kinase B)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway in cervical cancer cell lines. We provided evidence that activated Akt/mTOR pathway increased NPC1 degradation by ?50% in C33A cells when compared with SiHa or HeLa cells. NPC1 degradation in C33A cells was reversed when Akt/mTOR activation was blocked by specific inhibitors or when mTORC1 (mTOR complex 1) was disrupted by regulatory associated protein of mTOR (Raptor) knockdown. Importantly, inhibition of the Akt/mTOR pathway led to decreased NPC1 ubiquitination in C33A cells, pointing to a role of Akt/mTOR in the proteasomal degradation of NPC1. Moreover, we found that NPC1 depletion in several cancer cell lines inhibited cell proliferation and migration. Our results uncover Akt as a key regulator of NPC1 degradation and link NPC1 to cancer cell proliferation and migration. PMID:26283546

  8. Interactions of Xanthomonas type-III effector proteins with the plant ubiquitin and ubiquitin-like pathways

    PubMed Central

    Üstün, Suayib; Börnke, Frederik

    2014-01-01

    In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin–proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function. PMID:25566304

  9. Transforming growth factor-?3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

    PubMed

    Zhang, Xu; Lui, Wing-Yee

    2015-06-01

    Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-?) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-?3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-?3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-?3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-?3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-?3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-?3-induced JAM-B protein degradation. In addition, TGF-?3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-?3 promotes the degradation of JAM-B transcript and TGF-?3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-?3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES. PMID:25817991

  10. Overview of Proteasome Inhibitor-Based Anti-cancer Therapies: Perspective on Bortezomib and Second Generation Proteasome Inhibitors versus Future Generation Inhibitors of Ubiquitin-Proteasome System

    PubMed Central

    Dou, Q. Ping; Zonder, Jeffrey A.

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ-based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  11. Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system.

    PubMed

    Wang, Hongjuan; Zhu, Xiaonan; Li, Huan; Cui, Jing; Liu, Cheng; Chen, Xi; Zhang, Wei

    2014-01-01

    It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD. PMID:25103621

  12. Dual mode of degradation of Cdc25 A phosphatase

    PubMed Central

    Donzelli, Maddalena; Squatrito, Massimo; Ganoth, Dvora; Hershko, Avram; Pagano, Michele; Draetta, Giulio F.

    2002-01-01

    The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin–proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G1 is mediated by the anaphase-promoting complex or cyclosome (APC/C)Cdh1 ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment. PMID:12234927

  13. Prion degradation pathways: Potential for therapeutic intervention

    PubMed Central

    Goold, Rob; McKinnon, Chris; Tabrizi, Sarah J.

    2015-01-01

    Prion diseases are fatal neurodegenerative disorders. Pathology is closely linked to the misfolding of native cellular PrPC into the disease-associated form PrPSc that accumulates in the brain as disease progresses. Although treatments have yet to be developed, strategies aimed at stimulating the degradation of PrPSc have shown efficacy in experimental models of prion disease. Here, we describe the cellular pathways that mediate PrPSc degradation and review possible targets for therapeutic intervention. This article is part of a Special Issue entitled ‘Neuronal Protein’. PMID:25584786

  14. Natural small molecule FMHM inhibits lipopolysaccharide-induced inflammatory response by promoting TRAF6 degradation via K48-linked polyubiquitination

    PubMed Central

    Zeng, Ke-Wu; Liao, Li-Xi; Lv, Hai-Ning; Song, Fang-Jiao; Yu, Qian; Dong, Xin; Li, Jun; Jiang, Yong; Tu, Peng-Fei

    2015-01-01

    TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-?B inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-?B inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases. PMID:26423026

  15. Prion-promoted phosphorylation of heterologous amyloid is coupled with ubiquitin-proteasome system inhibition and toxicity.

    PubMed

    Yang, Zi; Stone, David E; Liebman, Susan W

    2014-09-01

    Many neurodegenerative diseases are associated with conversion of a soluble protein into amyloid deposits, but how this is connected to toxicity remains largely unknown. Here, we explore mechanisms of amyloid associated toxicity using yeast. [PIN(+)], the prion form of the Q/N-rich Rnq1 protein, was known to enhance aggregation of heterologous proteins, including the overexpressed Q/N-rich amyloid forming domain of Pin4 (Pin4C), and Pin4C aggregates were known to attract chaperones, including Sis1. Here we show that in [PIN(+)] but not [pin(-)] cells, overexpression of Pin4C is deadly and linked to hyperphosphorylation of aggregated Pin4C. Furthermore, Pin4C aggregation, hyperphosphorylation and toxicity are simultaneously reversed by Sis1 overexpression. Toxicity may result from proteasome overload because hyperphosphorylated Pin4C aggregation is associated with reduced degradation of a ubiquitin-protein degradation reporter. Finally, hyperphosphorylation of endogenous full-length Pin4 was also facilitated by [PIN(+)], revealing that a prion can regulate post-translational modification of another protein. PMID:25039275

  16. Elimination of C/EBPalpha through the ubiquitin-proteasome system promotes the development of liver cancer in mice.

    PubMed

    Wang, Guo-Li; Shi, Xiurong; Haefliger, Simon; Jin, Jingling; Major, Angela; Iakova, Polina; Finegold, Milton; Timchenko, Nikolai A

    2010-07-01

    Despite significant advancements in our understanding of cancer development, the molecular mechanisms that underlie the formation of liver cancer remain largely unknown. C/EBPalpha is a transcription factor that regulates liver quiescence. Phosphorylation of C/EBPalpha at serine 193 (S193-ph) is upregulated in older mice and is thought to contribute to age-associated liver dysfunction. Because development of liver tumors is associated with increasing age, we investigated the role of S193-ph in the development of liver cancer using knockin mice expressing a phospho-mimetic aspartic acid residue in place of serine at position 193 (S193D) of C/EBPalpha. The S193D isoform of C/EBPalpha was able to completely inhibit liver proliferation in vivo after partial hepatectomy. However, treatment of these mice with diethylnitrosamine/phenobarbital (DEN/PB), which induces formation of liver cancer, actually resulted in earlier development of liver tumors. DEN/PB treatment was associated with specific degradation of both the S193-ph and S193D isoforms of C/EBPalpha through activation of the ubiquitinproteasome system (UPS). The mechanism of UPS-mediated elimination of C/EBPalpha during carcinogenesis involved elevated levels of gankyrin, a protein that was found to interact with the S193-ph isoform of C/EBPalpha and target it for UPS-mediated degradation. This study identifies a molecular mechanism that supports the development of liver cancer in older mice and potential therapeutic targets for the prevention of liver cancer. PMID:20516642

  17. Redox control of protein degradation.

    PubMed

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H K; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-12-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  18. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  19. 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.

  20. Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin-proteasome-dependent proteolysis in cancer and septic rats.

    PubMed Central

    Combaret, Lydie; Tilignac, Thomas; Claustre, Agnès; Voisin, Laure; Taillandier, Daniel; Obled, Christiane; Tanaka, Keiji; Attaix, Didier

    2002-01-01

    The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-alpha (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14 kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19 S regulatory complex of the 26 S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub-proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis. PMID:11772390

  1. pVHL interacts with Ceramide kinase like (CERKL) protein and ubiquitinates it for oxygen dependent proteasomal degradation.

    PubMed

    Chen, Jiaxiang; Liu, Fei; Li, Hui; Archacki, Stephen; Gao, Meng; Liu, Ying; Liao, Shengjie; Huang, Mi; Wang, Jiuxiang; Yu, Shanshan; Li, Chang; Tang, Zhaohui; Liu, Mugen

    2015-11-01

    Mutations of Ceramide kinase-like (CERKL) gene are associated with retinitis pigmentosa (RP), an inherited degenerative eye disease. CERKL encodes an antioxidant protein which is critical to photoreceptor survival, its deficiency causes retinal degeneration as a result of oxidative damage. However, the regulation of CERKL in response to oxidative stress, and its contribution to photoreceptor survival remain unclear. pVHL, the substrate receptor of RING finger-type SCF like ECV ubiquitin ligase, binds and ubiquitinates a number of hydroxylated proteins for proteasomal degradation. Due to hydroxylated proteins which are modified by PHD1-3, pVHL dependent ubiquitin-proteasomal degradation pathway is blocked by PHD1-3 inhibitors (e.g. hypoxia or oxidative stress). In this study, we identified pVHL as an important regulator of CERKL. Western blot and in vivo ubiquitination assays showed hypoxia up-regulates CERKL at protein level by down-regulating its poly-ubiquitination. By Co-IP and domain mapping studies, we found CERKL complexes with ECV ligase via pVHL. Through overexpression and small RNA interference analysis, we demonstrated pVHL ubiquitinates CERKL for proteasomal degradation. Additionally, our work showed that the oxygen sensors PHD1 and PHD3 are involved in CERKL degradation. Collectively, our results indicated that pVHL interacts with CERKL and ubiquitinates it for oxygen dependent proteasomal degradation. PMID:26296657

  2. Molecular Design, Synthesis, and Evaluation of SNIPER(ER) That Induces Proteasomal Degradation of ER?.

    PubMed

    Okuhira, Keiichiro; Demizu, Yosuke; Hattori, Takayuki; Ohoka, Nobumichi; Shibata, Norihito; Kurihara, Masaaki; Naito, Mikihiko

    2016-01-01

    Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we recently developed a novel small molecule SNIPER(ER) that targets the estrogen receptor alpha (ER?) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds, and methods for the evaluation of their activity in cellular system. PMID:26585163

  3. The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina.

    PubMed

    Wiemer, Matthias; Osiewacz, Heinz D

    2014-01-01

    The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina. PMID:25520781

  4. The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina

    PubMed Central

    Wiemer, Matthias; Osiewacz, Heinz D.

    2014-01-01

    The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina. PMID:25520781

  5. ATR checkpoint kinase and CRL1?TRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

    PubMed Central

    Im, Jun-Sub; Keaton, Mignon; Lee, Kyung Yong; Kumar, Pankaj; Park, Jonghoon; Dutta, Anindya

    2014-01-01

    Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1?TRCP-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%–10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin. PMID:24700029

  6. Role of the ubiquitin-proteasome pathway in the inner ear : identification of an E3 ubiquitin ligase for Atoh1

    E-print Network

    Cheng, Yen-Fu

    2014-01-01

    Atoh1, the proneural basic-helix-loop-helix transcription factor, is critical for the differentiation of inner ear hair cells. Hair cells do not develop in mice that lack Atoh1, and overexpression of the transcription ...

  7. Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). T...

  8. Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes.

    PubMed

    Dimitrakis, Polychronis; Romay-Ogando, Maria-Iris; Timolati, Francesco; Suter, Thomas M; Zuppinger, Christian

    2012-11-01

    The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy. PMID:22864983

  9. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  10. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  11. Cancerous inhibitor of PP2A is targeted by natural compound celastrol for degradation in non-small-cell lung cancer

    PubMed Central

    Liu, Zi; Ma, Liang; Wen, Zhe-Sheng; Hu, Zheng; Wu, Fu-Qun; Li, Wei; Liu, Jinsong; Zhou, Guang-Biao

    2014-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed and inversely associated with prognosis in lung and many other human cancers. It modulates phospho-Akt and stabilizes c-Myc, and is required for cell proliferation and malignant transformation, indicating that CIP2A may play an important role in carcinogenesis. We reported here that a small compound celastrol could induce a rapid degradation of CIP2A, through the ubiquitin–proteasome pathway with the carboxyl terminus of Hsp70-interacting protein (CHIP) as the E3 ligase. Celastrol directly bound CIP2A protein and promoted CIP2A–CHIP interaction, leading to subsequent degradation of CIP2A in non-small-cell lung cancer cells. Furthermore, celastrol effectively inhibited cell proliferation and induced apoptosis in non-small-cell lung cancer cells, whereas CIP2A silencing enhanced these effects. Celastrol also suppressed tumor growth in xenograft murine models. In addition, celastrol potentiated the inhibitory effect of cytotoxic agent cisplatin on lung cancer cells in vitro and in vivo via inhibition of CIP2A–Akt pathway. These data indicate that celastrol is a CIP2A-targeting agent that may have therapeutic potentials in lung cancer. PMID:24293411

  12. Ubiquitin-protein ligase E3C promotes glioma progression by mediating the ubiquitination and degrading of Annexin A7.

    PubMed

    Pan, Si-Jian; Zhan, Shi-Kun; Ji, Wei-Zhong; Pan, Yi-Xin; Liu, Wei; Li, Dian-You; Huang, Peng; Zhang, Xiao-Xiao; Cao, Chun-Yan; Zhang, Jing; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang

    2015-01-01

    The ubiquitin-protein ligase E3C (UBE3C) belongs to the E3 ligase enzyme family and implicates in the ubiquitin-proteasome pathway, thus regulates physiological and cancer-related processes. Here, we investigated the expression and roles of UBE3C in glioma. We demonstrated that UBE3C was overexpressed in glioma tissues and cell lines. Inhibition of UBE3C expression in glioma cells significantly decreased cell migration and invasion in vitro. Mechanistically, we disclosed that UBE3C physically interacted with and ubiquitinated tumor suppressor gene annexin A7 (ANXA7), resulting in ubiquitination and degradation of ANXA7. Our results also revealed that increased UBE3C expression was accompanied by a reduction in ANXA7 protein expression in glioma tissues, but not ANXA7 mRNA. Importantly, the inhibition of ANXA7 expression in gliomas cells with UBE3C interference could rescue the cell invasion. Clinically, UBE3C overexpression significantly correlated with high-grade tumors (p?degradation of ANXA7, and thus presents a novel and promising target for glioma therapy. PMID:26067607

  13. Ubiquitin-protein ligase E3C promotes glioma progression by mediating the ubiquitination and degrading of Annexin A7

    PubMed Central

    Pan, Si-Jian; Zhan, Shi-Kun; Ji, Wei-Zhong; Pan, Yi-Xin; Liu, Wei; Li, Dian-You; Huang, Peng; Zhang, Xiao-Xiao; Cao, Chun-Yan; Zhang, Jing; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang

    2015-01-01

    The ubiquitin-protein ligase E3C (UBE3C) belongs to the E3 ligase enzyme family and implicates in the ubiquitin-proteasome pathway, thus regulates physiological and cancer-related processes. Here, we investigated the expression and roles of UBE3C in glioma. We demonstrated that UBE3C was overexpressed in glioma tissues and cell lines. Inhibition of UBE3C expression in glioma cells significantly decreased cell migration and invasion in vitro. Mechanistically, we disclosed that UBE3C physically interacted with and ubiquitinated tumor suppressor gene annexin A7 (ANXA7), resulting in ubiquitination and degradation of ANXA7. Our results also revealed that increased UBE3C expression was accompanied by a reduction in ANXA7 protein expression in glioma tissues, but not ANXA7 mRNA. Importantly, the inhibition of ANXA7 expression in gliomas cells with UBE3C interference could rescue the cell invasion. Clinically, UBE3C overexpression significantly correlated with high-grade tumors (p?degradation of ANXA7, and thus presents a novel and promising target for glioma therapy. PMID:26067607

  14. Participation of proteasome-ubiquitin protein degradation in autophagy and the activation of AMP-activated protein kinase.

    PubMed

    Jiang, Shaoning; Park, Dae Won; Gao, Yong; Ravi, Saranya; Darley-Usmar, Victor; Abraham, Edward; Zmijewski, Jaroslaw W

    2015-06-01

    Although activation of the AMP-activated protein kinase (AMPK) as well as of ubiquitin/proteasome degradative pathways play an essential role in the preservation of metabolic homeostasis, little is known concerning interactions between protein turnover and AMPK activity. In the present studies, we found that inhibition of the 26S proteasome resulted in rapid activation of AMPK in macrophages, epithelial and endothelial cells. This was associated with increased levels of non-degraded Ub-protein conjugates, in both cytosolic and mitochondrial fractions. Selective inhibitors of ubiquitination or siRNA-dependent knockdown of Ub-ligase E1 diminished AMPK activation in cells treated with MG132, a 26S proteasome inhibitor. In addition to inhibition of AMPK activation by Ub-ligase E1 inhibitors, deficiency in Park2 mitochondria-associated Ub-ligase E3 also reduced AMPK activation upon dissipation of mitochondrial membrane potential (??m). Accumulation of Ub-proteins was correlated with decreases in cellular bioenergetics, including mitochondria oxidative phosphorylation, and an increase in ROS formation. Antioxidants, such as N-acetyl-L-cysteine or mitochondria-targeted MitoTEMPO, effectively diminished MG132-induced AMPK activation. Glucose-dependent regulation of AMPK or AMPK-mediated autophagy was modulated by alterations in intracellular levels of Ub-protein conjugates. Our results indicate that accumulation of ubiquitinated proteins alter cellular bioenergetics and redox status, leading to AMPK activation. PMID:25728513

  15. Vitamin C Degradation Products and Pathways in the Human Lens*

    PubMed Central

    Nemet, Ina; Monnier, Vincent M.

    2011-01-01

    Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ?20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo. PMID:21885436

  16. Vitamin C degradation products and pathways in the human lens.

    PubMed

    Nemet, Ina; Monnier, Vincent M

    2011-10-28

    Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ?20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo. PMID:21885436

  17. Alkaline hydrogen peroxide pretreatment of softwood: hemicellulose degradation pathways.

    PubMed

    Alvarez-Vasco, Carlos; Zhang, Xiao

    2013-12-01

    This study investigated softwood hemicelluloses degradation pathways during alkaline hydrogen peroxide (AHP) pretreatment of Douglas fir. It was found that glucomannan is much more susceptible to alkaline pretreatment than xylan. Organic acids, including lactic, succinic, glycolic and formic acid are the predominant products from glucomannan degradation. At low treatment temperature (90°C), a small amount of formic acid is produced from glucomannan, whereas glucomannan degradation to lactic acid and succinic acid becomes the main reactions at 140°C and 180°C. The addition of H2O2 during alkaline pretreatment of D. fir led to a significant removal of lignin, which subsequently facilitated glucomannan solubilization. However, H2O2 has little direct effect on the glucomannan degradation reaction. The main degradation pathways involved in glucomannan conversion to organics acids are elucidated. The results from this study demonstrate the potential to optimize pretreatment conditions to maximize the value of biomass hemicellulose. PMID:24185034

  18. Understanding Degradation Pathways in Organic Photovoltaics (Poster)

    SciTech Connect

    Lloyd, M. T.; Olson, D. C.; Garcia, A.; Kauvar, I.; Kopidakis, N.; Reese, M. O.; Berry, J. J.; Ginley, D. S.

    2011-02-01

    Organic Photovoltaics (OPVs) recently attained power conversion efficiencies that are of interest for commercial production. Consequently, one of the most important unsolved issues facing a new industry is understanding what governs lifetime in organic devices and discovering solutions to mitigate degradation mechanisms. Historically, the active organic components are considered vulnerable to photo-oxidation and represent the primary degradation channel. However, we present several (shelf life and light soaking) studies pointing the relative stability of the active layers and instabilities in commonly used electrode materials. We show that engineering of the hole/electron layer at the electrode can lead to environmentally stable devices even without encapsulation.

  19. Degradation pathways of PCB upon gamma irradiation

    SciTech Connect

    Lepine, F. ); Masse, R. )

    1990-11-01

    In order to understand the modifications of the chromatographic profile of Aroclor 1260 upon gamma irradiation, a total of 14 pure polychlorinated biphenyl (PCB) congeners were separately irradiated in solution. Dechlorination was observed, and the generated products were investigated by gas chromatography and mass spectrometry. Degradation proceeds more rapidly in methanol/water mixture than in petroleum ether, but the relative amount of ortho-dechlorinated congeners formed upon irradiation was smaller in the former solvent Ortho chlorines are preferentially lost in petroleum ether except when they are involved in a 2,5 (or 3,6) substitution pattern, in which case para dechlorination becomes predominant. The precursors of some toxicologically important congeners such as congeners 77, 118, 167, and 189 have been identified. These data are useful to rationalize the modifications of the chromatographic profile of PCB complex mixture upon gamma irradiation.

  20. Pin1 Modulates ER? Levels in Breast Cancer through Inhibition of Phosphorylation-dependent Ubiquitination and Degradation

    PubMed Central

    Rajbhandari, Prashant; Schalper, Kurt A.; Solodin, Natalia M.; Ellison-Zelski, Stephanie J.; Lu, Kun Ping; Rimm, David L.; Alarid, Elaine T.

    2013-01-01

    Estrogen receptor-alpha (ER?) is an important biomarker used to classify and direct therapy decisions in breast cancer. Both ER? protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ER? protein and ESR1, which is currently unexplained. Cellular ER? protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway (UPP) through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ER?) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ER? transcriptional function. Here, we demonstrate that Pin1 can increase ER? protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ER? ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ER?. Quantitative in situ assessments of ER? protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ER? protein but not to ESR1 levels. These data show that ER? protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. Since Pin1 impacts both ER? protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ER?-positive breast cancer. PMID:23542176

  1. Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells

    PubMed Central

    Samuel, William; Kutty, R. Krishnan; Duncan, Todd; Vijayasarathy, Camasamudram; Kuo, Bryan C.; Chapa, Krysten M.; Redmond, T. Michael

    2014-01-01

    Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4 and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases. PMID:24357007

  2. ORGANOPHOSPHATE PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

    EPA Science Inventory

    Free chlorine has been found to react with organophosphate (OP) pesticides resulting in the more toxic oxon products. We will discuss OP pesticide degradation pathways and modeling in the presence of chlorine and chloramines, as well as present a relationship between structure a...

  3. Hydroxide Degradation Pathways for Substituted Trimethylammonium Cations: A DFT Study

    SciTech Connect

    Long, H.; Kim, K.; Pivovar, B. S.

    2012-05-03

    Substituted trimethylammonium cations serve as small molecule analogues for tetherable cations in anion exchange membranes. In turn, these membranes serve as the basis for alkaline membrane fuel cells by allowing facile conduction of hydroxide. As these cations are susceptible to hydroxide attack, they degrade over time and greatly limit the lifetime of the fuel cell. In this research, we performed density functional theory calculations to investigate the degradation pathways of substituted trimethylammonium cations to probe the relative durability of cation tethering strategies in alkyl and aromatic tethers. Our results show that significant changes in calculated energy barriers occur when substitution groups change. Specifically, we have found that, when available, the Hofmann elimination pathway is the most vulnerable pathway for degradation; however, this barrier is also found to depend on the carbon chain length and number of hydrogens susceptible to Hofmann elimination. S{sub N}2 barriers were also investigated for both methyl groups and substitution groups. The reported findings give important insight into potential tethering strategies for trimethylammonium cations in anion exchange membranes.

  4. Metabolic Pathways for Degradation of Aromatic Hydrocarbons by Bacteria.

    PubMed

    Ladino-Orjuela, Guillermo; Gomes, Eleni; da Silva, Roberto; Salt, Christopher; Parsons, John R

    2016-01-01

    The aim of this revision was to build an updated collection of information focused on the mechanisms and elements involved in metabolic pathways of aromatic hydrocarbons by bacteria. Enzymes as an expression of the genetic load and the type of electron acceptor available, as an environmental factor, were highlighted. In general, the review showed that both aerobic routes and anaerobic routes for the degradation of aromatic hydrocarbons are divided into two pathways. The first, named the upper pathways, from the original compound to central intermediate compounds still containing the aromatic ring but with the benzene nucleus chemically destabilized. The second, named the lower pathway, begins with ring de-aromatização and subsequent cleavage, resulting in metabolites that can be used by bacteria in the production of biomass. Under anaerobic conditions the five mechanisms of activation of the benzene ring described show the diversity of chemical reactions that take place. Obtaining carbon and energy from an aromatic hydrocarbon molecule is a process that exhibits the high complexity level of the metabolic apparatus of anaerobic microorganisms. The ability of these bacteria to express enzymes that catalyze reactions, known only in non-biological conditions, using final electron acceptors with a low redox potential, is a most interesting topic. The discovery of phylogenetic and functional characteristics of cultivable and non-cultivable hydrocarbon degrading bacteria has been made possible by improvements in molecular research techniques such as SIP (stable isotope probing) making trace of (13)C, (15)N and (18)O into nucleic acids and proteins. PMID:26613990

  5. Microbial degradation of organophosphorus xenobiotics: metabolic pathways and molecular basis.

    PubMed

    Karpouzas, Dimitrios G; Singh, Brajesh K

    2006-01-01

    Organophosphorus (OP) xenobiotics are used worldwide as pesticides and petroleum additives. OP compounds share the major portion of the pesticide market globally. Owing to large-scale use of OP compounds, contaminations of soil and water systems have been reported from all parts of the world. OP compounds possess very high mammalian toxicity and therefore early detection and subsequent decontamination and detoxification of the polluted environment is essential. Additionally, about 200,000 tons of extremely toxic OP chemical warfare agents are required to be destroyed by 2007 under Chemical Warfare Convention (1993). Chemical and physical methods of decontamination are not only expensive and time-consuming, but also in most cases they do not provide a complete solution. These approaches convert compounds from toxic into less toxic states, which in some cases can accumulate in the environment and still be toxic to a range of organisms. Bioremediation provides a suitable way to remove contaminants from the environment as, in most of the cases, OP compounds are totally mineralized by the microorganisms. Most OP compounds are degraded by microorganisms in the environment as a source of phosphorus or carbon or both. Several soil bacteria have been isolated and characterized, which can degrade OP compounds in laboratory cultures and in the field. The biochemical and genetic basis of microbial degradation has received considerable attention. Several genes/enzymes, which provide microorganisms with the ability to degrade OP compounds, have been identified and characterized. Some of these genes and enzymes have been engineered for better efficacy. Bacteria capable of complete mineralization are constructed by transferring the complete degradation pathway for specific compounds to one bacterium. In the present article, we review microbial degradation and metabolic pathways for some OP compounds. The biochemical and molecular basis of OP degradation by microbes and the evolution and distribution of genes/enzymes are also reviewed. This article also examines applications and future use of OP-degrading microbes and enzymes for bioremediation, treatment of OP poisoning, and as biosensors. PMID:17091564

  6. Degradation of methamidophos by Hyphomicrobium species MAP-1 and the biochemical degradation pathway.

    PubMed

    Wang, Li; Wen, Yang; Guo, Xinqing; Wang, Guangli; Li, Shunpeng; Jiang, Jiandong

    2010-07-01

    Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l(-1) methamidophos in 84 h under optimal conditions (pH 7.0, 30 degrees C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC-MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P-N bond to form O,S-dimethyl hydrogen thiophosphate and NH(3). Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P-O bond to release -OCH(3) and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P-S bond to release -SCH(3) and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid. PMID:19960233

  7. Parkin Promotes Degradation of the Mitochondrial Pro-Apoptotic ARTS Protein

    PubMed Central

    Kemeny, Stav; Dery, Dikla; Loboda, Yelena; Rovner, Marshall; Lev, Tali; Zuri, Dotan; Finberg, John P. M.; Larisch, Sarit

    2012-01-01

    Parkinson’s disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD. PMID:22792159

  8. Necdin promotes ubiquitin-dependent degradation of PIAS1 SUMO E3 ligase.

    PubMed

    Gur, Ibrahim; Fujiwara, Kazushiro; Hasegawa, Koichi; Yoshikawa, Kazuaki

    2014-01-01

    Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation. PMID:24911587

  9. Iodinated contrast media electro-degradation: process performance and degradation pathways.

    PubMed

    Del Moro, Guido; Pastore, Carlo; Di Iaconi, Claudio; Mascolo, Giuseppe

    2015-02-15

    The electrochemical degradation of six of the most widely used iodinated contrast media was investigated. Batch experiments were performed under constant current conditions using two DSA® electrodes (titanium coated with a proprietary and patented mixed metal oxide solution of precious metals such as iridium, ruthenium, platinum, rhodium and tantalum). The degradation removal never fell below 85% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) when perchlorate was used as the supporting electrolyte; however, when sulphate was used, the degradation performance was above 80% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) for all of the compounds studied. Three main degradation pathways were identified, namely, the reductive de-iodination of the aromatic ring, the reduction of alkyl aromatic amides to simple amides and the de-acylation of N-aromatic amides to produce aromatic amines. However, as amidotrizoate is an aromatic carboxylate, this is added via the decarboxylation reaction. The investigation did not reveal toxicity except for the lower current density used, which has shown a modest toxicity, most likely for some reaction intermediates that are not further degraded. In order to obtain total removal of the contrast media, it was necessary to employ a current intensity between 118 and 182 mA/cm(2) with energy consumption higher than 370 kWh/m(3). Overall, the electrochemical degradation was revealed to be a reliable process for the treatment of iodinated contrast media that can be found in contaminated waters such as hospital wastewater or pharmaceutical waste-contaminated streams. PMID:25433384

  10. Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways.

    PubMed

    Brugmans, M C P; S?ntjens, S H M; Cox, M A J; Nandakumar, A; Bosman, A W; Mes, T; Janssen, H M; Bouten, C V C; Baaijens, F P T; Driessen-Mol, A

    2015-11-01

    The emerging field of in situ tissue engineering (TE) of load bearing tissues places high demands on the implanted scaffolds, as these scaffolds should provide mechanical stability immediately upon implantation. The new class of synthetic supramolecular biomaterial polymers, which contain non-covalent interactions between the polymer chains, thereby forming complex 3D structures by self assembly. Here, we have aimed to map the degradation characteristics of promising (supramolecular) materials, by using a combination of in vitro tests. The selected biomaterials were all polycaprolactones (PCLs), either conventional and unmodified PCL, or PCL with supramolecular hydrogen bonding moieties (either 2-ureido-[1H]-pyrimidin-4-one or bis-urea units) incorporated into the backbone. As these materials are elastomeric, they are suitable candidates for cardiovascular TE applications. Electrospun scaffold strips of these materials were incubated with solutions containing enzymes that catalyze hydrolysis, or solutions containing oxidative species. At several time points, chemical, morphological, and mechanical properties were investigated. It was demonstrated that conventional and supramolecular PCL-based polymers respond differently to enzyme-accelerated hydrolytic or oxidative degradation, depending on the morphological and chemical composition of the material. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the investigated supramolecular materials, while, in contrast, the latter materials are more susceptible to oxidative degradation. Given the observed degradation pathways of the examined materials, we are able to tailor degradation characteristics by combining selected PCL backbones with additional supramolecular moieties. The presented combination of in vitro test methods can be employed to screen, limit, and select biomaterials for pre-clinical in vivo studies targeted to different clinical applications. PMID:26316031

  11. NP1EC Degradation Pathways Under Oxic and Microxic Conditions

    SciTech Connect

    Montgomery-Brown, John; Li, Yongmei; Ding, Wang-Hsien; Mong, Gary M.; Campbell, James A.; Reinhard, Martin

    2008-03-22

    The degradation pathway of nonylphenol ethoxyacetic acid (NP1EC) and the conditions favoring CAP1EC formation were studied in aerobic microcosms constructed with soil from the Mesa soil aquifer treatment (SAT) facility (Arizona, USA) and pristine sediments from Coyote Creek (California, USA). In the Mesa microcosms, para-NP1EC was transformed to para-NP, before being rapidly transformed to nonyl alcohols via ipso-hydroxylation. While the formation of NP from APEMs has been observed by several researchers under anaerobic conditions, this is the first time the transient formation of NP from APEMs has been observed under aerobic conditions. Unlike the Mesa microcosms, large quantities of CAP1ECs were observed in the Coyote Creek microcosms. Initially, CA8P1ECs were the dominant metabolites, but as biodegradation continued, CA6P1ECs became the dominant metabolites. Compared to the CA8P1ECs, the number of CA6P1ECs peaks observed was small (<6) even though their concentrations were high. This suggests that several CA8P1ECs are degraded to only a few CA6P1EC isomers (i.e., the degradation pathway converges) or that some CA6P1EC metabolites are significantly more recalcitrant than others. The different biodegradation pathways observed in the Mesa and Coyote Creek microcosms result from the limited availability of dissolved oxygen in the Coyote Creek microcosms. In both sets of microcosms, the ortho isomers were transformed more slowly than the para isomers and in the Coyote Creek microcosms several ortho-CAP1ECs were observed. In addition, several unknown metabolites were observed in the Coyote Creek microcosms that were not seen in the abiotic or Mesa microcosms; these metabolites appear to be CAP1EC metabolites, have a -CH2-C6H4- fragment, and contain one carboxylic acid. Nitro-nonylphenol was observed in the Mesa microcosms, however, further experimentation illustrated that it was the product of an abiotic reaction between nitrite and nonylphenol under acidic conditions.

  12. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    NASA Technical Reports Server (NTRS)

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  13. Degradation of sulphonated azo dye Red HE7B by Bacillus sp. and elucidation of degradative pathways.

    PubMed

    Thakur, Jyoti Kumar; Paul, Sangeeta; Dureja, Prem; Annapurna, K; Padaria, Jasdeep C; Gopal, Madhuban

    2014-08-01

    Bacteria capable of degrading the sulfonated azo dye Red HE7B were isolated from textile mill effluent contaminated soil. The most efficient isolate was identified as Bacillus sp. Azo1 and the isolate could successfully decolorize up to 89% of the dye. The decolorized cultural extract analyzed by HPLC confirmed degradation. Enzymatic analysis showed twofold and fourfold increase in the activity of azoreductase and laccase enzymes, respectively, indicating involvement of both reductive and oxidative enzymes in biodegradation of Red HE7B. Degraded products which were identified by GC/MS analysis included various metabolites like 8-nitroso 1-naphthol, 2-diazonium naphthalene. Mono azo dye intermediate was initially generated from the parent molecule. This mono azo dye was further degraded by the organism, into additional products, depending on the site of cleavage of R-N=N-R molecule. Based on the degradation products identified, three different pathways have been proposed. The mechanism of degradation in two of these pathways is different from that of the previously reported pathway for azo dye degradation. This is the first report of a microbial isolate following multiple pathways for azo dye degradation. Azo dye Red HE7B was observed to be phytotoxic, leading to decrease in root development, shoot length and seedling fresh weight. However, after biotreatment the resulting degradation products were non-phytotoxic. PMID:24682261

  14. Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

    PubMed Central

    2013-01-01

    Background n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance. Results By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols. Conclusions This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance. PMID:23552365

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  16. c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4.

    PubMed

    González-Prieto, Román; Cuijpers, Sabine Ag; Kumar, Ramesh; Hendriks, Ivo A; Vertegaal, Alfred Co

    2015-01-01

    c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation. PMID:25895136

  17. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  18. Disease-associated mutations of TDP-43 promote turnover of the protein through the proteasomal pathway.

    PubMed

    Araki, Wataru; Minegishi, Seiji; Motoki, Kazumi; Kume, Hideaki; Hohjoh, Hirohiko; Araki, Yumiko M; Tamaoka, Akira

    2014-12-01

    TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy. PMID:24477737

  19. Enzymes Involved in a Novel Anaerobic Cyclohexane Carboxylic Acid Degradation Pathway

    PubMed Central

    Kung, Johannes W.; Meier, Anne-Katrin; Mergelsberg, Mario

    2014-01-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

  20. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

    PubMed

    Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

    2009-03-01

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation. PMID:19122240

  1. On to the road to degradation: atherosclerosis and the proteasome

    PubMed Central

    Herrmann, Joerg; Lerman, Lilach O.; Lerman, Amir

    2010-01-01

    Protein metabolism is a central element of every living cell. The ubiquitin-proteasome system (UPS) is an integral part of the protein metabolism machinery mediating post-transcriptional processing and degradation of the majority of intracellular proteins. Over the past few years, remarkable progress has been made in our understanding of the role of the UPS in vascular biology and pathobiology, particularly atherosclerosis. This review reflects on the recent developments from the effects on endothelial cells and the initial stage of atherosclerosis to the effects on vascular smooth muscle and the progression stage of atherosclerosis and finally to the effects on cell viability and the complication stage of atherosclerosis. It will conclude with the integration of the available information in a synoptic view of the involvement of the UPS in atherosclerosis. PMID:19815565

  2. ORGANOPHOSPHORUS PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

    EPA Science Inventory

    The objective of this work was to investigate organophosphorus (OP) pesticide transformation pathways as a class in the presence of aqueous chlorine. Seven priority OP pesticides were examined for their reactivity with aqueous chlorine: chlorpyrifos (CP), parathion (PA), diazino...

  3. Scavenger receptor CD36 mediates inhibition of cholesterol synthesis via activation of the PPAR?/PGC-1? pathway and Insig1/2 expression in hepatocytes.

    PubMed

    Rodrigue-Way, Amélie; Caron, Véronique; Bilodeau, Stéphanie; Keil, Sarah; Hassan, Meryl; Lévy, Emile; Mitchell, Grant A; Tremblay, André

    2014-04-01

    The scavenger receptor CD36 plays a central role in lipid metabolism by promoting macrophage cholesterol efflux with the potential to reduce atherosclerotic lesions. However, the effect of CD36 on de novo cholesterol synthesis is not known. Here, we describe the cellular mechanism by which CD36 activation induces cholesterol depletion in HepG2 cells. Using the CD36 ligand hexarelin, we found a rapid phosphorylation of HMG-CoA reductase Ser-872 in treated cells, resulting in inactivation of the rate-limiting enzyme in sterol synthesis. Degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway was also enhanced by hexarelin, through an increased recruitment of the anchor proteins insulin-induced gene (Insig)-1 and Insig-2. Genes encoding key enzymes involved in cholesterol synthesis and under the control of transcription factor sterol regulatory element-binding protein (SREBP)-2 remained unresponsive to sterol depletion, due to retention of the SREBP-2 escort protein Scap by Insig-1/2. Insig1 and Insig2 gene expression was also increased through activation of nuclear receptor peroxisome-proliferator activating receptor ? (PPAR?) by CD36, which lifted the inhibitory effect of PPAR?1 Ser-84 phosphorylation. Recruitment of coactivator peroxisome proliferator-activated receptor-? coactivator 1? (PGC1?) to activated AMPK? was also promoted, resulting in PGC-1? transcriptional activation through Sirt1-mediated deacetylation, increased recruitment of PPAR?, and up-regulation of Insig-1/2, revealing a regulatory role of CD36 on PGC-1? signaling. Our data identify CD36 as a novel regulator of HMG-CoA reductase function and Insig-1/2 expression, 2 critical steps regulating cholesterol synthesis in hepatocytes. PMID:24371122

  4. Characterization of a novel ?-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of ?-cypermethrin.

    PubMed

    Deng, Weiqin; Lin, Derong; Yao, Kai; Yuan, Huaiyu; Wang, Zhilong; Li, Jianlong; Zou, Likou; Han, Xinfeng; Zhou, Kang; He, Li; Hu, Xinjie; Liu, Shuliang

    2015-10-01

    Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) sequence. The strain could degrade 54.83 % of ?-cypermethrin (?-CY; 50 mg L(-1)) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L(-1)) in 22 h. The half-lives of ?-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of ?-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of ?-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. ?-CY degradation products were analyzed. Results indicated that YAT strain transformed ?-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)-contaminated environments and fermented food. PMID:26022858

  5. PHOSPHOLIPIDS OF FIVE PSEUDOMONAD ARCHETYPES FOR DIFFERENT TOLUENE DEGRADATION PATHWAYS

    EPA Science Inventory

    Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine phospholipid profiles for five reference pseudomonad strains harboring distinct toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, B...

  6. AEROBIC DEGRADATION OF DINITROTOLUENES AND PATHWAY FOR BACTERIAL DEGRADATION OF 2,6-DINITROTOLUENE

    EPA Science Inventory

    An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkhoderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize dinitrotoluene (2,6-DNT) by a different pathway. Burkhoderia ...

  7. Proteasome Inhibition Enhances the Induction and Impairs the Maintenance of Late-Phase Long-Term Potentiation

    ERIC Educational Resources Information Center

    Dong, Chenghai; Upadhya, Sudarshan C.; Ding, Lan; Smith, Thuy K.; Hegde, Ashok N.

    2008-01-01

    Protein degradation by the ubiquitin-proteasome pathway plays important roles in synaptic plasticity, but the molecular mechanisms by which proteolysis regulates synaptic strength are not well understood. We investigated the role of the proteasome in hippocampal late-phase long-term potentiation (L-LTP), a model for enduring synaptic plasticity.…

  8. EXPRESSION OF K6W-UBIQUITIN IN LENS EPITHELIAL CELLS LEADS TO UP-REGULATION OF A BROAD SPECTRUM OF MOLECULAR CHAPERONES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases. The ubiquitin-proteasome pathway (UPP) is one of the protein quality control mechanisms that selectively degrade damaged or obsolete proteins. The other arm of the protein quality control me...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Regulation of Ubiquitination-Mediated Protein Degradation by Survival Kinases in Cancer

    PubMed Central

    Yamaguchi, Hirohito; Hsu, Jennifer L.; Hung, Mien-Chie

    2011-01-01

    The ubiquitin–proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, AKT, extracellular signal-regulated kinase, and I?B kinase, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, ?-catenin, myeloid cell leukemia-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic. PMID:22649777

  11. Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 - elucidating the downstream pathway.

    PubMed

    Ricken, Benjamin; Fellmann, Oliver; Kohler, Hans-Peter E; Schäffer, Andreas; Corvini, Philippe François-Xavier; Kolvenbach, Boris Alexander

    2015-12-25

    Microbacterium sp. strain BR1 is among the first bacterial isolates which were proven to degrade sulfonamide antibiotics. The degradation is initiated by an ipso-substitution, initiating the decay of the molecule into sulfur dioxide, the substrate specific heterocyclic moiety as a stable metabolite and benzoquinone imine. The latter appears to be instantaneously reduced to p-aminophenol, as that in turn was detected as the first stable intermediate. This study investigated the downstream pathway of sulfonamide antibiotics by testing the strain's ability to degrade suspected intermediates of this pathway. While p-aminophenol was degraded, degradation products could not be identified. Benzoquinone was shown to be degraded to hydroquinone and hydroquinone in turn was shown to be degraded to 1,2,4-trihydroxybenzene. The latter is assumed to be the potential substrate for aromatic ring cleavage. However, no products from the degradation of 1,2,4-trihydroxybenzene could be identified. There are no signs of accumulation of intermediates causing oxidative stress, which makes Microbacterium sp. strain BR1 an interesting candidate for industrial waste water treatment. PMID:25796473

  12. Characterization of the novel dimethyl sulfide-degrading bacterium Alcaligenes sp. SY1 and its biochemical degradation pathway.

    PubMed

    Sun, Yiming; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Chen, Jianmeng

    2016-03-01

    Recently, the biodegradation of volatile organic sulfur compounds (VOSCs) has become a burgeoning field, with a growing focus on the reduction of VOSCs. The reduction of VOSCs encompasses both organic emission control and odor control. Herein, Alcaligenes sp. SY1 was isolated from active sludge and found to utilize dimethyl sulfide (DMS) as a growth substrate in a mineral salt medium. Response surface methodology (RSM) analysis was applied to optimize the incubation conditions. The following conditions for optimal degradation were identified: temperature 27.03°C; pH 7.80; inoculum salinity 0.84%; and initial DMS concentration 1585.39?M. Under these conditions, approximately 99% of the DMS was degraded within 30h of incubation. Two metabolic compounds were detected and identified by gas chromatography-mass spectrometry (GC-MS): dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). The DMS degradation kinetics for different concentrations were evaluated using the Haldane-Andrews model and the pseudo first-order model. The maximum specific growth rate and degradation rate of Alcaligenes sp. SY1 were 0.17h(-1) and 0.63gsgx(-1)h(-1). A possible degradation pathway is proposed, and the results suggest that Alcaligenes sp. SY1 has the potential to control odor emissions under aerobic conditions. PMID:26623933

  13. Proteomic analysis of the benzoate degradation pathway in Acinetobacter sp. KS-1.

    PubMed

    Kim, Seung Il; Song, Seung Youl; Kim, Kyung Wook; Ho, Eun Mi; Oh, Kye Heon

    2003-12-01

    The purpose of this study was to perform proteome analysis of Acinetobacter sp. KS-1, a bacterium capable of degrading benzoate as a sole carbon source. In order to understand the benzoate degradation pathway used by strain KS-1, proteomes of benzoate-cultured and succinate-cultured KS-1 were comparatively analyzed by two dimensional gel electrophoresis (2-DE). Eighteen protein spots proteins were exclusively induced from the benzoate-cultured strain KS-1. Of these 18 spots, two benzoate-degrading enzymes (catechol 1,2-dioxygenase and beta-ketoadipate succinyl-CoA transferase) were identified by MS/MS analysis by MALDI-TOF/TOF mass spectrometry, which suggests that strain KS-1 degrades benzoate by the beta-ketoadipate pathway. DEAE-chromatography suggested that strain KS-1 induced only one type of catechol 1,2-dioxygenase during benzoate degradation. The catechol 1,2-dioxygenase was purified using three steps of ammonium sulfate precipitation, DEAE-sepharose, and Mono-Q chromatography. The purified catechol 1,2-dioxygenase of strain KS-1 had strong dioxygenase activity for 4-methylcatechol as well as catechol. Sequencing analysis using N-terminal and internal amino acid sequences showed that this catechol 1,2-dioxygenase is highly homologous with catechol 1,2-dioxygenase of Acinetobacter radioresistens. These results suggest that comparative proteomic analysis of biodegrading bacteria cultured under different conditions may be a useful initial step toward the elucidation of the aromatic compound degradation pathway. PMID:14643408

  14. Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-LR

    EPA Science Inventory

    This study focuses on the identification of reaction intermediates formed during the photocatalytic degradation of the cyanotoxin microcystin-LR with immobilized TiO2 Tphotocatalysts at neutral pH. To differentiate between impurities already existing in the MC-LR stand...

  15. New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

    PubMed Central

    Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

    2014-01-01

    Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

  16. Modelling Pathways to Rubisco Degradation: A Structural Equation Network Modelling Approach

    PubMed Central

    Tétard-Jones, Catherine; Gatehouse, Angharad M. R.; Cooper, Julia; Leifert, Carlo; Rushton, Steven

    2014-01-01

    ‘Omics analysis (transcriptomics, proteomics) quantifies changes in gene/protein expression, providing a snapshot of changes in biochemical pathways over time. Although tools such as modelling that are needed to investigate the relationships between genes/proteins already exist, they are rarely utilised. We consider the potential for using Structural Equation Modelling to investigate protein-protein interactions in a proposed Rubisco protein degradation pathway using previously published data from 2D electrophoresis and mass spectrometry proteome analysis. These informed the development of a prior model that hypothesised a pathway of Rubisco Large Subunit and Small Subunit degradation, producing both primary and secondary degradation products. While some of the putative pathways were confirmed by the modelling approach, the model also demonstrated features that had not been originally hypothesised. We used Bayesian analysis based on Markov Chain Monte Carlo simulation to generate output statistics suggesting that the model had replicated the variation in the observed data due to protein-protein interactions. This study represents an early step in the development of approaches that seek to enable the full utilisation of information regarding the dynamics of biochemical pathways contained within proteomics data. As these approaches gain attention, they will guide the design and conduct of experiments that enable ‘Omics modelling to become a common place practice within molecular biology. PMID:24498339

  17. Impact of degrading permafrost on subsurface solute transport pathways and travel times

    NASA Astrophysics Data System (ADS)

    Frampton, Andrew; Destouni, Georgia

    2015-09-01

    Subsurface solute transport under surface warming and degrading permafrost conditions is studied using a physically based model of coupled cryotic and hydrogeological flow processes combined with a particle tracking method. Changes in the subsurface water and inert solute pathways and travel times are analyzed for different modeled geological configurations. For all simulated cases, the minimum and mean travel times increase nonlinearly with warming irrespective of geological configuration and heterogeneity structure. The timing of the start of increase in travel time depends on heterogeneity structure, combined with the rate of permafrost degradation that also depends on material thermal and hydrogeological properties. The travel time changes depend on combined warming effects of: i) increase in pathway length due to deepening of the active layer, ii) reduced transport velocities due to a shift from horizontal saturated groundwater flow near the surface to vertical water percolation deeper into the subsurface, and iii) pathway length increase and temporary immobilization caused by cryosuction-induced seasonal freeze cycles.

  18. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 ?m). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  19. A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi

    PubMed Central

    Nicolás, Francisco E.; Vila, Ana; Moxon, Simon; Dalmay, Tamas; Torres-Martínez, Santiago; Garre, Victoriano; Ruiz-Vázquez, Rosa M.

    2015-01-01

    The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing. PMID:25875805

  20. Novel degradation pathway of 4-chloro-2-aminophenol via 4-chlorocatechol in Burkholderia sp. RKJ 800.

    PubMed

    Arora, Pankaj Kumar; Srivastava, Alok; Singh, Vijay Pal

    2014-02-01

    Burkholderia sp. RKJ 800 utilized 4-chloro-2-aminophenol (4C2AP) as the sole carbon and energy source and degraded it with release of chloride and ammonium ions. The metabolic pathway of degradation of 4C2AP was studied and a novel intermediate, 4-chlorocatechol was identified as a major degradation product of 4C2AP using high-performance liquid chromatography and gas chromatography-mass spectrometry. Enzyme activities for 4C2AP-deaminase and 4-chlorocatechol-1,2-dioxygenase were detected in the crude extracts of the 4C2AP-induced cells of strain RKJ 800. The activity of the 4C2AP-deaminase confirmed the formation of 4-chlorocatechol from 4C2AP and the 4-chlorocatechol-1,2-dioxygenase activity suggested the cleavage of 4-chlorocatechol into 3-chloro-cis,cis-muconate. On the basis of the identified metabolites, we have proposed a novel degradation pathway of 4C2AP for Burkholderia sp. RKJ 800. Furthermore, the potential of Burkholderia sp. RKJ 800 to degrade 4C2AP in soil was also investigated using microcosm studies under laboratory conditions. The results of microcosm studies conclude that Burkholderia sp. RKJ 800 was able to degrade 4C2AP in soil and may be used to remediate 4C2AP-contaminated site. This is the first report of (1) the formation of 4-chlorocatechol and 3-chloro-cis,cis-muconate in the degradation pathway of 4C2AP and (2) bioremediation of 4C2AP by any bacterium. PMID:24057966

  1. Degradation kinetics and pathway of phenol by Pseudomonas and Bacillus species

    PubMed Central

    Hasan, Syed Adnan; Jabeen, Suraiya

    2015-01-01

    This article elucidates that strain Pseudomonas aeruginosa (IES-Ps-1) is a versatile toxic organic compound degrader. With the degradation of malathion and cypermethrin (studied by other researchers previously), this strain was able to degrade phenol. Two other indigenous soil flora (i.e., Pseudomonas sp. (IES-S) and Bacillus subtilis (IES-B)) were also found to be potential phenol degraders. Phenol was degraded with Monod kinetics during growth in nutrient broth and mineral salts medium. Before entering into the growth inhibition phase, strains IES-Ps-1, IES-S and IES-B could tolerate up to 400, 700 and 500 mg/L phenol, respectively, when contained in nutrient broth. However, according to the Luong–Levenspiel model, the growth of strains IES-Ps-1, IES-S and IES-B would cease at 2000, 2174 and 2190 mg/L phenol, respectively. Strain IES-Ps-1 degraded 700, 900 and 1050 mg/L phenol contained in mineral salts medium with the specific rates of 0.034, 0.075 and 0.021 h?1, respectively. All these strains grew by making clusters when exposed to phenol in order to prevent damages due to high substrate concentration. These strains transformed phenol into catechol, which was then degraded via ortho-cleavage pathway.

  2. Genomic and metabolic analysis of fluoranthene degradation pathway in Celeribacter indicus P73T

    PubMed Central

    Cao, Junwei; Lai, Qiliang; Yuan, Jun; Shao, Zongze

    2015-01-01

    Celeribacter indicus P73T, isolated from deep-sea sediment from the Indian Ocean, is capable of degrading a wide range of polycyclic aromatic hydrocarbons (PAHs) and is the first fluoranthene-degrading bacterium within the family Rhodobacteraceae. Here, the complete genome sequence of strain P73T is presented and analyzed. Besides a 4.5-Mb circular chromosome, strain P73T carries five plasmids, and encodes 4827 predicted protein-coding sequences. One hundred and thirty-eight genes, including 14 dioxygenase genes, were predicted to be involved in the degradation of aromatic compounds, and most of these genes are clustered in four regions. P73_0346 is the first fluoranthene 7,8-dioxygenase to be discovered and the first fluoranthene dioxygenase within the toluene/biphenyl family. The degradative genes in regions B and D in P73T are absent in Celeribacter baekdonensis B30, which cannot degrade PAHs. Four intermediate metabolites [acenaphthylene-1(2H)-one, acenaphthenequinone, 1,2-dihydroxyacenaphthylene, and 1,8-naphthalic anhydride] of fluoranthene degradation by strain P73T were detected as the main intermediates, indicating that the degradation of fluoranthene in P73T was initiated by dioxygenation at the C-7,8 positions. Based on the genomic and metabolitic results, we propose a C-7,8 dioxygenation pathway in which fluoranthene is mineralized to TCA cycle intermediates. PMID:25582347

  3. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity.

    PubMed

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the ?-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of ?-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  4. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity

    PubMed Central

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the ?-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of ?-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  5. Degradation of ibuprofen by hydrodynamic cavitation: Reaction pathways and effect of operational parameters.

    PubMed

    Musmarra, Dino; Prisciandaro, Marina; Capocelli, Mauro; Karatza, Despina; Iovino, Pasquale; Canzano, Silvana; Lancia, Amedeo

    2016-03-01

    Ibuprofen (IBP) is an anti-inflammatory drug whose residues can be found worldwide in natural water bodies resulting in harmful effects to aquatic species even at low concentrations. This paper deals with the degradation of IBP in water by hydrodynamic cavitation in a convergent-divergent nozzle. Over 60% of ibuprofen was degraded in 60min with an electrical energy per order (EEO) of 10.77kWhm(-3) at an initial concentration of 200?gL(-1) and a relative inlet pressure pin=0.35MPa. Five intermediates generated from different hydroxylation reactions were identified; the potential mechanisms of degradation were sketched and discussed. The reaction pathways recognized are in line with the relevant literature, both experimental and theoretical. By varying the pressure upstream the constriction, different degradation rates were observed. This effect was discussed according to a numerical simulation of the hydroxyl radical production identifying a clear correspondence between the maximum kinetic constant kOH and the maximum calculated OH production. Furthermore, in the investigated experimental conditions, the pH parameter was found not to affect the extent of degradation; this peculiar feature agrees with a recently published kinetic insight and has been explained in the light of the intermediates of the different reaction pathways. PMID:26584987

  6. Degradation kinetics and pathways of three calcium channel blockers under UV irradiation.

    PubMed

    Zhu, Bing; Zonja, Bozo; Gonzalez, Oscar; Sans, Carme; Pérez, Sandra; Barceló, Damia; Esplugas, Santiago; Xu, Ke; Qiang, Zhimin

    2015-12-01

    Calcium channel blockers (CCBs) are a group of pharmaceuticals widely prescribed to lower blood pressure and treat heart diseases. They have been frequently detected in wastewater treatment plant (WWTP) effluents and downstream river waters, thus inducing a potential risk to aquatic ecosystems. However, little is known about the behavior and fate of CCBs under UV irradiation, which has been adopted as a primary disinfection method for WWTP effluents. This study investigated the degradation kinetics and pathways of three commonly-used CCBs, including amlodipine (AML), diltiazem (DIL), and verapamil (VER), under UV (254 nm) irradiation. The chemical structures of transformation byproducts (TBPs) were first identified to assess the potential ecological hazards. On that basis, a generic solid-phase extraction method, which simultaneously used four different cartridges, was adopted to extract and enrich the TBPs. Thereafter, the photo-degradation of target CCBs was performed under UV fluences typical for WWTP effluent disinfection. The degradation of all three CCBs conformed to the pseudo-first-order kinetics, with rate constants of 0.031, 0.044 and 0.011min(-1) for AML, DIL and VER, respectively. By comparing the MS(2) fragments and the evolution (i.e., formation or decay) trends of identified TBPs, the degradation pathways were proposed. In the WWTP effluent, although the target CCBs could be degraded, several TBPs still contained the functional pharmacophores and reached peak concentrations under UV fluences of 40-100 mJ cm(-2). PMID:26003333

  7. New metabolic pathway for degradation of 2-nitrobenzoate by Arthrobacter sp. SPG

    PubMed Central

    Arora, Pankaj K.; Sharma, Ashutosh

    2015-01-01

    Arthrobacter sp. SPG utilized 2-nitrobenzoate as its sole source of carbon and energy and degraded it with accumulation of stoichiometric amounts of nitrite ions. Salicylate and catechol were detected as metabolites of the 2-nitrobenzoate degradation using high performance liquid chromatography and gas chromatography–mass spectrometry. Enzyme activities for 2-nitrobenzoate-2-monooxygenase, salicylate hydroxylase, and catechol-1,2-dioxygenase were detected in the crude extracts of the 2-nitrobenzoate-induced cells of strain SPG. The 2-nitrobenzoate-monooxygenase activity resulted in formation of salicylate and nitrite from 2-nitrobenzoate, whereas salicylate hydroxylase catalyzed the conversion of salicylate to catechol. The ring-cleaving enzyme, catechol-1,2-dioxygenase cleaved catechol to cis,cis-muconic acid. Cells of strain SPG were able to degrade 2-nitrobenzoate in sterile as well as non-sterile soil microcosms. The results of microcosm studies showed that strain SPG degraded more than 90% of 2-nitrobenzoate within 10–12 days. This study clearly shows that Arthrobacter sp. SPG degraded 2-nitrobenzoate via a new pathway with formation of salicylate and catechol as metabolites. Arthrobacter sp. SPG may be used for bioremediation of 2-nitrobenzoate-contaminated sites due to its ability to degrade 2-nitrobenzoate in soil. PMID:26082768

  8. Phenylacetate catabolism in Rhodococcus sp. strain RHA1: a central pathway for degradation of aromatic compounds.

    PubMed

    Navarro-Llorens, Juana María; Patrauchan, Marianna A; Stewart, Gordon R; Davies, Julian E; Eltis, Lindsay D; Mohn, William W

    2005-07-01

    In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria. PMID:15968060

  9. Thermally induced degradation pathways of three different antibody-based drug development candidates.

    PubMed

    Fincke, Anja; Winter, Jonas; Bunte, Thomas; Olbrich, Carsten

    2014-10-01

    Protein-based medicinal products are prone to undergo a variety of chemical and physical degradation pathways. One of the most important exogenous stress condition to consider during manufacturing, transport and storage processes is temperature, because antibody-based therapeutics are only stable in a limited temperature range. In this study, three different formats of antibody-based molecules (IgG1, a bispecific scFv and a fab fragment) were exposed to thermal stress conditions occurring during transport and storage. For evaluation, an analytical platform was developed for the detection and characterization of relevant degradation pathways of different antibody-based therapeutics. The effect of thermal stress conditions on the stability of the three antibody-based formats was therefore investigated using visual inspection, different spectroscopic measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electrophoresis, asymmetric flow field-flow fractionation (AF4) and surface plasmon resonance technology (SPR). In summary, thermal stress led to heterogeneous chemical and physical degradation pathways of all three antibody-based formats used. In addition, identical exogenous stress conditions resulted in different kinds and levels of aggregates and fragmentation products. This knowledge is fundamental for a systematic and successful stabilization of protein-based therapeutics by the use of formulation additives. PMID:24878389

  10. Degradation of methiocarb by monochloramine in water treatment: kinetics and pathways.

    PubMed

    Qiang, Zhimin; Tian, Fang; Liu, Wenjun; Liu, Chao

    2014-03-01

    The micropollution of drinking water sources with pesticides has become a global concern. This work investigated the degradation of methiocarb (MC), a most commonly-used carbamate pesticide, by monochloramine (NH2Cl) under simulated water treatment conditions. Results indicate that the reaction was of first-order in MC and varied orders in NH2Cl depending on water pH. The observed rate constant of MC degradation decreased quickly with either a decrease in the molar ratio of chlorine to ammonia (Cl2:N) or an increase in water pH. The apparent activation energy of the reaction was determined to be 34 kJ mol(-1). The MC degradation pathways also exhibited a strong pH dependence: at pH 6.5, MC was first oxidized by NH2Cl to methiocarb sulfoxide (MCX), and then hydrolyzed to methiocarb sulfoxide phenol (MCXP); while at pH 8.5, MCX, MCXP and methiocarb sulfone phenol (MCNP) were formed successively through either oxidation or hydrolysis reactions. Based on the identified byproducts and their concentrations evolution, the proposed pathways of MC degradation in the presence of NH2Cl were further validated through kinetic model simulations. PMID:24380738

  11. Comparative genomic analysis of nine Sphingobium strains: Insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

    DOE PAGESBeta

    Verma, Helianthous; Kumar, Roshan; Oldach, Phoebe; Sangwan, Naseer; Khurana, Jitendra P.; Gilbert, Jack A.; Lal, Rup

    2014-11-23

    Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6). Results: Efficient HCH degraders phylogeneticallymore »clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. In addition, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity. In conclusion, the bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.« less

  12. Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

    SciTech Connect

    Shen Jianying; Hisaeda, Hajime; Chou Bin; Yu Qingsheng; Tu Liping; Himeno, Kunisuke

    2008-01-25

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8{sup +} T cells. In this study, we exploited UPS to induce CD8{sup +} T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-{gamma}-producing CD8{sup +} T cells compared with those from the latter group of mice.

  13. Carbon Nanotube Degradation in Macrophages: Live Nanoscale Monitoring and Understanding of Biological Pathway.

    PubMed

    Elgrabli, Dan; Dachraoui, Walid; Ménard-Moyon, Cécilia; Liu, Xiao Jie; Bégin, Dominique; Bégin-Colin, Sylvie; Bianco, Alberto; Gazeau, Florence; Alloyeau, Damien

    2015-10-27

    Despite numerous applications, the cellular-clearance mechanism of multiwalled carbon nanotubes (MWCNTs) has not been clearly established yet. Previous in vitro studies showed the ability of oxidative enzymes to induce nanotube degradation. Interestingly, these enzymes have the common capacity to produce reactive oxygen species (ROS). Here, we combined material and life science approaches for revealing an intracellular way taken by macrophages to degrade carbon nanotubes. We report the in situ monitoring of ROS-mediated MWCNT degradation by liquid-cell transmission electron microscopy. Two degradation mechanisms induced by hydroxyl radicals were extracted from these unseen dynamic nanoscale investigations: a non-site-specific thinning process of the walls and a site-specific transversal drilling process on pre-existing defects of nanotubes. Remarkably, similar ROS-induced structural injuries were observed on MWCNTs after aging into macrophages from 1 to 7 days. Beside unraveling oxidative transformations of MWCNT structure, we elucidated an important, albeit not exclusive, biological pathway for MWCNT degradation in macrophages, involving NOX2 complex activation, superoxide production, and hydroxyl radical attack, which highlights the critical role of oxidative stress in cellular processing of MWCNTs. PMID:26331631

  14. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.

  15. Degradation of retinoid X receptor ? by TPA through proteasome pathway in gastric cancer cells

    PubMed Central

    Ye, Xiao-Feng; Liu, Su; Wu, Qiao; Lin, Xiao-Feng; Zhang, Bing; Wu, Jia-Fa; Zhang, Ming-Qing; Su, Wen-Jin

    2003-01-01

    AIM: To investigate and determine the mechanism and signal pathway of tetradecanoylphorbol-1, 3-acetate (TPA) in degradation of RXR?. METHODS: Gastric cancer cell line, BGC-823 was used in the experiments. The expression level of RXR? protein was detected by Western blot. Nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of RXR? were observed under laser-scanning confocal microscope through labeling specific anti-RXR? antibody and corresponding immunofluorescent antibody as secondary antibody. Different inhibitors were used as required. RESULTS: In BGC-823 cells, RXR? was expressed in the nucleus. When cells were treated with TPA, expression of RXR? was repressed in a time-dependent and TPA-concentration-dependent manner. Meanwhile, translocation of RXR? from the nucleus to the cytoplasm occurred, also in a time-dependent manner. When cells were pre-incubated with proteasome inhibitor MG132 for 3 hrs, followed by TPA for another 12 hrs, TPA-induced RXR? degradation was inhibited. Further observation of RXR? translocation in the presence of MG132 showed that MG-132 could block TPA-induced RXR? redistribution. Conversely, when RXR? translocation was inhibited by LMB, an inhibitor for blocking protein export from the nucleus, TPA could not repress expression of RXR?. CONCLUSION: TPA could induce the degradation of RXR? protein in BGC-823 cells, and this degradation is time- and TPA-concentration-dependent. Furthermore, the degradation of RXR? by TPA is via a proteasome pathway and associated with RXR? translocation from the nucleus to the cytoplasm. PMID:12970875

  16. Enzymes of the benzoyl-coenzyme A degradation pathway in the hyperthermophilic archaeon Ferroglobus placidus.

    PubMed

    Schmid, Georg; René, Sandra Bosch; Boll, Matthias

    2015-09-01

    The Fe(III)-respiring Ferroglobus placidus is the only known archaeon and hyperthermophile for which a complete degradation of aromatic substrates to CO2 has been reported. Recent genome and transcriptome analyses proposed a benzoyl-coenzyme A (CoA) degradation pathway similar to that found in the phototrophic Rhodopseudomonas palustris, which involves a cyclohex-1-ene-1-carboxyl-CoA (1-enoyl-CoA) forming, ATP-dependent key enzyme benzoyl-CoA reductase (BCR). In this work, we demonstrate, by first in vitro studies, that benzoyl-CoA is ATP-dependently reduced by two electrons to cyclohexa-1,5-dienoyl-CoA (1,5-dienoyl-CoA), which is further degraded by hydration to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA (6-OH-1-enoyl-CoA); upon addition of NAD(+) , the latter was subsequently converted to ?-oxidation intermediates. The four candidate genes of BCR were heterologously expressed, and the enriched, oxygen-sensitive enzyme catalysed the two-electron reduction of benzoyl-CoA to 1,5-dienoyl-CoA. A gene previously assigned to a 2,3-didehydropimeloyl-CoA hydratase was heterologously expressed and shown to act as a typical 1,5-dienoyl-CoA hydratase that does not accept 1-enoyl-CoA. A gene previously assigned to a 1-enoyl-CoA hydratase was heterologously expressed and identified to code for a bifunctional crotonase/3-OH-butyryl-CoA dehydrogenase. In summary, the results consistently provide biochemical evidence that F.?placidus and probably other archaea predominantly degrade aromatics via the Thauera/Azoarcus type and not or only to a minor extent via the predicted R.?palustris-type benzoyl-CoA degradation pathway. PMID:25630364

  17. Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation

    PubMed Central

    Tao, Ting; Shi, Hui; Guan, Yihong; Huang, Delai; Chen, Ye; Lane, David P; Chen, Jun; Peng, Jinrong

    2013-01-01

    p53 protein turnover through the ubiquitination pathway is a vital mechanism in the regulation of its transcriptional activity; however, little is known about p53 turnover through proteasome-independent pathway(s). The digestive organ expansion factor (Def) protein is essential for the development of digestive organs. In zebrafish, loss of function of def selectively upregulates the expression of p53 response genes, which raises a question as to what is the relationship between Def and p53. We report here that Def is a nucleolar protein and that loss of function of def leads to the upregulation of p53 protein, which surprisingly accumulates in the nucleoli. Our extensive studies have demonstrated that Def can mediate the degradation of p53 protein and that this process is independent of the proteasome pathway, but dependent on the activity of Calpain3, a cysteine protease. Our findings define a novel nucleolar pathway that regulates the turnover function of p53, which will advance our understanding of p53's role in organogenesis and tumorigenesis. PMID:23357851

  18. Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis[W

    PubMed Central

    Jung, Benjamin; Flörchinger, Martin; Kunz, Hans-Henning; Traub, Michaela; Wartenberg, Ruth; Jeblick, Wolfgang; Neuhaus, H. Ekkehard; Möhlmann, Torsten

    2009-01-01

    Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding Km values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter ?-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development. PMID:19293370

  19. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

    2013-01-01

    Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

  20. Unraveling the Specific Regulation of the Central Pathway for Anaerobic Degradation of 3-Methylbenzoate*

    PubMed Central

    Juárez, Javier F.; Liu, Huixiang; Zamarro, María T.; McMahon, Stephen; Liu, Huanting; Naismith, James H.; Eberlein, Christian; Boll, Matthias; Carmona, Manuel; Díaz, Eduardo

    2015-01-01

    The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the ?-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB1, and P3R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds. PMID:25795774

  1. A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7.

    PubMed

    Hasegawa, Y; Muraki, T; Tokuyama, T; Iwaki, H; Tatsuno, M; Lau, P C

    2000-09-15

    A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed. PMID:11034277

  2. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

    SciTech Connect

    Chou, Bin; Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 ; Hiromatsu, Kenji; Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi; Murata, Shigeo; Tanaka, Keiji; Himeno, Kunisuke

    2010-02-12

    Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +} T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  3. Molecular characterization of the Akt-TOR signaling pathway in rainbow trout: potential role in muscle growth/degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Akt-TOR signaling pathway plays a key role in cellular metabolism and muscle growth. Hormone, nutrition and stress factors affect the Akt-TOR pathway by regulating gene transcription, protein synthesis and degradation. In addition, we previously showed that energetic demands elevate during vit...

  4. Parenteral formulation and thermal degradation pathways of a potent rebeccamycin based indolocarbazole topoisomerase I inhibitor.

    PubMed

    Sato, Yuichi; Breslin, David; Kitada, Hideyuki; Minagawa, Wataru; Nomoto, Takashi; Qin, Xue-Zhi; Karki, Shyam B

    2010-05-10

    The development of a practical and pharmaceutically acceptable parenteral dosage form of 1 is described. A cosolvent formulation strategy was selected to achieve the necessary human dose of 1 for administration via intravenous infusion. The final market formulation of 1 chosen for commercial development and Phase II clinical supplies was the topoisomerase inhibitor dissolved in a 50% aqueous propylene glycol solution vehicle with 50mM citrate buffered to pH 4. The thermal degradation pathways of 1 in this aqueous propylene glycol vehicle in the pH range of 3-5 were determined by relative kinetics and degradation product identification using LC/MS, LC/MS/MS, and NMR analysis. The primary mode of degradation of 1 in this aqueous cosolvent formulation is hydrolysis affording the anhydride 2 (in equilibrium with the dicarboxylic acid 3) and release of the hydrazine diol side chain 11. Subsequent oxidative degradation of 11 occurs in several chemical steps which yield a complicated mixture of secondary reaction products that have been structurally identified. PMID:20074629

  5. Sulfamethoxazole degradation by ultrasound/ozone oxidation process in water: kinetics, mechanisms, and pathways.

    PubMed

    Guo, Wan-Qian; Yin, Ren-Li; Zhou, Xian-Jiao; Du, Juan-Shan; Cao, Hai-Ou; Yang, Shan-Shan; Ren, Nan-Qi

    2015-01-01

    In this research, sulfamethoxazole (SMX) degradation was investigated using ultrasound (US), ozone (O3) and ultrasound/ozone oxidation process (UOOP). It was proved that ultrasound significantly enhanced SMX ozonation by assisting ozone in producing more hydroxyl radicals in UOOP. Ultrasound also made the rate constants improve by kinetics analysis. When ultrasound was added to the ozonation process, the reaction rate increased by 6-26% under different pH conditions. Moreover, main intermediates oxidized by US, O3 and UOOP system were identified. Although the main intermediates in ozonation and UOOP were similar, the introduction of ultrasound in UOOP had well improved the cleavage of S-N bond. In this condition SMX become much easier to be attacked, which led to enhanced SMX removal rate in UOOP compared to the other two examined processes. Finally, the SMX degradation pathways were proposed. PMID:25107668

  6. Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic ?-cells

    SciTech Connect

    Lee, Sanghwan; Hur, Eu-gene; Ryoo, In-geun; Jung, Kyeong-Ah; Kwak, Jiyeon; Kwak, Mi-Kyoung

    2012-11-01

    The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2?related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma ?-cell line, ?TC-6. shRNA-mediated silencing of Nrf2 expression in ?TC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ER stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in ?TC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated?like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic ?-cells by enhancing proteasome-mediated ERAD. -- Highlights: ? Nrf2 silencing in pancreatic ?-cells enhanced tunicamycin-mediated ER stress. ? Expression of the proteasome was inducible by Nrf2 signaling. ? Nrf2 activator D3T protected ?-cells from tunicamycin-mediated ER stress. ? Protective effect of D3T was associated with Nrf2-dependent proteasome induction.

  7. Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

    PubMed

    Rincon-Heredia, Ruth; Flores-Benitez, David; Flores-Maldonado, Catalina; Bonilla-Delgado, José; García-Hernández, Vicky; Verdejo-Torres, Odette; Castillo, Aida M; Larré, Isabel; Poot-Hernández, Augusto C; Franco, Martha; Gariglio, Patricio; Reyes, José L; Contreras, Rubén G

    2014-01-01

    In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (?300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway. PMID:24140471

  8. Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.

    PubMed

    Wall, N R; Mohammad, R M; Reddy, K B; Al-Katib, A M

    2000-02-01

    The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression. PMID:10639596

  9. A stress-responsive system for mitochondrial protein degradation

    PubMed Central

    Heo, Jin-Mi; Livnat-Levanon, Nurit; Taylor, Eric B.; Jones, Kevin T.; Dephoure, Noah; Ring, Julia; Xie, Jianxin; Brodsky, Jeffrey L.; Madeo, Frank; Gygi, Steven P.; Ashrafi, Kaveh; Glickman, Michael H.; Rutter, Jared

    2010-01-01

    We show that Ydr049 (renamed VCP/ Cdc48-associated Mitochondrial Stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress and decreased chronological lifespan. Both yeast and mammalian Vms1 stably interact with Cdc48/ VCP/ p97, a component of the ubiquitin/ proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular and organismal viability. PMID:21070972

  10. ERManI (Endoplasmic Reticulum Class I ?-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-01

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) ?-mannosidases, ERManI, and ER-degradation enhancing ?-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four ?-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 ?-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. PMID:26205822

  11. Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse.

    PubMed Central

    Papes, F; Kemper, E L; Cord-Neto, G; Langone, F; Arruda, P

    1999-01-01

    Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels. PMID:10567240

  12. Methyl-mercury degradation pathways: A comparison among three mercury impacted ecosystems

    USGS Publications Warehouse

    Marvin-DiPasquale, M.; Agee, J.; Mcgowan, C.; Oremland, R.S.; Thomas, M.; Krabbenhoft, D.; Gilmour, C.C.

    2000-01-01

    We examined microbial methylmercury (MeHg) degradation in sediment of the Florida Everglades, Carson River (NV), and San Carlos Creek (CA), three freshwater environments that differ in the extent and type of mercury contamination and sediment biogeochemistry. Degradation rate constant (k(deg)) values increased with total mercury (Hg(t)) contamination both among and within ecosystems. The highest k(deg)'s (2.8-5.8 d-1) were observed in San Carlos Creek, at acid mine drainage impacted sites immediately downstream of the former New Idria mercury mine, where Hg(t) ranged from 4.5 to 21.3 ppm (dry wt). A reductive degradation pathway (presumably mer-detoxification) dominated degradation at these sites, as indicated by the nearly exclusive production of 14CH4 from 14C-MeHg, under both aerobic and anaerobic conditions. At the upstream control site, and in the less contaminated ecosystems (e.g. the Everglades), k(deg)'s were low (???0.2 d-1) and oxidative demethylation (OD) dominated degradation, as evident from 14CO2 production. k(deg) increased with microbial CH4 production, organic content, and reduced sulfur in the Carson River system and increased with decreasing pH in San Carlos Creek. OD associated CO2 production increased with pore-water SO42- in Everglades samples but was not attributable to anaerobic methane oxidation, as has been previously proposed. This ecosystem comparison indicates that severely contaminated sediments tend to have microbial populations that actively degrade MeHg via mer-detoxification, whereas OD occurs in heavily contaminated sediments as well but dominates in those less contaminated.We examined microbial methylmercury (MeHg) degradation in sediment of the Florida Everglades, Carson River (NV), and San Carlos Creek (CA), three freshwater environments that differ in the extent and type of mercury contamination and sediment biogeochemistry. Degradation rate constant (kdeg) values increased with total mercury (Hgt) contamination both among and within ecosystems. The highest kdeg???s (2.8-5.8 d-1) were observed in San Carlos Creek, at acid mine drainage impacted sites immediately downstream of the former New Idria mercury mine, where Hgt ranged from 4.5 to 21.3 ppm (dry wt). A reductive degradation pathway (presumably mer-detoxification) dominated degradation at these sites, as indicated by the nearly exclusive production of 14CH4 from 14C-MeHg, under both aerobic and anaerobic conditions. At the upstream control site, and in the less contaminated ecosystems (e.g. the Everglades), kdeg???s were low (???0.2 d-1) and oxidative demethylation (OD) dominated degradation, as evident from 14CO2 production. kdeg in increased with microbial CH4 production, organic content, and reduced sulfur in the Carson River system and increased with decreasing pH in San Carlos Creek. OD associated CO2 production increased with pore-water SO42- in Everglades samples but was not attributable to anaerobic methane oxidation, as has been previously proposed. This ecosystem comparison indicates that severely contaminated sediments tend to have microbial populations that actively degrade MeHg via mer-detoxification, whereas OD occurs in heavily contaminated sediments as well but dominates in those less contaminated.Methylmercury degradation dynamics, in terms of methane and carbon dioxide end-products, were investigated in three Hg-contaminated ecosystems: the Florida everglades, San Carlos Creek, CA, and Carson River, NV. The everglades represented a moderately contaminated ecosystem, while the other two exhibited significantly higher Hg levels. Sediment samples were collected from each study site and analyzed. Results showed that the degradation rate constants increased with total Hg contamination both among and within the ecosystems, with highest rates observed in San Carlos Creek. The degradation dynamics were dominated by a reductive degradation pathway at San Carlos Creek, while in the everglades, oxidative demethylation was imp

  13. Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway? †

    PubMed Central

    Varghese, Bentley; Barriere, Herve; Carbone, Christopher J.; Banerjee, Anamika; Swaminathan, Gayathri; Plotnikov, Alexander; Xu, Ping; Peng, Junmin; Goffin, Vincent; Lukacs, Gergely L.; Fuchs, Serge Y.

    2008-01-01

    The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling. PMID:18573876

  14. Candida albicans Utilizes a Modified ?-Oxidation Pathway for the Degradation of Toxic Propionyl-CoA*

    PubMed Central

    Otzen, Christian; Bardl, Bettina; Jacobsen, Ilse D.; Nett, Markus; Brock, Matthias

    2014-01-01

    Propionyl-CoA arises as a metabolic intermediate from the degradation of propionate, odd-chain fatty acids, and some amino acids. Thus, pathways for catabolism of this intermediate have evolved in all kingdoms of life, preventing the accumulation of toxic propionyl-CoA concentrations. Previous studies have shown that fungi generally use the methyl citrate cycle for propionyl-CoA degradation. Here, we show that this is not the case for the pathogenic fungus Candida albicans despite its ability to use propionate and valerate as carbon sources. Comparative proteome analyses suggested the presence of a modified ?-oxidation pathway with the key intermediate 3-hydroxypropionate. Gene deletion analyses confirmed that the enoyl-CoA hydratase/dehydrogenase Fox2p, the putative 3-hydroxypropionyl-CoA hydrolase Ehd3p, the 3-hydroxypropionate dehydrogenase Hpd1p, and the putative malonate semialdehyde dehydrogenase Ald6p essentially contribute to propionyl-CoA degradation and its conversion to acetyl-CoA. The function of Hpd1p was further supported by the detection of accumulating 3-hydroxypropionate in the hpd1 mutant on propionyl-CoA-generating nutrients. Substrate specificity of Hpd1p was determined from recombinant purified enzyme, which revealed a preference for 3-hydroxypropionate, although serine and 3-hydroxyisobutyrate could also serve as substrates. Finally, virulence studies in a murine sepsis model revealed attenuated virulence of the hpd1 mutant, which indicates generation of propionyl-CoA from host-provided nutrients during infection. PMID:24497638

  15. Characterization of a novel cometabolic degradation carbazole pathway by a phenol-cultivated Arthrobacter sp. W1.

    PubMed

    Shi, Shengnan; Qu, Yuanyuan; Zhou, Hao; Ma, Qiao; Ma, Fang

    2015-10-01

    Arthrobacter sp. W1 was used to characterize the pathways involved in cometabolic degradation of carbazole (CA) with phenol as the primary substrate. To clarify the upper pathway of cometabolic degradation CA, Escherichia coli strain BL21 expressing phenol hydroxylase from strain W1 (PHIND) was investigated to degrade CA. Firstly, CA was initially monohydroxylated at C-2 and C-4 positions to produce 2- and 4-hydroxycarbazole, followed by successively hydroxylated to the corresponding 1,2- and 3,4-dihydroxycarbazole, of which 3,4-dihydroxycarbazole was unequivocally identified for the first time. To characterize the downstream cometabolic degradation CA pathway, purified 3,4-dihydroxycarbazole was used as the substrate for phenol-grown W1, and a series of novel indole derivatives were identified. These results suggested that a novel pathway of CA catabolism was employed by strain W1 via a successive hydroxylation and meta-cleavage pathway. These findings provide new insights into the cometabolic degradation CA process and have potential applications in biotechnology and bioremediation. PMID:26142994

  16. Investigation of the ubiquitin proteasome system in Schizosaccharomyces pombe 

    E-print Network

    Glover, James S. A.

    2010-01-01

    Ubiquitin is an essential 76 amino acid protein which can be conjugated to lysine residues on a variety of substrates via its C-terminal diglycine motif. This conjugation allows the protein to act as a molecular tag in ...

  17. Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp. ONBA-17 and deduction on its metabolic pathway

    PubMed Central

    Yu, Fang-Bo; Li, Xiao-Dan; Ali, Shinawar Waseem; Shan, Sheng-Dao; Luo, Lin-Ping; Guan, Li-Bo

    2014-01-01

    A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation. PMID:25763034

  18. Autophagic degradation contributes to muscle wasting in cancer cachexia.

    PubMed

    Penna, Fabio; Costamagna, Domiziana; Pin, Fabrizio; Camperi, Andrea; Fanzani, Alessandro; Chiarpotto, Elena M; Cavallini, Gabriella; Bonelli, Gabriella; Baccino, Francesco M; Costelli, Paola

    2013-04-01

    Muscle protein wasting in cancer cachexia is a critical problem. The underlying mechanisms are still unclear, although the ubiquitin-proteasome system has been involved in the degradation of bulk myofibrillar proteins. The present work has been aimed to investigate whether autophagic degradation also plays a role in the onset of muscle depletion in cancer-bearing animals and in glucocorticoid-induced atrophy and sarcopenia of aging. The results show that autophagy is induced in muscle in three different models of cancer cachexia and in glucocorticoid-treated mice. In contrast, autophagic degradation in the muscle of sarcopenic animals is impaired but can be reactivated by calorie restriction. These results further demonstrate that different mechanisms are involved in pathologic muscle wasting and that autophagy, either excessive or defective, contributes to the complicated network that leads to muscle atrophy. In this regard, particularly intriguing is the observation that in cancer hosts and tumor necrosis factor ?-treated C2C12 myotubes, insulin can only partially blunt autophagy induction. This finding suggests that autophagy is triggered through mechanisms that cannot be circumvented by using classic upstream modulators, prompting us to identify more effective approaches to target this proteolytic system. PMID:23395093

  19. Carbon and chlorine isotope analysis to identify abiotic degradation pathways of 1,1,1-trichloroethane.

    PubMed

    Palau, Jordi; Shouakar-Stash, Orfan; Hunkeler, Daniel

    2014-12-16

    This study investigates dual C-Cl isotope fractionation during 1,1,1-TCA transformation by heat-activated persulfate (PS), hydrolysis/dehydrohalogenation (HY/DH) and Fe(0). Compound-specific chlorine isotope analysis of 1,1,1-TCA was performed for the first time, and transformation-associated isotope fractionation ? bulk C and ? bulk Cl values were -4.0 ± 0.2‰ and no chlorine isotope fractionation with PS, -1.6 ± 0.2‰ and -4.7 ± 0.1‰ for HY/DH, -7.8 ± 0.4‰ and -5.2 ± 0.2‰ with Fe(0). Distinctly different dual isotope slopes (??13C/??37Cl): ? with PS, 0.33 ± 0.04 for HY/DH and 1.5 ± 0.1 with Fe(0) highlight the potential of this approach to identify abiotic degradation pathways of 1,1,1-TCA in the field. The trend observed with PS agreed with a C-H bond oxidation mechanism in the first reaction step. For HY/DH and Fe(0) pathways, different slopes were obtained although both pathways involve cleavage of a C-Cl bond in their initial reaction step. In contrast to the expected larger primary carbon isotope effects relative to chlorine for C-Cl bond cleavage, ? bulk C < ? bulk Cl was observed for HY/DH and in a similar range for reduction by Fe(0), suggesting the contribution of secondary chlorine isotope effects. Therefore, different magnitude of secondary chlorine isotope effects could at least be partly responsible for the distinct slopes between HY/DH and Fe(0) pathways. Following this dual isotope approach, abiotic transformation processes can unambiguously be identified and quantified. PMID:25379605

  20. The identification of degradation products and degradation pathway of malvidin-3-glucoside and malvidin-3,5-diglucoside under microwave treatment.

    PubMed

    Zhao, Mengyao; Luo, Yinghua; Li, Yuan; Liu, Xin; Wu, Jihong; Liao, Xiaojun; Chen, Fang

    2013-12-01

    Understanding the degradation of anthocyanins (Acys) during microwave treatment is required for the application of this novel technology to fruit and vegetable processing. The pH 3.5 buffer solutions containing 50mg/l malvidin-3-glucoside (Mv-3-glu) and malvidin-3,5-diglucoside (Mv-3,5-diglu), respectively, were treated with microwaves at 700 W. In comparison, the samples were heated at 98±2 °C in a water bath. In total, nine degradation products were detected (using LC-UV/Vis-MS/MS) for each Acys after microwave treatment. Anthocyanone A, hydroxycoumarins and dihydroxy phenylacetaldehyde were characterised as the main degradation products of both Acys under microwave conditions, which were not detected in the thermal treatment. In addition, syringic acid, 2,4,6-trihydroxybenzaldehyde, coumarin glucosides and 2,6-dimethoxyphenol were found simultaneously under the microwave and thermal treatments. During the microwave treatment, hydrogen peroxide was formed and resulted in the Baeyer-Villiger-type oxidation of the Acys, which was the main degradation pathway. At the same time, the conventional thermal degradation pathway of Acys also occurred. PMID:23871085

  1. Morpholine Degradation Pathway of Mycobacterium aurum MO1: Direct Evidence of Intermediates by In Situ 1H Nuclear Magnetic Resonance

    PubMed Central

    Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.; Poupin, P.; Truffaut, N.

    1998-01-01

    Resting Mycobacterium aurum MO1 cells were incubated with morpholine, a waste from the chemical industry. The kinetics of biodegradation was monitored by using in situ nuclear magnetic resonance (NMR). The incubation medium was directly analyzed by 1H NMR. This technique allowed the unambiguous identification of two intermediates of the metabolic pathway involved in the biodegradation process, glycolate and 2-(2-aminoethoxy)acetate. The latter compound, which was not commercially available, was synthesized, in three steps, from 2-(2-aminoethoxy)ethanol. Quantitative analysis of the kinetics of degradation of morpholine was performed by integrating the signals of the different metabolites in 1H-NMR spectra. Morpholine was degraded within 10 h. The intermediates increased during the first 10 h and finally disappeared after 20 h incubation. Assays of degradation were also carried out with glycolate and ethanolamine, hypothetical intermediates of the morpholine degradation pathway. They were degraded within 4 and 8 h, respectively. Until now, no tool for direct detection of intermediates or even morpholine has been available, consequently, only hypothetical pathways have been proposed. The approach described here gives both qualitative and quantitative information about the metabolic routes used in morpholine degradation by M. aurum MO1. It could be used to investigate many biodegradative processes. PMID:9435073

  2. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  3. The Whole Genome Sequence of Sphingobium chlorophenolicum L-1: Insights into the Evolution of the Pentachlorophenol Degradation Pathway

    SciTech Connect

    Copley, Shelley D.; Rokicki, Joseph; Turner, Pernilla; Daligault, Hajnalka E.; Nolan, Matt; Land, Miriam L

    2012-01-01

    Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria.

  4. The Whole Genome Sequence of Sphingobium chlorophenolicum L-1: Insights into the Evolution of the Pentachlorophenol Degradation Pathway

    PubMed Central

    Copley, Shelley D.; Rokicki, Joseph; Turner, Pernilla; Daligault, Hajnalka; Nolan, Matt; Land, Miriam

    2012-01-01

    Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria. PMID:22179583

  5. Reaction pathway of the degradation of the p-hydroxybenzoic acid by sulfate radical generated by ionizing radiations

    NASA Astrophysics Data System (ADS)

    Criquet, Justine; Leitner, Nathalie Karpel Vel

    2015-01-01

    The degradation of p-hydroxybenzoic acid (HBA) in aqueous solutions by ionizing radiation was studied. The phenolic pollutant was easily removed by the electron beam irradiation, as more than 80% of the initial 100 ?M introduced was degraded for a dose of 600 Gy. It was shown that the addition of persulfate, producing the sulfate radical as additional reactive species, induced a change in the reaction pathway. LC-MS analyses were performed in order to identify the different by-products formed. In the absence of persulfate, the main by-product formed was 3,4-dihydroxybenzoic acid, while in presence of persulfate, 1,4-benzoquinone was detected and the hydroxylated by-products were not present. A reaction pathway of HBA degradation by hydroxyl and sulfate radicals was proposed from the identification of the chemical structure of the different by-products detected. The influences of pH and dissolved oxygen were also studied. A high decline of HBA degradation was observed at pH 11 compared to pH 4.5, this decrease was minimized in the presence of persulfate. The dissolved oxygen concentration was found to be a limiting parameter of HBA degradation, however an excess of dissolved oxygen in solution did not improve the degradation to a large extent.

  6. Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator

    PubMed Central

    Wang, Wei; Yang, Shihui; Hunsinger, Glendon B.; Pienkos, Philip T.; Johnson, David K.

    2014-01-01

    To produce lignocellulosic biofuels economically, the complete release of monomers from the plant cell wall components, cellulose, hemicellulose, and lignin, through pre-treatment and hydrolysis (both enzymatic and chemical), and the efficient utilization of these monomers as carbon sources, is crucial. In addition, the identification and development of robust microbial biofuel production strains that can tolerate the toxic compounds generated during pre-treatment and hydrolysis is also essential. In this work, Cupriavidus necator was selected due to its capabilities for utilizing lignin monomers and producing polyhydroxylbutyrate (PHB), a bioplastic as well as an advanced biofuel intermediate. We characterized the growth kinetics of C. necator in pre-treated corn stover slurry as well as individually in the pre-sence of 11 potentially toxic compounds in the saccharified slurry. We found that C. necator was sensitive to the saccharified slurry produced from dilute acid pre-treated corn stover. Five out of 11 compounds within the slurry were characterized as toxic to C. necator, namely ammonium acetate, furfural, hydroxymethylfurfural (HMF), benzoic acid, and p-coumaric acid. Aldehydes (e.g., furfural and HMF) were more toxic than the acetate and the lignin degradation products benzoic acid and p-coumaric acid; furfural was identified as the most toxic compound. Although toxic to C. necator at high concentration, ammonium acetate, benzoic acid, and p-coumaric acid could be utilized by C. necator with a stimulating effect on C. necator growth. Consequently, the lignin degradation pathway of C. necator was reconstructed based on genomic information and literature. The efficient conversion of intermediate catechol to downstream products of cis,cis-muconate or 2-hydroxymuconate-6-semialdehyde may help improve the robustness of C. necator to benzoic acid and p-coumaric acid as well as improve PHB productivity. PMID:24904560

  7. Oxidative degradation pathways of cellular DNA: product formation and mechanistic insights.

    PubMed

    Cadet, Jean

    2014-10-01

    More than 100 oxidized purine and pyrimidine nucleosides including hydroperoxides and diastereomers have been characterized so far in extensive model studies. However, much less information is currently available on the oxidatively generated base damage to cellular DNA at the exception however of the overwhelming modifications produced by singlet oxygen ((1)O2). This is mostly due to analytical difficulties that are now, at least, partly overcome with the advent of the accurate and sensitive high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). Hydroxyl radical ((•)OH) and one-electron oxidants that may be either endogenously formed through oxidative metabolism, phagocytosis, inflammation and other pathological conditions are predominantly at the origin of oxidatively generated damage to cellular DNA. It is worth mentioning that exposure of cells to exogenous physical agents (UVA light, high intensity UV laser, ionizing radiation) and chemicals such as bromate may also induce oxidatively generated damage to DNA. Emphasis is placed in this presentation on the critical survey of the main recently available information concerning the formation of (1)O2, (•)OH and one-electron oxidant-mediated single and more complex DNA damage (tandem lesions, intra- and interstrand cross-links, DNA-protein cross-links) arising from one radical hit. Evidence was provided that (•)OH and one-electron oxidants, through the generation of neutral radicals and radical cations respectively from nucleobases, are able to induce partly common degradation pathways. In addition selective oxidative reactions giving rise to specific degradation products of (•)OH and one-electron oxidation reactions that can be used as representative biomarkers of these oxidants have been identified. Emphasis was recently placed on the detection of oxidatively generated damage to cytosine and 5-methylcytosine in human cells. PMID:26461303

  8. Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator.

    PubMed

    Wang, Wei; Yang, Shihui; Hunsinger, Glendon B; Pienkos, Philip T; Johnson, David K

    2014-01-01

    To produce lignocellulosic biofuels economically, the complete release of monomers from the plant cell wall components, cellulose, hemicellulose, and lignin, through pre-treatment and hydrolysis (both enzymatic and chemical), and the efficient utilization of these monomers as carbon sources, is crucial. In addition, the identification and development of robust microbial biofuel production strains that can tolerate the toxic compounds generated during pre-treatment and hydrolysis is also essential. In this work, Cupriavidus necator was selected due to its capabilities for utilizing lignin monomers and producing polyhydroxylbutyrate (PHB), a bioplastic as well as an advanced biofuel intermediate. We characterized the growth kinetics of C. necator in pre-treated corn stover slurry as well as individually in the pre-sence of 11 potentially toxic compounds in the saccharified slurry. We found that C. necator was sensitive to the saccharified slurry produced from dilute acid pre-treated corn stover. Five out of 11 compounds within the slurry were characterized as toxic to C. necator, namely ammonium acetate, furfural, hydroxymethylfurfural (HMF), benzoic acid, and p-coumaric acid. Aldehydes (e.g., furfural and HMF) were more toxic than the acetate and the lignin degradation products benzoic acid and p-coumaric acid; furfural was identified as the most toxic compound. Although toxic to C. necator at high concentration, ammonium acetate, benzoic acid, and p-coumaric acid could be utilized by C. necator with a stimulating effect on C. necator growth. Consequently, the lignin degradation pathway of C. necator was reconstructed based on genomic information and literature. The efficient conversion of intermediate catechol to downstream products of cis,cis-muconate or 2-hydroxymuconate-6-semialdehyde may help improve the robustness of C. necator to benzoic acid and p-coumaric acid as well as improve PHB productivity. PMID:24904560

  9. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

    PubMed

    Antunes, Ana T; Goos, Yvonne J; Pereboom, Tamara C; Hermkens, Dorien; Wlodarski, Marcin W; Da Costa, Lydie; MacInnes, Alyson W

    2015-07-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  10. The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

    PubMed Central

    Zhou, Tao; Dang, Ying

    2014-01-01

    ABSTRACT The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4+ T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ?64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. IMPORTANCE The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins are folded in the ER and secreted through the classical secretory pathway. The Env folding process involves extensive cross-linking of 10 Cys residues by disulfide bond formation and heavy N-glycosylation on ?30 Asn residues. Currently, it is still unclear how this process is regulated. Here, we studied this mechanism in the HIV nonpermissive human CD4+ T cell line CEM.NKR. We found that Env proteins were rapidly degraded through a cellular pathway that specifically targets misfolded proteins, resulting in inhibition of Env expression. Importantly, we have identified a mitochondrial translocator protein, TSPO, which could trigger this degradation by interfering with the Env folding process. Further characterization of TSPO antiviral activity will reveal a novel antiretroviral mechanism that targets the Env protein. PMID:24403586

  11. 2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris.

    PubMed

    Pelletier, D A; Harwood, C S

    2000-05-01

    A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway. PMID:10781543

  12. Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity

    PubMed Central

    Lee, Jung Hoon; Shin, Seung Kyun; Jiang, Yanxialei; Choi, Won Hoon; Hong, Chaesun; Kim, Dong-Eun; Lee, Min Jae

    2015-01-01

    The ubiquitin-proteasome system (UPS) is the primary mechanism by which intracellular proteins, transcription factors, and many proteotoxic proteins with aggregation-prone structures are degraded. The UPS is reportedly downregulated in various neurodegenerative disorders, with increased proteasome activity shown to be beneficial in many related disease models. Proteasomes function under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a proteasome-associated deubiquitinating enzyme (DUB). We identified three specific RNA aptamers of USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich regions upstream, and similar secondary structures. They efficiently elevated proteasomal activity, as determined by the increased degradation of small fluorogenic peptide substrates and physiological polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated in the presence of the UPS14 aptamers in vitro. Our results indicate that these novel inhibitory UPS14 aptamers can be used to enhance proteasome activity, and to facilitate the degradation of proteotoxic proteins, thereby protecting cells from various neurodegenerative stressors. PMID:26041011

  13. Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways.

    PubMed

    Yu, Hao; Tang, Hongzhi; Li, Yangyang; Xu, Ping

    2015-12-15

    Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppAS and vppAL [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5? and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase. PMID:26407884

  14. Ozonation of ofloxacin in water: by-products, degradation pathway and ecotoxicity assessment.

    PubMed

    Tay, Kheng Soo; Madehi, Norfazrina

    2015-07-01

    Application of ozonation in water treatment involves complex oxidation pathways that could lead to the formation of various by-products, some of which may be harmful to living organisms. In this work, ozonation by-products of ofloxacin (OFX), a frequently detected pharmaceutical pollutant in the environment, were identified and their ecotoxicity was estimated using the Ecological Structure Activity Relationships (ECOSAR) computer program. In order to examine the role of ozone (O3) and hydroxyl radicals (?OH) in the degradation of ofloxacin, ozonation was performed at pH2, 7 and 12. In this study, 12 new structures have been proposed for the ozonation by-products detected during the ozonation of ofloxacin. According to the identified ozonation by-products, O3 and ?OH were found to react with ofloxacin during ozonation. The reaction between ofloxacin and O3 proceeded via hydroxylation and breakdown of heterocyclic ring with unsaturated double-bond. The reaction between ofloxacin and ·OH generated various by-products derived from the breakdown of heterocyclic ring. Ecotoxicity assessment indicated that ozonation of OFX could yield by-products of greater toxicity compared with parent compounds. PMID:25791053

  15. Phototransformation of mefenamic acid induced by nitrite ions in water: mechanism, toxicity, and degradation pathways.

    PubMed

    Chen, Ping; Lv, Wenying; Chen, Zhiming; Ma, Jingshuai; Li, Ruobai; Yao, Kun; Liu, Guoguang; Li, Fuhua

    2015-08-01

    Here, we evidenced the photo-induced degradation of mefenamic acid, a nonsteroidal anti-inflammatory drug, through the 254-nm light excitation of nitrite. The results demonstrated that the photodegradation of mefenamic acid was enhanced, and the mefenamic acid photodegradation rate significantly increased, from 0.00627 to 0.0350 min(-1) as the nitrite was increased from 0 to 0.5 mmol L(-1). The photodegradation rate increased from 0.0287 to 0.0512 min(-1) as the pH was elevated, from 5.0 to 10.0. The actual second-order rate constant for the reaction of mefenamic acid with ·OH was investigated to 1.079?×?10(10) M(-1) s(-1) according to steady-state ·OH concentration of 3.5?×?10(-14) mmol L(-1) and the contribution to the rate of ·OH of 67.1%. The photoproducts were identified using HPLC/MS/MS, and possible nitrite-induced photodegradation pathways were proposed by hydroxylation, dehydrogenation, hydration, nitrosylation, and ketonized reactions. The toxicity of the phototransformation products was evaluated using the Microtox test, which revealed that the photoproducts were more toxic than mefenamic acid for the generation of nitrosation aromatic compounds. PMID:25907630

  16. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus

    PubMed Central

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-01-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates. PMID:23902518

  17. Endocytosis and ligand dissociation and degradation mediated by the hepatic galactosyl receptor occur via two different pathways

    SciTech Connect

    Weigel, P.H.; Clarke, B.L.; Oka, J.A.

    1986-05-01

    Isolated rat hepatocytes express two distinct populations of surface Galactosyl receptor activity, measured by the binding of /sup 125/I-asialo-orosomucoid (ASOR), which they designate State 1 and State 2. Freshly isolated cells express only state 1 receptors. Cells equilibrated at 37/sup 0/C also express State 2 receptors, which represent 50-80% of the total surface activity. In the absence of ligand, State 2 receptor activity is reversibly decreased by metabolic energy poisons, low temperature and microtubule drugs, whereas State 1 receptor activity is unaffected. Endocytosis of /sup 125/I-ASOR by State 1 receptors is followed by a slow dissociation of /sup 125/I-ASOR from receptor but the immediate release of acid soluble degradation products. In contrast, State 2 receptors mediate endocytosis which involves a rapid dissociation step but a 20 min lag, prior to the release of degradation products. Both pathways follow first order kinetics and are functional under steady state conditions indicating coordinated receptor recycling. Degradation mediated by both pathways is inhibited by leupeptin and chloroquine. The State 1 and 2 pathways can be further differentiated by the greater sensitivity of the latter to microtubule drugs. These results suggest that there are either structurally different native receptors or that identical receptors are directed into two different functional pathways, for example by interaction with different types of coated pits.

  18. Development of hybrid small molecules that induce degradation of estrogen receptor-alpha and necrotic cell death in breast cancer cells.

    PubMed

    Okuhira, Keiichiro; Demizu, Yosuke; Hattori, Takayuki; Ohoka, Nobumichi; Shibata, Norihito; Nishimaki-Mogami, Tomoko; Okuda, Haruhiro; Kurihara, Masaaki; Naito, Mikihiko

    2013-11-01

    Manipulation of protein stability with small molecules has a great potential for both basic research and clinical therapy. Recently, we have developed a series of hybrid small molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser) that induces degradation of target proteins via ubiquitin-proteasome system. Here we report the activities of SNIPER(ER) that targets estrogen receptor alpha (ER?) for degradation. SNIPER(ER) induced degradation of ER? and inhibited estrogen-dependent expression of pS2 gene in an estrogen-dependent breast cancer cell line MCF-7. A proteasome inhibitor MG132 and siRNA-mediated downregulation of cIAP1 abrogated the SNIPER(ER)-induced ER? degradation, suggesting that the ER? is degraded by proteasome subsequent to cIAP1-mediated ubiquitylation. Intriguingly, after the ER? degradation, the SNIPER(ER)-treated MCF-7 cells undergo rapid cell death. Detailed analysis indicated that SNIPER(ER) caused necrotic cell death accompanied by a release of HMGB1, a marker of necrosis, from the cells. Following the ER? degradation, reactive oxygen species (ROS) was produced in the SNIPER(ER)-treated MCF-7 cells, and an anti-oxidant N-acetylcysteine inhibited the necrotic cell death. These results indicate that SNIPER(ER) induces ER? degradation, ROS production and necrotic cell death, implying a therapeutic potential of SNIPER(ER) as a lead for the treatment of ER?-positive breast cancers. PMID:23992566

  19. Chemotaxis and degradation of organophosphate compound by a novel moderately thermo-halo tolerant Pseudomonas sp. strain BUR11: evidence for possible existence of two pathways for degradation

    PubMed Central

    Pailan, Santanu

    2015-01-01

    An organophosphate (OP) degrading chemotactic bacterial strain BUR11 isolated from an agricultural field was identified as a member of Pseudomonas genus on the basis of its 16S rRNA gene sequence. The strain could utilize parathion, chlorpyrifos and their major hydrolytic intermediates as sole source of carbon for its growth and exhibited positive chemotactic response towards most of them. Optimum concentration of parathion for its growth was recorded to be 200 ppm and 62% of which was degraded within 96 h at 37 °C. Growth studies indicated the strain to be moderately thermo-halo tolerant in nature. Investigation based on identification of intermediates of parathion degradation by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC) and liquid chromatography mass spectrometry (LC-MS/MS) provided evidence for possible existence of two pathways. The first pathway proceeds via 4-nitrophenol (4-NP) while the second proceeds through formation of 4-aminoparathion (4-APar), 4-aminophenol (4-AP) and parabenzoquinone (PBQ). This is the first report of chemotaxis towards organophosphate compound by a thermo-halo tolerant bacterium. PMID:26587344

  20. Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway.

    PubMed Central

    Falnes, P O; Olsnes, S

    1998-01-01

    The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process. PMID:9430652

  1. Functional compartments of sulphatide metabolism in cultured living cells: evidence for the involvement of a novel sulphatide-degrading pathway.

    PubMed Central

    Tempesta, M C; Salvayre, R; Levade, T

    1994-01-01

    The modes of uptake and degradation of radiolabelled cerebroside sulphate (CS or sulphatide) were investigated in cultured living skin fibroblasts and Epstein-Barr virus-transformed lymphoblastoid cell lines established from control individuals and patients affected with metachromatic leucodystrophy (cerebroside sulphatase deficiency), multiple sulphatase deficiency and low-density-lipoprotein-receptor-negative familial hypercholesterolaemia. In both cell types, CS was taken up through a non-receptor-mediated process. In fibroblasts, CS degradation occurred intralysosomally as was evident from the findings that fibroblasts from metachromatic leucodystrophic patients accumulated the sulphatide and that chloroquine inhibited its degradation by normal cells. In contrast, under similar conditions of CS availability, lymphoblastoid cell lines from patients with metachromatic leucodystrophy could degrade the incorporated sulphatide exactly as their normal counterparts. This metabolic pathway was also fully active in lymphoblastoid cells from patients with multiple sulphatase deficiency and was not inhibited by chloroquine treatment. These data are consistent with a non-lysosomal type of hydrolysis. In addition to the lysosomal and non-lysosomal compartments, a third compartment was identified in the two cell types which is probably formed by the pool of the sulphatide molecules incorporated into the plasma membrane. This is the first report on the existence of a CS-degrading pathway in intact cells with deficient lysosomal cerebroside sulphatase activity. PMID:7906514

  2. Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway

    PubMed Central

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Lavire, Céline; Hommais, Florence

    2014-01-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)–CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

  3. Coxsackievirus B5 induced apoptosis of HeLa cells: Effects on p53 and SUMO

    SciTech Connect

    Gomes, Rogerio; Guerra-Sa, Renata; Arruda, Eurico

    2010-01-20

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  4. PROTEASOME ACTIVITY DECLINES IN AGED MACROPHAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway is involved in regulation of a variety of biologically important processes including antigen presentation by macrophages. Age-related decrease in proteasome activity has been reported in other tissues. However, the effect of aging on the ubiquitin-proteasome pathway ...

  5. Protein aggregation and degradation mechanisms in neurodegenerative diseases

    PubMed Central

    Takalo, Mari; Salminen, Antero; Soininen, Hilkka; Hiltunen, Mikko; Haapasalo, Annakaisa

    2013-01-01

    Neurodegenerative diseases are characterized by selective neuronal vulnerability and neurodegeneration in specific brain regions. The pathogenesis of these disorders centrally involves abnormal accumulation and aggregation of specific proteins, which are deposited in intracellular inclusions or extracellular aggregates that are characteristic for each disease. Increasing evidence suggests that genetic mutations or environmental factors can instigate protein misfolding and aggregation in these diseases. Consequently, neurodegenerative diseases are often considered as conformational diseases. This idea is further supported by studies implicating that impairment of the protein quality control (PQC) and clearance systems, such as the ubiquitin-proteasome system and autophagosome-lysosome pathway, may lead to the abnormal accumulation of disease-specific proteins. This suggests that similar pathological mechanisms may underlie the pathogenesis of the different neurodegenerative disorders. Interestingly, several proteins that are known to associate with neurodegenerative diseases have been identified as important regulators of PQC and clearance systems. In this review, we summarize the central features of abnormal protein accumulation in different common neurodegenerative diseases and discuss some aspects of specific disease-associated proteins regulating the PQC and clearance mechanisms, such as ubiquilin-1. PMID:23516262

  6. A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

    SciTech Connect

    Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko; Endo, Toshiya; Nishikawa, Shuh-ichi

    2010-03-12

    Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

  7. The relationship between protein synthesis and protein degradation in object recognition memory.

    PubMed

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor ?-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, ?-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. PMID:26200717

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

    PubMed

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

    2015-05-29

    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 PKC?. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation. PMID:25897075

  9. HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation

    SciTech Connect

    Noy, Tahel; Suad, Oded; Taglicht, Daniel; Ciechanover, Aaron

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HUWE1 ubiquitinates MyoD in vitro and in cells. Black-Right-Pointing-Pointer The ubiquitination by HUWE1 targets MyoD for proteasomal degradation. Black-Right-Pointing-Pointer HUWE1 can modify MyoD on its N-terminal residue. -- Abstract: MyoD is a tissue-specific transcriptional activator that acts as a master switch for muscle development. It activates a broad array of muscle-specific genes, which leads to conversion of proliferating myoblasts into mature myotubes. The ubiquitin proteasome system (UPS) plays an important role in controlling MyoD. Both its N-terminal residue and internal lysines can be targeted by ubiquitin, and both modifications appear to direct it for proteasomal degradation. The protein is short-lived and has a half-life of {approx}45 min in different cells. It was reported that MyoD can be ubiquitinated by MAFbx/AT-1, but accumulating lines of experimental evidence showed that other ligase(s) may also participate in its targeting. Here we describe the involvement of HUWE1 in the ubiquitination and proteasomal degradation of MyoD. Furthermore, we show that the ligase can ubiquitinate the protein in its N-terminal residue.

  10. Extracellular Signal-regulated Kinase (ERK) Regulates Cortactin Ubiquitination and Degradation in Lung Epithelial Cells*

    PubMed Central

    Zhao, Jing; Wei, Jianxin; Mialki, Rachel; Zou, Chunbin; Mallampalli, Rama K.; Zhao, Yutong

    2012-01-01

    Cortactin, an actin-binding protein, is essential for cell growth and motility. We have shown that cortactin is regulated by reversible phosphorylation, but little is known regarding cortactin protein stability. Here, we show that lipopolysaccharide (LPS)-induced cortactin degradation is mediated by extracellular regulated signal kinase (ERK). LPS induces cortactin serine phosphorylation, ubiquitination, and degradation in mouse lung epithelia, an effect abrogated by ERK inhibition. Serine phosphorylation sites mutant, cortactinS405A/S418A, enhances its protein stability. Cortactin is polyubiquitinated and degraded within the proteasome, whereas a cortactinK79R mutant exhibited proteolytic stability during cyclohexamide (CHX) or LPS treatment. The E3 ligase subunit ?-Trcp interacts with cortactin, and its overexpression reduced cortactin protein levels, an effect attenuated by ERK inhibition. Overexpression of ?-Trcp was sufficient to reduce the protective effects of exogenous cortactin on epithelial cell barrier integrity, an effect not observed after expression of a cortactinK79R mutant. These results provide evidence that LPS modulation of cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network. PMID:22514278

  11. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    SciTech Connect

    Lee, Sang-Myeong; Kleiboeker, Steven B. . E-mail: KleiboekerS@Missouri.edu

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradation of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  12. Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases.

    PubMed

    Sainsbury, Paul D; Mineyeva, Yelena; Mycroft, Zoe; Bugg, Timothy D H

    2015-06-01

    Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15?M) and D3 for MhpB (IC50 110?M). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the ?-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde. PMID:25984987

  13. Comparative genomic analysis of nine Sphingobium strains: Insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

    SciTech Connect

    Verma, Helianthous; Kumar, Roshan; Oldach, Phoebe; Sangwan, Naseer; Khurana, Jitendra P.; Gilbert, Jack A.; Lal, Rup

    2014-11-23

    Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6). Results: Efficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. In addition, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity. In conclusion, the bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

  14. Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.

    PubMed

    Pérez, Moisés; García-Limones, Carmen; Zapico, Inés; Marina, Anabel; Schmitz, M Lienhard; Muñoz, Eduardo; Calzado, Marco A

    2012-10-01

    The ubiquitin E3 ligase SIAH2 is an important regulator of the hypoxic response as it leads to the ubiquitin/proteasomal degradation of prolyl hydroxylases such as PHD3, which in turn increases the stability of hypoxia-inducible factor (HIF)-1?. In the present study, we identify the serine/threonine kinase DYRK2 as SIAH2 interaction partner that phosphorylates SIAH2 at five residues (Ser16, Thr26, Ser28, Ser68, and Thr119). Phosphomimetic and phospho-mutant forms of SIAH2 exhibit different subcellular localizations and consequently change in PHD3 degrading activity. Accordingly, phosphorylated SIAH2 is more active than the wild-type E3 ligase and shows an increased ability to trigger the HIF-1?-mediated transcriptional response and angiogenesis. We also found that SIAH2 knockdown increases DYRK2 stability, whereas SIAH2 expression facilitates DYRK2 polyubiquitination and degradation. Hypoxic conditions cause a SIAH2-dependent DYRK2 polyubiquitination and degradation which ultimately also results in an impaired SIAH2 phosphorylation. Similarly, DYRK2-mediated phosphorylation of p53 at Ser46 is impaired under hypoxic conditions, suggesting a molecular mechanism underlying chemotherapy resistance in solid tumors. PMID:22878263

  15. FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex

    PubMed Central

    Sjögren, Benita; Swaney, Steven; Neubig, Richard R.

    2015-01-01

    The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation. PMID:25970626

  16. Degradation pathways study of the natriuretic and ?-adrenoceptor antagonist tienoxolol using liquid chromatography-electrospray ionization multistage mass spectrometry.

    PubMed

    Gana, Ines; Dugay, Annabelle; Henriet, Théo; Rietveld, Ivo B; Bernard, Mélisande; Guechot, Christophe; Teulon, Jean-Marie; Safta, Fathi; Yagoubi, Najet; Céolin, René; Do, Bernard

    2014-08-01

    Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to combine a diuretic and a ?-adrenoreceptor antagonist into a single molecule. Its degradation products generated in several stress media have been determined by high-pressure liquid chromatography (HPLC) coupled to a hybrid mass spectrometer with a triple quadrupole-linear trap. A Polaris(®) column with a C18-A stationary phase and a linear gradient mobile phase composed of a mixture of trifluoroacetic acid 1% (v/v) and acetonitrile allowed for optimal separation. Structural elucidation of the degradation products has been based on MS/MS techniques, by comparing their fragmentation patterns to the precursor's data. Up to seven degradation products of the active ingredient, resulting from hydrolysis, oxidation, dehydration and transamidation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics have been studied to assess the molecule's shelf life and to identify the most important degradation factor. PMID:24726889

  17. Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

    PubMed Central

    Khan, Fazlurrahman; Pal, Deepika; Vikram, Surendra; Cameotra, Swaranjit Singh

    2013-01-01

    2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium. PMID:23614030

  18. Activation of FGF-23 Mediated Vitamin D Degradative Pathways by Cholecalciferol

    PubMed Central

    Alshayeb, Hala; Showkat, Arif; Wall, Barry M.; Gyamlani, Geeta G.; David, Valentin

    2014-01-01

    Context: The optimal circulating concentration of 25(OH) vitamin D is controversial. Objective: The aim was to investigate if FGF-23 and 24,25(OH)2D can guide cholecalciferol replacement. Design: Oral cholecalciferol (10,000 IU weekly) administered to subjects with 25(OH)D levels < 20 ?g/mL and eGFR > 60 mL/min/1.73 m2 (n = 25), chronic kidney disease (CKD) (n = 27), or end stage renal disease (ESRD) (n = 14). Setting: The study was conducted at the Veterans Affairs clinics. Main Outcome Measure: Serum FGF-23, PTH, 25(OH)D, 1,25(OH)2D, 24,25(OH)2D, calcium, and phosphorous concentrations, and urinary excretion of calcium and phosphorus at baseline and after 8 weeks of treatment. Results: Cholecalciferol treatment increased concentrations of serum 25(OH)D by (19.3 ± 8 ?g/mL, P = .001; 12.2 ± 9 ?g/mL, P = .0001) and 24,25(OH)2D (1.14 ± 0.89 ?g/mL, P = .0024; 1.0 ± 0.72 ?g/mL P = .0002), and reduced serum PTH (?11 ± 21 pg/mL, P = .0292; ?42 ± 68 pg/mL, P = .0494) in normal and CKD subjects, respectively. Cholecalciferol increased serum FGF-23 levels only in normal subjects (44 ± 57 ?g/mL, P = .01). Increments in serum 25(OH)D positively correlated with serum FGF-23 and 24,25(OH)2D and negatively correlated with PTH. In ESRD, cholecalciferol administration increased 25(OH)D by (16.6 ± 6.6 ?g/mL P ? .05) without changing 24,25(OH)2D, FGF-23 or PTH levels. Conclusion: Modest elevations of serum 25(OH)D levels after cholecalciferol treatment are sufficient to induce compensatory degradative pathways in patients with sufficient renal reserves, suggesting that optimal circulating 25(OH)D levels are approximately 20 ?g/mL. In addition, catabolism of 25(OH)D may also contribute to the low circulating vitamin D levels in CKD, since elevations of FGF-23 in CKD are associated with increased 24,25(OH)2D after cholecalciferol administration. PMID:24960544

  19. Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. Strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, ...

  20. REACTION PATHWAY OF THE DIKETONITRILE DEGRADATE OF ISOXAFLUTOLE (BALANCE(TM)) WITH HYPOCHLORITE IN WATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isoxaflutole (IXF; Balance(TM)) belongs to the new class of isoxazole herbicides. Isoxaflutole has a very short half-life in soil and rapidly degrades to a stable and phytotoxic degradate, diketonitrile (DKN). DKN was previously discovered to rapidly react with hypochlorite (OCl-) in tap water, yie...

  1. Smurf2 regulates the degradation of YY1.

    PubMed

    Jeong, Hyung Min; Lee, Sung Ho; Yum, Jinah; Yeo, Chang-Yeol; Lee, Kwang Youl

    2014-09-01

    Transcription factor YY1 plays important roles in cell proliferation and differentiation. For example, YY1 represses the expression of muscle-specific genes and the degradation of YY1 is required for myocyte differentiation. The activity of YY1 can be regulated by various post-translational modifications; however, little is known about the regulatory mechanisms for YY1 degradation. In this report, we attempted to identify potential E3 ubiquitin ligases for YY1, and found that Smurf2 E3 ubiquitin ligase can negatively regulate YY1 protein level, but not mRNA level. Smurf2 interacted with YY1, induced the poly-ubiquitination of YY1 and shortened the half-life of YY1 protein. Conversely, an E3 ubiquitin ligase-defective mutant form of Smurf2 or knockdown of Smurf2 increased YY1 protein level. PPxY motif is a typical target recognition site for Smurf2, and the PPxY motif in YY1 was important for Smurf2 interaction and Smurf2-induced degradation of YY1 protein. In addition, Smurf2 reduced the YY1-mediated activation of a YY1-responsive reporter whereas Smurf2 knockdown increased it. Finally, Smurf2 relieved the suppression of p53 activity by YY1. Taken together, our results suggest a novel regulatory mechanism for YY1 function by Smurf2 in which the protein stability and transcriptional activity of YY1 are regulated by Smurf2 through the ubiquitin-proteasome-mediated degradation of YY1. PMID:24803334

  2. Ubiquitin-specific protease 14 modulates degradation of cellular prion protein

    PubMed Central

    Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Fuse, Takayuki; Mori, Tsuyoshi; Satoh, Katsuya; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrPC). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrPSc, forms amyloid fibrils and could be infectious. It has been suggested that PrPSc is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrPC, as well as PrPSc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrPSc, whereas wildtype USP14 increased PrPSc in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrPSc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases. PMID:26061634

  3. PROTEASOME ACTIVITY DECLINES IN AGED MACROPHAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway is involved in regulation of a variety of biologically important processes including antigen presentation by macrophages (Mf). Age-related decrease in proteasome activity has been reported in other tissues. However, the effect of aging on the ubiquitin-proteasome pat...

  4. Evidence of distinct pathways for bacterial degradation of the steroid compound cholate suggests the potential for metabolic interactions by interspecies cross-feeding.

    PubMed

    Holert, Johannes; Yücel, Onur; Suvekbala, Vemparthan; Kuli?, Zarko; Möller, Heiko; Philipp, Bodo

    2014-05-01

    The distribution and the metabolic pathways of bacteria degrading steroid compounds released by eukaryotic organisms were investigated using the bile salt cholate as model substrate. Cholate-degrading bacteria could be readily isolated from freshwater environments. All isolated strains transiently released steroid degradation intermediates into culture supernatants before their further degradation. Cholate degradation could be initiated via two different reaction sequences. Most strains degraded cholate via a reaction sequence known from the model organism Pseudomonas sp. strain Chol1 releasing intermediates with a 3-keto-?(1,4) -diene structure of the steroid skeleton. The actinobacterium Dietzia sp. strain Chol2 degraded cholate via a different and yet unexplored reaction sequence releasing intermediates with a 3-keto-?(4,6) -diene-7-deoxy structure of the steroid skeleton such as 3,12-dioxo-4,6-choldienoic acid (DOCDA). Using DOCDA as substrate, two Alphaproteobacteria, strains Chol10-11, were isolated that produced the same cholate degradation intermediates as strain Chol2. With DOCDA as substrate for Pseudomonas sp. strain Chol1 only the side chain was degraded while the ring system was transformed into novel steroid compounds accumulating as dead-end metabolites. These metabolites could be degraded by the DOCDA-producing strains Chol10-11. These results indicate that bacteria with potentially different pathways for cholate degradation coexist in natural habitats and may interact via interspecies cross-feeding. PMID:24447610

  5. Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxine

    PubMed Central

    Kobayashi, Jun; Yoshida, Hiromi; Chu, Huy Nhat; Yoshikane, Yu; Kamitori, Shigehiro; Yagi, Toshiharu

    2009-01-01

    ?-(N-Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B6 and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9?Å, ? = 103.4°, and diffraction data were collected to 2.7?Å resolution. The V M value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit. PMID:19652351

  6. NOVEL PATHWAY OF TOLUENE CATABOLISM IN THE TRICHLOROETHYLENE DEGRADING BACTERIUM G4

    EPA Science Inventory

    o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. he absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities sug...

  7. Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12?†

    PubMed Central

    Prakash, Dhan; Kumar, Ravi; Jain, R. K.; Tiwary, B. N.

    2011-01-01

    The organism Acinetobacter sp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidative ortho dehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2 per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and 18O2 indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by the ortho ring cleavage catechol-1,2-dioxygenase to cis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA? derivative and a 2C4NBA+ transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ?55-kb transmissible plasmid present in RKJ12. PMID:21803909

  8. Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages.

    PubMed

    Min, Mingwei; Mevissen, Tycho E T; De Luca, Maria; Komander, David; Lindon, Catherine

    2015-12-01

    The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)-directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome-for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner. PMID:26446837

  9. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RAR? protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RAR? oncoprotein by As2O3, which results in initiation of PML-RAR? degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RAR? fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  10. Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway

    PubMed Central

    Xing, Weirong; Pourteymoor, Sheila

    2011-01-01

    Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs. PMID:21467157

  11. Crystallization and preliminary X-ray analysis of pyridoxine 4-oxidase, the first enzyme in pyridoxine degradation pathway I

    PubMed Central

    Mugo, Andrew Njagi; Kobayashi, Jun; Mikami, Bunzo; Ohnishi, Kouhei; Yagi, Toshiharu

    2012-01-01

    Vitamin B6-degradation pathway I has recently been identified in Mesorhizobium loti MAFF303099. Pyridoxine 4-oxidase, an FAD-dependent enzyme, is the first enzyme in this pathway and catalyzes the irreversible oxidation of pyridoxine to pyridoxal. The enzyme was overexpressed in Escherichia coli with a His6 tag and purified. The recombinant enzyme was crystallized at 277?K by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. The crystal, which belonged to space group P212121 with unit-cell parameters a = 62.38, b = 79.44, c = 136.43?Å, diffracted to 2.2?Å resolution. The calculated V M value (3.19?Å3 Da–1) suggested that the asymmetric unit contained one molecule. PMID:22232175

  12. Regulation of the Intracellular Localization of Foxo3a by Stress-Activated Protein Kinase Signaling Pathways in Skeletal Muscle Cells ?

    PubMed Central

    Clavel, Stephan; Siffroi-Fernandez, Sandrine; Coldefy, Anne Sophie; Boulukos, Kim; Pisani, Didier F.; Dérijard, Benoît

    2010-01-01

    Muscle atrophy is a debilitating process associated with many chronic wasting diseases, like cancer, diabetes, sepsis, and renal failure. Rapid loss of muscle mass occurs mainly through the activation of protein breakdown by the ubiquitin proteasome pathway. Foxo3a transcription factor is critical for muscle atrophy, since it activates the expression of ubiquitin ligase Atrogin-1. In several models of atrophy, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway induces nuclear import of Foxo3a through an Akt-dependent process. This study aimed to identify signaling pathways involved in the control of Foxo3a nuclear translocation in muscle cells. We observed that after nuclear import of Foxo3a by PI3K/Akt pathway inhibition, activation of stress-activated protein kinase (SAPK) pathways induced nuclear export of Foxo3a through CRM1. This mechanism involved the c-Jun NH2-terminal kinase (JNK) signaling pathway and was independent of Akt. Likewise, we showed that inhibition of p38 induced a massive nuclear relocalization of Foxo3a. Our results thus suggest that SAPKs are involved in the control of Foxo3a nucleocytoplasmic translocation in C2C12 cells. Moreover, activation of SAPKs decreases the expression of Atrogin-1, and stable C2C12 myotubes, in which the p38 pathway is constitutively activated, present partial protection against atrophy. PMID:19917721

  13. Sonolysis of natural phenolic compounds in aqueous solutions: degradation pathways and biodegradability.

    PubMed

    Vassilakis, Constantinos; Pantidou, Ariadni; Psillakis, Elefteria; Kalogerakis, Nicolas; Mantzavinos, Dionissios

    2004-07-01

    The sonochemical degradation in water of p-coumaric acid and p-hydroxybenzaldehyde, compounds that are commonly found in effluents of agricultural origin, was investigated using an 80 kHz horn-type sonicator. Experiments were performed at initial concentrations varying between 10 and 100 mg L(-1), an applied power varying between 75 and 150 W and liquid bulk temperatures varying between ambient and 70 degrees C. At the conditions in question, substrate removal was found to increase with increasing power and decreasing initial concentration and temperature. Addition of 1-butanol as a radical scavenger at a concentration of 1000 mg L(-1) nearly completely suppressed p-coumaric acid degradation. Conversely, addition of radical promoters (Fe2+ ions or H2O2) had a positive effect on removal, thus implying that degradation proceeds predominantly via hydroxyl radical-induced reactions. Reaction intermediates were determined using liquid and gas chromatography and a reaction network for p-coumaric acid degradation is suggested. Shake flask tests with activated sludge were performed to assess the aerobic biodegradability (in terms of chemical oxygen demand removal) before and after sonochemical treatment. At the conditions under consideration, the use of ultrasound enhanced the aerobic degradability of the substrates in question. PMID:15261550

  14. The putative role of proteolytic pathways in the pathogenesis of Type 1 diabetes mellitus: the 'autophagy' hypothesis.

    PubMed

    Fierabracci, Alessandra

    2014-05-01

    Autoimmune diseases are a heterogeneous group of disorders affecting different organs and tissues. New tools, such as genome-wide association studies, have provided evidence for new susceptibility loci and candidate genes in the disease process including common susceptibility genes involved in the immunological synapse and T cell activation. Close linkages have been found in a number of diseases, including ankylosing spondylitis, multiple sclerosis, Crohn's disease and insulin-dependent diabetes mellitus (Type 1 diabetes mellitus). Evidence for some association with Type 1 diabetes was previously found in the region containing 5q15/ERAP1 (endoplasmic reticulum aminopeptidase 1) (rs30187, ARTS1). Recent data suggest that in eukaryotic cells in addition to the ubiquitin/proteasome system another proteolytic pathway may have a significant role in the autoimmunity process, i.e. the autophagic pathway which constitutes the principal regulated catabolic process mediated by lysosomes. Autophagy could play a role in MHC class I and class II self-antigen presentation at the basis of the autoimmunity process. Furthermore cross-talk among different proteolytic pathways was recently highlighted i.e. components processed in the ubiquitin/proteasome system possibly engaged in autophagic pathways. T1D is an autoimmune disease characterised by the destruction of pancreatic beta cells by autoreactive T cells. Immunological abnormalities can precede months to years the initial symptoms and clinical diagnosis. Our hypothesis suggests that in the autoimmune process autophagy can intervene at different levels, during the thymic selection process of T lymphocytes causing escape of autoreactive T cells, at the initiation stage of the disease, in the preclinical period or subsequently to the disease onset having a role at the level of perpetuation of the autoimmunity process. Supporting evidence derives from the already reported discovery of polymorphisms in autophagy-related genes in patients affected by several autoimmune conditions such as Systemic Lupus Erithematosus. In addition deregulated autophagy was detected in T cells from lupus-prone mice and also found in T cells from patients. Autophagy was found activated in osteoclasts from RA patients as demonstrated by the increased expression of Atg7 and Beclin-1. Our hypothesis to be unraveled could have, if correct, relevant implications for the management of autoimmune conditions such as Type 1 diabetes. In principle, novel therapeutic approaches could be established by targeting deregulated autophagy offering novel opportunities to personalized medicine in patients affected by the disease. PMID:24582332

  15. Mutations in NGLY1 Cause an Inherited Disorder of the Endoplasmic Reticulum-Associated Degradation (ERAD) Pathway

    PubMed Central

    Enns, Gregory M.; Shashi, Vandana; Bainbridge, Matthew; Gambello, Michael J.; Zahir, Farah R.; Bast, Thomas; Crimian, Rebecca; Schoch, Kelly; Platt, Julia; Cox, Rachel; Bernstein, Jonathan; Scavina, Mena; Walter, Rhonda S.; Bibb, Audrey; Jones, Melanie; Hegde, Madhuri; Graham, Brett H.; Need, Anna C.; Oviedo, Angelica; Schaaf, Christian P.; Boyle, Sean; Butte, Atul J.; Chen, Rong; Clark, Michael J.; Haraksingh, Rajini; Cowan, Tina M.; He, Ping; Langlois, Sylvie; Zoghbi, Huda Y.; Snyder, Michael; Gibbs, Richard; Freeze, Hudson H.; Goldstein, David B.

    2014-01-01

    Purpose The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for the translocation of misfolded proteins across the ER membrane into the cytosol for subsequent degradation by the proteasome. In order to understand the spectrum of clinical and molecular findings in a complex neurological syndrome, we studied a series of eight patients with inherited deficiency of N-glycanase 1 (NGLY1), a novel disorder of cytosolic ERAD dysfunction. Methods Whole-genome, whole-exome or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data. Results All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypo- or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele. Conclusions NGLY1 deficiency is a novel autosomal recessive disorder of the ERAD pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a more broad range of mutations are detected. PMID:24651605

  16. Relationship between the proteasomal system and autophagy

    PubMed Central

    Lilienbaum, Alain

    2013-01-01

    Two major pathways degrade most cellular proteins in eukaryotic cells: the ubiquitin–proteasome system (UPS), which usually degrades the majority of proteins, and autophagy, primarily responsible for the degradation of most long-lived or aggregated proteins and cellular organelles. Disruption of these processes can contribute to pathology of a variety of diseases. Further, both pathways are critical for the maintenance of several aspects of cellular homeostasis, but, until recently, were thought to be largely distinct. Recent advances in this field, however, now strongly suggest that their activities are carefully orchestrated through several interfacing elements that are presented and discussed in this review. PMID:23638318

  17. 13C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H

    PubMed Central

    Ostermann, Steffen; Richhardt, Janine; Bringer, Stephanie; Bott, Michael; Wiechert, Wolfgang; Oldiges, Marco

    2015-01-01

    Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically 13C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from 13C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with 13C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments. PMID:26404385

  18. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  19. Pathways of nitrobenzene degradation in horizontal subsurface flow constructed wetlands: Effect of intermittent aeration and glucose addition.

    PubMed

    Kirui, Wesley K; Wu, Shubiao; Kizito, Simon; Carvalho, Pedro N; Dong, Renjie

    2016-01-15

    Intermittent aeration and addition of glucose were applied to horizontal subsurface flow constructed wetlands in order to investigate the effect on pathways of nitrobenzene (NB) degradation and interactions with microbial nitrogen and sulphur transformations. The experiment was carried out in three phases A, B and C consisting of different NB loading and glucose dosing. For each phase, the effect of aeration was assessed by intermittently aerating one wetland and leaving one unaerated. Regardless of whether or not the wetland was aerated, at an influent NB concentration of 140 mg/L, both wetlands significantly reduced NB to less than 2 mg/L, a reduction efficiency of 98%. However, once the influent NB concentration was increased to 280 mg/L, the aerated wetland had a higher removal performance 82% compared to that of the unaerated wetland 71%. Addition of glucose further intensified the NB removal to 95% in the aerated wetlands and 92% in the unaerated. Aeration of wetlands enhanced NB degradation, but also resulted in higher NB volatilization of 6 mg m(-2) d(-1). The detected high concentration of sulphide 20-60 mg/L in the unaerated wetland gave a strong indication that NB may act as an electron donor to sulphate-reducing bacteria, but this should be further investigated. Aeration positively improved NB removal in constructed wetlands, but resulted in higher NB volatilization. Glucose addition induced co-metabolism to enhance NB degradation. PMID:26468606

  20. Elucidation of the upper pathway of alicyclic musk Romandolide degradation in OECD screening tests with activated sludge.

    PubMed

    Seyfried, M; Boschung, A; Miffon, F; Ohleyer, E; Chaintreau, A

    2014-01-01

    The degradation of Romandolide ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO? evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for Romandolide degradation were identified using extracts at different time points of incubation. Early metabolism of Romandolide yielded ester hydrolysis products, including Cyclademol (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from Cyclademol by sludge was confirmed in subsequent experiments using Cyclademol as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of Cyclademol with concomitant degradation of the resulting acids, (2) conversion of Cyclademol into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage. PMID:24277432

  1. Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli

    PubMed Central

    Clarke, Justin E.; Kime, Louise; Romero A., David; McDowall, Kenneth J.

    2014-01-01

    Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5?-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5?-sensor mutant of RNase E in vitro that, contrary to current models, 5?-monophosphate-independent, ‘direct entry’ cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5?-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5?-monophosphorylated ends was also revealed. PMID:25237058

  2. Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli.

    PubMed

    Clarke, Justin E; Kime, Louise; Romero A, David; McDowall, Kenneth J

    2014-10-01

    Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed. PMID:25237058

  3. Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

    PubMed Central

    Kmiec, Beata; Teixeira, Pedro F.; Berntsson, Ronnie P.-A.; Murcha, Monika W.; Branca, Rui M. M.; Radomiljac, Jordan D.; Regberg, Jakob; Svensson, Linda M.; Bakali, Amin; Langel, Ülo; Lehtiö, Janne; Whelan, James; Stenmark, Pål; Glaser, Elzbieta

    2013-01-01

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8–1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å3. The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts. PMID:24043784

  4. Toll-Like Receptor Signaling Induces Nrf2 Pathway Activation through p62-Triggered Keap1 Degradation.

    PubMed

    Yin, Shasha; Cao, Wangsen

    2015-08-01

    Toll-like receptors (TLRs) induce inflammation and tissue repair through multiple signaling pathways. The Nrf2 pathway plays a key role in defending against the tissue damage incurred by microbial infection or inflammation-associated diseases. The critical event that mediates TLR-induced Nrf2 activation is still poorly understood. In this study, we found that lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists activate Nrf2 signaling and the activation is due to the reduction of Keap1, the key Nrf2 inhibitor. TLR signaling-induced Keap1 reduction promoted Nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. TLR agonists modulated Keap1 at the protein posttranslation level through autophagy. TLR signaling increased the expression of autophagy protein p62 and LC3-II and induced their association with Keap1 in the autophagosome-like structures. We also characterized the interaction between p62 and Keap1 and found that p62 is indispensable for TLR-mediated Keap1 reduction: TLR signaling had no effect on Keap1 if cells lacked p62 or if cells expressed a mutant Keap1 that could not interact with p62. Our study indicates that p62-mediated Keap1 degradation through autophagy represents a critical linkage for TLR signaling regulation of the major defense network, the Nrf2 signaling pathway. PMID:26012548

  5. Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

    SciTech Connect

    Neel, Benjamin D.; Gillet, Germain . E-mail: g.gillet@ibcp.fr

    2005-11-15

    Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60 {sup v-src} tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60 {sup v-src}, we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60 {sup v-src} inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60 {sup v-src} inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

  6. Characterization of the metabolic pathway and catabolic gene expression in biphenyl degrading marine bacterium Pseudomonas aeruginosa JP-11.

    PubMed

    Chakraborty, Jaya; Das, Surajit

    2016-02-01

    Metabolic pathway of biphenyl assimilation and the catabolic gene expression in a marine bacterium Pseudomonas aeruginosa JP-11, isolated from the coastal sediments of Odisha, India have been studied. This strain utilized 98.86% ± 2.29% of biphenyl within 72 h when supplied as the sole source of carbon, however, preferential utilization of glucose was observed over catechol and biphenyl when grown in a complex medium. Combination of chromatographic and spectrophotometric techniques confirmed the catechol pathway and identified 2-Hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate as the intermediate metabolic product. Assimilation of biphenyl was initiated by its dioxygenation, forming cis-2, 3-dihydro-2, 3-dihydroxybiphenyl subsequently transformed to 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate. In the lower pathway, cis-1, 6-dihydroxy-2, 4-cyclohexadiene-1-carboxylic acid was detected which formed catechol before entering into the Krebs cycle. Detection of key enzyme catechol-1, 2-dioxygenase in the cell-free extract of P. aeruginosa JP-11 supported the proposed degradation pathway. The primary enzyme for biphenyl assimilation, biphenyl dioxygenase encoded by bphA gene was found in the genome of the isolate. On increasing biphenyl stress (50, 100, 150 and 200 mg L(-1)), bphA gene showed a significant (P < 0.01) up-regulation upto 43.5 folds. Production of biosurfactant was confirmed and the rhamnolipid synthesizing gene rhlAB was amplified. This gene also showed a significant (P < 0.01) up-regulation upto 258 folds on increasing biphenyl stress. PMID:26519802

  7. Population sinks resulting from degraded habitats of an obligate life-history pathway.

    PubMed

    Hickford, Michael J H; Schiel, David R

    2011-05-01

    Many species traverse multiple habitats across ecosystems to complete their life histories. Degradation of critical, life stage-specific habitats can therefore lead to population bottlenecks and demographic deficits in sub-populations. The riparian zone of waterways is one of the most impacted areas of the coastal zone because of urbanisation, deforestation, farming and livestock grazing. We hypothesised that sink populations can result from alterations of habitats critical to the early life stages of diadromous fish that use this zone, and tested this with field-based sampling and experiments. We found that for Galaxias maculatus, one of the most widely distributed fishes of the southern hemisphere, obligate riparian spawning habitat was very limited and highly vulnerable to disturbance across 14 rivers in New Zealand. Eggs were laid only during spring tides, in the highest tidally influenced vegetation of waterways. Egg survival increased to >90% when laid in three riparian plant species and where stem densities were great enough to prevent desiccation, compared to no survival where vegetation was comprised of other species or was less dense. Experimental exclusion of livestock, one of the major sources of riparian degradation in rural waterways, resulted in quick regeneration, a tenfold increase in egg laying by fish and a threefold increase in survival, compared to adjacent controls. Overall, there was an inverse relationship between river size and egg production. Some of the largest rivers had little or no spawning habitat and very little egg production, effectively becoming sink populations despite supporting large adult populations, whereas some of the smallest pristine streams produced millions of eggs. We demonstrate that even a wide-ranging species with many robust adult populations can be compromised if a stage-specific habitat required to complete a life history is degraded by localised or more diffuse impacts. PMID:21076966

  8. Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin.

    PubMed

    Nabhan, Joseph F; Gooch, Renea L; Piatnitski Chekler, Eugene L; Pierce, Betsy; Bulawa, Christine E

    2015-01-01

    Friedreich's Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein. PMID:26671574

  9. Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin

    PubMed Central

    Nabhan, Joseph F.; Gooch, Renea L.; Piatnitski Chekler, Eugene L.; Pierce, Betsy; Bulawa, Christine E.

    2015-01-01

    Friedreich’s Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein. PMID:26671574

  10. Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots

    PubMed Central

    Zhang, Hongtao; Gannon, Lucy; Powers, Stephen J; Lilley, Kathryn S; Theodoulou, Frederica L

    2015-01-01

    According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719). PMID:25728785

  11. Coupled Metabolic and Photolytic Pathway for Degradation of Pyridinedicarboxylic Acids, Especially Dipicolinic Acid

    PubMed Central

    Amador, José A.; Taylor, Barrie F.

    1990-01-01

    Three isomers of pyridinedicarboxylic acid (PDCA) (2,3-, 2,5-, and 2,6-PDCA) were partially oxidized by marine bacteria when grown aerobically on the corresponding phthalate analogs. The metabolites, unlike the parent PDCAs, absorbed light in the solar actinic range (wavelengths greater than 300 nm) and were readily degraded in sunlight. The principal product from 2,6-PDCA (dipicolinic acid) metabolism was extracted from a culture fluid, purified by column chromatography, and analyzed by UV-visible, infrared, and 13C nuclear magnetic resonance spectroscopy. The compound was identified as 2,3-dihydroxypicolinic acid (2,3-DHPA). 2,3-DHPA was photolyzed in aqueous solution (pH 8.0) with a half-life of 100 min. Eight photoproducts, three of which were photolabile, were detected by high-performance liquid chromatography. Ammonia was also photoproduced from 2,3-DHPA. Analysis of the photoproducts by UV-visible spectroscopy and by high-performance liquid chromatography of 2,4-dinitrophenylhydrazones indicated that the products were conjugated carbonyls and carboxylic acids. Six of the photoproducts were readily consumed by bacterial strain CC9M. In illuminated aquatic environments, coupled bio- and photodegradative mechanisms probably contribute to the degradation of PDCAs. PMID:16348188

  12. Electrochemical degradation of sulfonamides at BDD electrode: kinetics, reaction pathway and eco-toxicity evaluation.

    PubMed

    Fabia?ska, Aleksandra; Bia?k-Bieli?ska, Anna; Stepnowski, Piotr; Stolte, Stefan; Siedlecka, Ewa Maria

    2014-09-15

    The investigation dealt with electrochemical oxidation of five sulfonamides (SNs): sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMN) and sulfadimethoxine (SDM) in aqueous solution at boron-doped diamond (BDD) electrode. All studied sulfonamides were degraded according to a pseudo first order kinetics. The structure of SNs had no significant effect on the values of pseudo first order rate constants. Increased degradation efficiency was observed in higher temperature and in acidic pH. Due to the presence of chlorine and nitrate SNs were more effectively oxidized from municipal wastewater treatment plant (WWTP) effluents than from pure supporting electrolyte Na2SO4. The intermediates identified by LC-MS and GC-MS analysis suggested that the hydroxyl radicals attack mainly the SN bond, but also the aromatic ring systems (aniline, pyrimidine or triazole) of SNs. Finally, the toxicity of the SNs solutions and effluents after electrochemical treatment was assessed through the measurement of growth inhibition of green algae (Scenedesmus vacualatus) and duckweed (Lemna minor). Toxicity of SMR, STZ, SMN solutions before and after electrochemical oxidation and SDM solution after the process in L. minor test was observed. No significant toxicity of studied SNs was observed in algae test. PMID:25215656

  13. Characterization of the KstR2 regulator responsible of the lower cholesterol degradative pathway in Mycobacterium smegmatis.

    PubMed

    García-Fernández, Julia; Galán, Beatriz; Medrano, Francisco J; García, José L

    2015-02-01

    The interaction of KstR2-dependent promoters of the divergon constituted by the MSMEG_6000-5999 and MSMEG_6001-6004 operons of Mycobacterium smegmatis which encode the genes involved in the lower cholesterol degradative pathway has been characterized. Footprint analyses have demonstrated experimentally for the first time that KstR2 specifically binds to an operator region of 29 nucleotides containing the palindromic sequence AAGCAAGNNCTTGCTT. This region overlaps with the -10 and -35 boxes of the putative P(6000) and P(6001) divergent promoters, suggesting that KstR2 represses their transcription by preventing the binding of the ribonucleic acid polymerase. A three-dimensional model of the KstR2 protein revealed a typical TetR-type regulator folding with two domains, a deoxyribonucleic acid (DNA)-binding N-terminal domain and a regulator-binding C-terminal domain composed by three and six helices respectively. KstR2 is an all alpha protein as confirmed by circular dichroism. We have determined that M. smegmatis is able to grow using sitolactone (HIL) as the only carbon source and that this compound induces the kstR2 regulon in vivo. HIL or its open form 5OH-HIP were unable to release in vitro the KstR2-DNA operator interaction, suggesting that 5OH-HIP-CoA or a further derivative would induce the lower cholesterol catabolic pathway. PMID:25511435

  14. Sources and Input Pathways of Glyphosate and its Degradation Product AMPA

    NASA Astrophysics Data System (ADS)

    Bischofberger, S.; Hanke, I.; Wittmer, I.; Singer, H.; Stamm, C.

    2009-04-01

    Despite being the pesticide used in the largest quantities worldwide, the environmental relevance of glyphosate has been considered low for many years. Reasons for this assessment were the observations that glyphosate degrades quickly into its degradation product AMPA and that it sorbs strongly to soil particles. Hence, little losses to water bodies had been expected. Research during the last few years however contradicts this expectation. Although glyphosate is a dominant pesticide used in agriculture, recent studies on other pesticides revealed that urban sources may play a significant role for water quality. Therefore this study compares glyphosate input into streams from agricultural and urban sources. For that purpose, a catchment of an area of 25 km2 was selected. It has by about 12'000 inhabitants and about 15 % of the area is used as arable land. Four sampling sites were selected in the river system in order to reflect different urban and agricultural sources. Additionally, we sampled a combined sewer overflow, a rain sewer and the outflow of a waste water treatment plant. At each site discharge was measured continuously from March to November 2007. During 16 rain events samples were taken by automatic devices at a high temporal resolution. To analyze the concentration of glyphosate and its degradation product AMPA, the samples were derivatized with FMOC-Cl at low pH conditions and then filtrated. The solid phase extraction was conducted with Strata-X sorbent cartridge. Glyphosate and AMPA were detected with API 4000 after the chromatography with X bridge column C18. To assure the data quality, interne standards of Glyphosate and AMPA were added to every sample. The limit of detection and quantification for glyphosate and AMPA are bellow 1ng/l. We analyzed two rain events at a high resolution for all stations and several events at the outlet of the catchment. We measured high glyphosate concentration in urban and agriculture dominated catchments with up to 3'600ng/l in the rainwater sewer and 2'500ng/l from agricultural origin. The highest glyphosate concentrations were detected during peak flow. The input of the waste water treatment plant (WWTP) was up to 500ng/l. We detected glyphosate above the detection limit in all samples throughout the sampling period. Even after the vegetation period in November, glyphosate peak flow concentration in the outlet of the entire catchment was 137 ng/l exceeding the Swiss water quality criteria for single pesticides. The AMPA concentrations were generally lower than those of Glyphosate except for the WWTP. Generally, AMPA concentrations varied less during a rain event than glyphosate concentrations. Despite the strong sorption to soil particles and short half-life, glyphosate occurs in high concentrations in surface waters affected by urban and agricultural sources. Concentrations were even higher than those of other widely used herbicides like atrazine and mecoprop.

  15. Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation.

    PubMed

    Cruz Viggi, Carolina; Rossetti, Simona; Fazi, Stefano; Paiano, Paola; Majone, Mauro; Aulenta, Federico

    2014-07-01

    Interspecies electron transfer mechanisms between Bacteria and Archaea play a pivotal role during methanogenic degradation of organic matter in natural and engineered anaerobic ecosystems. Growing evidence suggests that in syntrophic communities electron transfer does not rely exclusively on the exchange of diffusible molecules and energy carriers such as hydrogen or formate, rather microorganisms have the capability to exchange metabolic electrons in a more direct manner. Here, we show that supplementation of micrometer-size magnetite (Fe3O4) particles to a methanogenic sludge enhanced (up to 33%) the methane production rate from propionate, a key intermediate in the anaerobic digestion of organic matter and a model substrate to study energy-limited syntrophic communities. The stimulatory effect most probably resulted from the establishment of a direct interspecies electron transfer (DIET), based on magnetite particles serving as electron conduits between propionate-oxidizing acetogens and carbon dioxide-reducing methanogens. Theoretical calculations revealed that DIET allows electrons to be transferred among syntrophic partners at rates which are substantially higher than those attainable via interspecies H2 transfer. Besides the remarkable potential for improving anaerobic digestion, which is a proven biological strategy for renewable energy production, the herein described conduction-based DIET could also have a role in natural methane emissions from magnetite-rich soils and sediments. PMID:24901501

  16. Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells

    PubMed Central

    Perez-Neut, Mathew; Shum, Andrew; Cuevas, Bruce D.; Miller, Richard; Gentile, Saverio

    2015-01-01

    Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators. PMID:25596745

  17. Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

    PubMed Central

    Sapir, Amir; Tsur, Assaf; Koorman, Thijs; Ching, Kaitlin; Mishra, Prashant; Bardenheier, Annabelle; Podolsky, Lisa; Bening-Abu-Shach, Ulrike; Boxem, Mike; Chou, Tsui-Fen; Broday, Limor; Sternberg, Paul W.

    2014-01-01

    Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin–proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies. PMID:25187565

  18. Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity

    PubMed Central

    Arnhold, Florian; Gührs, Karl-Heinz

    2015-01-01

    Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton. PMID:25699204

  19. Proteogenomic Elucidation of the Initial Steps in the Benzene Degradation Pathway of a Novel Halophile, Arhodomonas sp. Strain Rozel, Isolated from a Hypersaline Environment

    PubMed Central

    Dalvi, Sonal; Azetsu, Sei; Patrauchan, Marianna A.; Aktas, Deniz F.

    2012-01-01

    Lately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles. PMID:22885747

  20. Finely Tuned Regulation of the Aromatic Amine Degradation Pathway in Escherichia coli

    PubMed Central

    Zeng, Ji

    2013-01-01

    FeaR is an AraC family regulator that activates transcription of the tynA and feaB genes in Escherichia coli. TynA is a periplasmic topaquinone- and copper-containing amine oxidase, and FeaB is a cytosolic NAD-linked aldehyde dehydrogenase. Phenylethylamine, tyramine, and dopamine are oxidized by TynA to the corresponding aldehydes, releasing one equivalent of H2O2 and NH3. The aldehydes can be oxidized to carboxylic acids by FeaB, and (in the case of phenylacetate) can be further degraded to enter central metabolism. Thus, phenylethylamine can be used as a carbon and nitrogen source, while tyramine and dopamine can be used only as sources of nitrogen. Using genetic, biochemical and computational approaches, we show that the FeaR binding site is a TGNCA-N8-AAA motif that occurs in 2 copies in the tynA and feaB promoters. We show that the coactivator for FeaR is the product rather than the substrate of the TynA reaction. The feaR gene is upregulated by carbon or nitrogen limitation, which we propose reflects regulation of feaR by the cyclic AMP receptor protein (CRP) and the nitrogen assimilation control protein (NAC), respectively. In carbon-limited cells grown in the presence of a TynA substrate, tynA and feaB are induced, whereas in nitrogen-limited cells, only the tynA promoter is induced. We propose that tynA and feaB expression is finely tuned to provide the FeaB activity that is required for carbon source utilization and the TynA activity required for nitrogen and carbon source utilization. PMID:24013633

  1. Finely tuned regulation of the aromatic amine degradation pathway in Escherichia coli.

    PubMed

    Zeng, Ji; Spiro, Stephen

    2013-11-01

    FeaR is an AraC family regulator that activates transcription of the tynA and feaB genes in Escherichia coli. TynA is a periplasmic topaquinone- and copper-containing amine oxidase, and FeaB is a cytosolic NAD-linked aldehyde dehydrogenase. Phenylethylamine, tyramine, and dopamine are oxidized by TynA to the corresponding aldehydes, releasing one equivalent of H2O2 and NH3. The aldehydes can be oxidized to carboxylic acids by FeaB, and (in the case of phenylacetate) can be further degraded to enter central metabolism. Thus, phenylethylamine can be used as a carbon and nitrogen source, while tyramine and dopamine can be used only as sources of nitrogen. Using genetic, biochemical and computational approaches, we show that the FeaR binding site is a TGNCA-N8-AAA motif that occurs in 2 copies in the tynA and feaB promoters. We show that the coactivator for FeaR is the product rather than the substrate of the TynA reaction. The feaR gene is upregulated by carbon or nitrogen limitation, which we propose reflects regulation of feaR by the cyclic AMP receptor protein (CRP) and the nitrogen assimilation control protein (NAC), respectively. In carbon-limited cells grown in the presence of a TynA substrate, tynA and feaB are induced, whereas in nitrogen-limited cells, only the tynA promoter is induced. We propose that tynA and feaB expression is finely tuned to provide the FeaB activity that is required for carbon source utilization and the TynA activity required for nitrogen and carbon source utilization. PMID:24013633

  2. UCH-L1 involved in regulating the degradation of EGFR and promoting malignant properties in drug-resistant breast cancer

    PubMed Central

    Jin, Yiting; Zhang, Wei; Xu, Jiawen; Wang, Hongying; Zhang, Zijing; Chu, Chengyu; Liu, Xiuping; Zou, Qiang

    2015-01-01

    Ubiquitin carboxy terminal hydrolase-L1 (UCHL1) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. Our previous research showed that UCH-L1 and EGFR could regulate the expression of P-gp, CD147 and MMPs in multi-drug resistance (MDR) breast cancer cells, respectively. But it is still unclear whether direct regulation exists between the UCH-L1 and EGFR in MDR breast cancer. In order to clarify this, MDR human breast carcinoma cell line MCF7/Adr, that expresses relatively high UCH-L1, and its parental cell line MCF7, that expresses relatively low UCH-L1, were chosen for this study. We added ubiquitin proteasome inhibitor MG-132 into the culture of MCF7/Adr cells and transfected pIRES2-UCH-L1-EGFP plasmid into MCF7 cells, respectively. Using quantitative real-time polymerase chain reaction and western blot analyses, we found accompanying over-expression of UCH-L1, EGFR was up-regulated in both MCF7/ADR and MCF7 cells. Preliminary results indicated the degradation of EGFR might be regulated by ubiquitin level. So we speculated that up-regulated UCH-L1 could promote expression level of EGFR, thereby enhance the invasion and metastasis abilities of tumor cells. Moreover, to further explore the role of UCH-L1 and EGFR, we investigated the expression of UCH-L1, EGFR and P-gp in 65 local advanced breast cancer cases by immunohistochemistry assay. The result showed that the patients not responding to chemotherapy had higher UCH-L1, EGFR and P-gp expression levels and more lymph nodes metastasis. The Kaplan-Meier survival analysis showed that the patients with elevated UCH-L1 expression after chemotherapy presented shorter overall survival and disease free survival times than those with down-regulated or unchanged expression of UCH-L1. Our findings suggest that UCH-L1 may be an indicator of chemotherapy-response and poor-survival in breast cancer. UCH-L1 might be an appropriate target for improving chemo-resistant breast cancer therapy. PMID:26722437

  3. (Per)Chlorate-Reducing Bacteria Can Utilize Aerobic and Anaerobic Pathways of Aromatic Degradation with (Per)Chlorate as an Electron Acceptor

    PubMed Central

    Carlström, Charlotte I.; Loutey, Dana; Bauer, Stefan; Clark, Iain C.; Rohde, Robert A.; Iavarone, Anthony T.; Lucas, Lauren

    2015-01-01

    ABSTRACT The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2?), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms. PMID:25805732

  4. An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K{sup +} ions

    SciTech Connect

    Ajiro, Kozo; Bortner, Carl D.; Westmoreland, Jim; Cidlowski, John A.

    2008-04-01

    Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca{sup 2+} on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca{sup 2+}-dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca{sup 2+} in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca{sup 2+} is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca{sup 2+}-induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K{sup +} inhibit both Ca{sup 2+}-dependent DNA degradation and histone release suggesting that a reduction of intracellular K{sup +} is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.

  5. Cathepsin?L Ameliorates Cardiac Hypertrophy Through Activation of the Autophagy–Lysosomal Dependent Protein Processing Pathways

    PubMed Central

    Sun, Mei; Ouzounian, Maral; de Couto, Geoffrey; Chen, Manyin; Yan, Ran; Fukuoka, Masahiro; Li, Guohua; Moon, Mark; Liu, Youan; Gramolini, Anthony; Wells, George J.; Liu, Peter P.

    2013-01-01

    Background Autophagy is critical in the maintenance of cellular protein quality control, the final step of which involves the fusion of autophagosomes with lysosomes. Cathepsin?L (CTSL) is a key member of the lysosomal protease family that is expressed in the murine and human heart, and it may play an important role in protein turnover. We hypothesized that CTSL is important in regulating protein processing in the heart, particularly under pathological stress. Methods and Results Phenylephrine?induced cardiac hypertrophy in vitro was more pronounced in CTSL?deficient neonatal cardiomyocytes than in in controls. This was accompanied by a significant accumulation of autophagosomes, increased levels of ubiquitin?conjugated protein, as well as impaired protein degradation and decreased cell viability. These effects were partially rescued with CTSL1 replacement via adeno?associated virus–mediated gene transfer. In the in vivo murine model of aortic banding (AB), a deficiency in CTSL markedly exacerbated cardiac hypertrophy, worsened cardiac function, and increased mortality. Ctsl?/? AB mice demonstrated significantly decreased lysosomal activity and increased sarcomere?associated protein aggregation. Homeostasis of the endoplasmic reticulum was also altered by CTSL deficiency, with increases in Bip and GRP94 proteins, accompanied by increased ubiquitin–proteasome system activity and higher levels of ubiquitinated proteins in response to AB. These changes ultimately led to a decrease in cellular ATP production, enhanced oxidative stress, and increased cellular apoptosis. Conclusions Lysosomal CTSL attenuates cardiac hypertrophy and preserves cardiac function through facilitation of autophagy and proteasomal protein processing. PMID:23608608

  6. Factors Affecting Biodefluorination of Fluorotelomer Alcohols (FTOHs): Degradative Microorganisms, Transformation Metabolites and Pathways, and Effects of Co-substrates 

    E-print Network

    Kim, Myung Hee 1982-

    2012-12-11

    responsible for biotransformation of FTOHs. This study deciphered factors affecting biodefluorination of FTOHs and their metabolites, and developed three effective FTOH-degrading consortia. Two alkane-degrading Pseudomonas strains (P. oleovorans and P...

  7. SIAH-1 Interacts with the Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORF45 Protein and Promotes Its Ubiquitylation and Proteasomal Degradation?

    PubMed Central

    Abada, Rinat; Dreyfuss-Grossman, Tsofia; Herman-Bachinsky, Yifat; Geva, Haim; Masa, Shiri-Rivka; Sarid, Ronit

    2008-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection. PMID:18077711

  8. Exchange Factor TBL1 and Arginine Methyltransferase PRMT6 Cooperate in Protecting G Protein Pathway Suppressor 2 (GPS2) from Proteasomal Degradation.

    PubMed

    Huang, Jiawen; Cardamone, M Dafne; Johnson, Holly E; Neault, Mathieu; Chan, Michelle; Floyd, Z Elizabeth; Mallette, Frédérick A; Perissi, Valentina

    2015-07-31

    G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNF? pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment. PMID:26070566

  9. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity

    PubMed Central

    Belogurov, Alexey; Kuzina, Ekaterina; Kudriaeva, Anna; Kononikhin, Alexey; Kovalchuk, Sergey; Surina, Yelena; Smirnov, Ivan; Lomakin, Yakov; Bacheva, Anna; Stepanov, Alexey; Karpova, Yaroslava; Lyupina, Yulia; Kharybin, Oleg; Melamed, Dobroslav; Ponomarenko, Natalia; Sharova, Natalia; Nikolaev, Eugene; Gabibov, Alexander

    2015-01-01

    Recent findings indicate that the ubiquitin–proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and ?1ihigh immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, ?1i is increased in resident CNS cells, whereas ?5i is imported by infiltrating lymphocytes through the blood–brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived ?1ihigh immunoproteasomes in vitro (kobs/[I] = 240 M?1s?1), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the ?1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.—Belogurov Jr., A., Kuzina, E., Kudriaeva, A., Kononikhin, A., Kovalchuk, S., Surina, Y., Smirnov, I., Lomakin, Y., Bacheva, A., Stepanov, A., Karpova, Y., Lyupina, Y., Kharybin, O., Melamed, D., Ponomarenko, N., Sharova, N., Nikolaev, E., Gabibov, A. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity. PMID:25634956

  10. Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

    PubMed Central

    Zattas, Dimitrios; Hochstrasser, Mark

    2014-01-01

    The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

  11. Intracellular signaling pathways modulated by phenolic compounds: application for new anti-inflammatory drugs discovery.

    PubMed

    Costa, G; Francisco, V; Lopes, M C; Cruz, M T; Batista, M T

    2012-01-01

    Extensive research within the last two decades revealed that most chronic illnesses, including cancer, neurological, autoimmune and cardiovascular diseases are mediated through chronic inflammation. Thus, suppressing chronic inflammation has the potential to delay, prevent, and treat those diseases. However, side effects and high costs of current anti-inflammatory drugs force the development of new drugs. Natural products represent an important source of new bioactive compounds. Among them, phenolic compounds, which are widely distributed in plants, have been described as having many therapeutic effects. Several reviews have addressed the anti-inflammatory activity of phenols, attributing their properties not only to the antioxidant capacity, but also to inflammatory mediators' modulation, namely cytokines and pro-inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase-2. Signal transduction pathways precede changes in inflammatory mediators' expression. However, only a restricted number of studies have addressed the effect of phenols on a specific signal transduction pathway. The present review attempts to summarize and highlight a broad range of inflammation-associated signaling pathways modulated by phenols namely: nuclear factor (NF)-?B, activator protein (AP)-1, peroxisome proliferator-activated receptor (PPAR) and nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factors; mitogen-activated protein kinases (MAPKs); protein tyrosine kinases (PTKs); tyrosine phosphatidylinositol 3-kinase (PI3K)/Akt and ubiquitin-proteasome system. As a consequence of phenols effect on signaling pathways, described above, their action on inflammatory mediators' production is mentioned. Finally, it is established that the structure-activity relationships of phenolic compounds are a valuable information source on the development of new anti-inflammatory drugs from natural products. PMID:22519398

  12. A Systems Biology Approach to Identify Molecular Pathways Altered by HDAC Inhibition in Osteosarcoma

    PubMed Central

    Wittenburg, Luke A.; Ptitsyn, Andrey A.; Thamm, Douglas H.

    2015-01-01

    Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose-intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti-tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT-PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin-proteasome among those pathways most affected by HDACi. The mitomycin C-bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi-containing combination therapies in OS. PMID:21976144

  13. Use of 13C NMR and ftir for elucidation of degradation pathways during natural litter decomposition and composting I. early stage leaf degradation

    USGS Publications Warehouse

    Wershaw, R.L.; Leenheer, J.A.; Kennedy, K.R.; Noyes, T.I.

    1996-01-01

    Oxidative degradation of plant tissue leads to the formation of natural dissolved organic carbon (DOC) and humus. Infrared (IR) and 13C nuclear magnetic resonance (NMR) spectrometry have been used to elucidate the chemical reactions of the early stages of degradation that give rise to DOC derived from litter and compost. The results of this study indicate that oxidation of the lignin components of plant tissue follows the sequence of O-demethylation, and hydroxylation followed by ring-fission, chain-shortening, and oxidative removal of substituents. Oxidative ring-fission leads to the formation of carboxylic acid groups on the cleaved ends of the rings and, in the process, transforms phenolic groups into aliphatic alcoholic groups. The carbohydrate components are broken down into aliphatic hydroxy acids and aliphatic alcohols.

  14. Removal of Acid Orange 7 in simulated wastewater using a three-dimensional electrode reactor: removal mechanisms and dye degradation pathway.

    PubMed

    Zhao, Hua-Zhang; Sun, Yan; Xu, Li-Na; Ni, Jin-Ren

    2010-01-01

    The removal of Acid Orange 7 (AO7) in simulated wastewater was experimentally investigated using a three-dimensional electrode reactor with granular activated carbon as the particle electrode, ACF (activated carbon fiber)/Fe as the anode, and ACF/Ti as the cathode. Particular attention was paid to the reaction mechanisms and the dye degradation pathway in the system. The removal of AO7 in the system was mainly dependent on the oxidation by the produced active substances (()OH, etc.) and the coagulation by Fe(II) or Fe(III) dissolved from the anode. The former mechanism was predominant. A possible pathway for AO7 degradation was proposed by monitoring the temporal evolution of intermediates in the solution, with the use of some techniques including GC/MS, FTIR and HPLC. The AO7 molecule was observed to be firstly decomposed to aromatic intermediates, further degraded to ring opening products and finally mineralized to CO(2), H(2)O and inorganic salts. The intermediates increased the biodegradability of the wastewater, which was proved by the increase of the BOD/COD value after electrolysis treatment. The three-dimensional electrode method can be considered an effective alternative to dye wastewater pretreatment prior to the biological process. PMID:19897229

  15. I?B Kinase-Independent I?B? Degradation Pathway: Functional NF-?B Activity and Implications for Cancer Therapy

    PubMed Central

    Tergaonkar, Vinay; Bottero, Virginie; Ikawa, Masahito; Li, Qiutang; Verma, Inder M.

    2003-01-01

    Antiapoptotic activity of NF-?B in tumors contributes to acquisition of resistance to chemotherapy. Degradation of I?B is a seminal step in activation of NF-?B. The I?B kinases, IKK1 and IKK2, have been implicated in both I?B degradation and subsequent modifications of NF?B. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2?/?), we document a novel I?B degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of I?B?. Degradation of I?B? is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-?B generated by DoxR-induced I?B degradation in IKK1/2?/? cells is able to activate chromatin based NF-?B reporter gene and expression of the endogenous target gene, I?B?. These results also imply that modification of NF-?B by IKK1 or IKK2 either prior or subsequent to its release from I?B is not essential for NF-?B-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2?/? MEFs is enhanced by simultaneous inhibition of NF-?B activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-?B during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy. PMID:14585967

  16. Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

    PubMed Central

    Wan, Pin-Jun; Yuan, San-Yue; Tang, Yao-Hua; Li, Kai-Long; Yang, Lu; Fu, Qiang; Li, Guo-Qing

    2015-01-01

    Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens. PMID:26000452

  17. The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway*

    PubMed Central

    Peña, Maria Marjorette O.; Melo, Sandra P.; Xing, Yang-Yang; White, Kenneth; Barbour, Karen W.; Berger, Franklin G.

    2009-01-01

    The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an ?-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N?-acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed. PMID:19797058

  18. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of ?-secretase

    PubMed Central

    Singh, Amir Kumar; Pati, Uttam

    2015-01-01

    In patient with Alzheimer’s disease (AD), deposition of amyloid-beta A?, a proteolytic cleavage of amyloid precursor protein (APP) by ?-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin–proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates A? generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing A?. CHIPUbox domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53’s DNA-binding conformation and its binding upon 5? UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP–BACE1–p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD. PMID:25773675

  19. IGF-1 promotes the growth and metastasis of hepatocellular carcinoma via the inhibition of proteasome-mediated cathepsin B degradation

    PubMed Central

    Lei, Tian; Ling, Xie

    2015-01-01

    AIM: To investigate the molecular mechanisms of the high IGF-1 level linking diabetes and cancers, which is a risk factor. METHODS: We used cell growth, wound healing and transwell assay to evaluate the proliferation and metastasis ability of the hepatocellular carcinoma (HCC) cells. Western blot and reverse transcription polymerase chain reaction were used to assess a previously identified lysosomal protease, cathepsin B (CTSB) expression in the HCC cell lines. C57 BL/6J and KK-Ay diabetic mice are used to detect the growth and metastasis of HCC cells that were depleted with or without CTSB shRNA in vivo. Statistical significance was determined by Student’s t-test. RESULTS: IGF-1 promoted the growth and metastasis of the HCC cell lines via its ability to enhance CTSB expression in both a time-dependent and concentration-dependent manner. HCC cells grew much faster in diabetic KK-Ay mice than in C57 BL/6J mice. Additionally, more metastatic nodules were found in the lungs of KK-Ay mice than the lungs of C57 BL/6J mice. CTSB depletion protects against the tumor-promoting actions of IGF-1 in HCC cells, as well tumor growth and metastasis both in vitro and in vivo. IGF-1 did not change the mRNA levels of CTSB but prolonged the half-life of cathepsin B in Hepa 1-6 and H22 cells. Our results showed that IGF-1 promotes the growth and metastasis of the HCC cells most likely by hindering CTSB degradation mediated by the ubiquitin-proteasome system (UPS), but not autophagy. Overexpression of proteasome activator 28, a family of activators of the 20S proteasome, could not only restore IGF-1-inhibited UPS activity but also decrease IGF-1-induced CTSB accumulation. CONCLUSION: Our study demonstrates that IGF-1 promotes the growth and metastasis of hepatocellular carcinoma by inhibition of proteasome-mediated CTSB degradation. PMID:26401078

  20. Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation

    SciTech Connect

    Salinero, Kennan Kellaris; Keller, Keith; Feil, William S.; Feil, Helene; Trong, Stephan; Di Bartolo, Genevieve; Lapidus, Alla

    2008-11-17

    Initial interest in Dechloromonas aromatica strain RCB arose from its ability to anaerobically degrade benzene. It is also able to reduce perchlorate and oxidize chlorobenzoate, toluene, and xylene, creating interest in using this organism for bioremediation. Little physiological data has been published for this microbe. It is considered to be a free-living organism. The a priori prediction that the D. aromatica genome would contain previously characterized 'central' enzymes involved in anaerobic aromatic degradation proved to be false, suggesting the presence of novel anaerobic aromatic degradation pathways in this species. These missing pathways include the benzyl succinyl synthase (bssABC) genes (responsible for formate addition to toluene) and the central benzoylCoA pathway for monoaromatics. In depth analyses using existing TIGRfam, COG, and InterPro models, and the creation of de novo HMM models, indicate a highly complex lifestyle with a large number of environmental sensors and signaling pathways, including a relatively large number of GGDEF domain signal receptors and multiple quorum sensors. A number of proteins indicate interactions with an as yet unknown host, as indicated by the presence of predicted cell host remodeling enzymes, effector enzymes, hemolysin-like proteins, adhesins, NO reductase, and both type III and type VI secretory complexes. Evidence of biofilm formation including a proposed exopolysaccharide complex with the somewhat rare exosortase (epsH), is also present. Annotation described in this paper also reveals evidence for several metabolic pathways that have yet to be observed experimentally, including a sulphur oxidation (soxFCDYZAXB) gene cluster, Calvin cycle enzymes, and nitrogen fixation (including RubisCo, ribulose-phosphate 3-epimerase, and nif gene families, respectively). Analysis of the D. aromatica genome indicates there is much to be learned regarding the metabolic capabilities, and life-style, for this microbial species. Examples of recent gene duplication events in signaling as well as dioxygenase clusters are present, indicating selective gene family expansion as a relatively recent event in D. aromatica's evolutionary history. Gene families that constitute metabolic cycles presumed to create D. aromatica's environmental 'foot-print' indicate a high level of diversification between its predicted capabilities and those of its close relatives, A. aromaticum str EbN1 and Azoarcus BH72.

  1. Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture.

    PubMed

    Duetz, W A; Marqués, S; Wind, B; Ramos, J L; van Andel, J G

    1996-02-01

    In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h-1) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway. When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source. PMID:8593060

  2. Effects of 1-MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage.

    PubMed

    Cheng, Yudou; Dong, Yu; Yan, Hongbo; Ge, Wenya; Shen, Chengguo; Guan, Junfeng; Liu, Liqin; Zhang, Yingying

    2012-11-15

    The peel yellowing is an important pigment physiological process of green fruit ripening, which mainly results from chlorophyll degradation in the fruit peel. In this work, two typical cultivars with different ripening speed, a slow ripening pear 'Emerald' (Pyrus bretschneideri Rehd. cv. Emerald) and a fast ripening 'Jingbai' (Pyrus ussuriensis Maxim. cv. Jingbai) were used to investigate the molecular mechanism of chlorophyll degradation in pear yellowing/ripening during postharvest storage. The fruits after harvest were treated with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor at 1.0 ?Ll(-1) to determine its effect on chloroplast ultrastructure and the expression of chlorophyll degradation associated genes in peel tissues. Our results show that the pears treated with 1-MCP had a lower ethylene production rate and higher chlorophyll content compared to those of untreated fruit. The more intact chloroplasts with well-organised grana thylakoids and small plastoglobuli were maintained in the peel of 1-MCP treated fruit for up to 30 and 15 d in 'Emerald' and 'Jingbai', respectively. The expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO), non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR) in both cultivar fruits treated with 1-MCP. These results suggest that 1-MCP can delay chlorophyll degradation by inhibiting ethylene production and suppressing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway. PMID:22868108

  3. The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l-Phenylalanine, l-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

    PubMed Central

    Arias-Barrau, Elsa; Olivera, Elías R.; Luengo, José M.; Fernández, Cristina; Galán, Beatriz; García, José L.; Díaz, Eduardo; Miñambres, Baltasar

    2004-01-01

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position ?16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds. PMID:15262943

  4. Skeletal muscle wasting with disuse atrophy is multi-dimensional: the response and interaction of myonuclei, satellite cells and signaling pathways

    PubMed Central

    Brooks, Naomi E.; Myburgh, Kathryn H.

    2014-01-01

    Maintenance of skeletal muscle is essential for health and survival. There are marked losses of skeletal muscle mass as well as strength and physiological function under conditions of low mechanical load, such as space flight, as well as ground based models such as bed rest, immobilization, disuse, and various animal models. Disuse atrophy is caused by mechanical unloading of muscle and this leads to reduced muscle mass without fiber attrition. Skeletal muscle stem cells (satellite cells) and myonuclei are integrally involved in skeletal muscle responses to environmental changes that induce atrophy. Myonuclear domain size is influenced differently in fast and slow twitch muscle, but also by different models of muscle wasting, a factor that is not yet understood. Although the myonuclear domain is 3-dimensional this is rarely considered. Apoptosis as a mechanism for myonuclear loss with atrophy is controversial, whereas cell death of satellite cells has not been considered. Molecular signals such as myostatin/SMAD pathway, MAFbx, and MuRF1 E3 ligases of the ubiquitin proteasome pathway and IGF1-AKT-mTOR pathway are 3 distinctly different contributors to skeletal muscle protein adaptation to disuse. Molecular signaling pathways activated in muscle fibers by disuse are rarely considered within satellite cells themselves despite similar exposure to unloading or low mechanical load. These molecular pathways interact with each other during atrophy and also when various interventions are applied that could alleviate atrophy. Re-applying mechanical load is an obvious method to restore muscle mass, however how nutrient supplementation (e.g., amino acids) may further enhance recovery (or reduce atrophy despite unloading or ageing) is currently of great interest. Satellite cells are particularly responsive to myostatin and to growth factors. Recently, the hibernating squirrel has been identified as an innovative model to study resistance to atrophy. PMID:24672488

  5. Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Model

    PubMed Central

    Genevini, Paola; Papiani, Giulia; Ruggiano, Annamaria; Cantoni, Lavinia; Navone, Francesca; Borgese, Nica

    2014-01-01

    VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3?, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3? or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency. PMID:25409455

  6. Degradation pathway and kinetic analysis for p-xylene removal by a novel Pandoraea sp. strain WL1 and its application in a biotrickling filter.

    PubMed

    Wang, Xiangqian; Wang, Qiaoli; Li, Sujing; Li, Wei

    2015-05-15

    In this study, a novel Pandoraea sp. strain WL1 capable of mineralizing p-xylene as sole carbon and energy source was isolated from the activated sludge of a pharmaceutical wastewater treatment plant. A nearly complete degradation of 16.6?99.4 mg L(-1)p-xylene in the liquid-phase was achieved within 6?18 h accompanied by 15.9?56.3 mg dry cell weight (DCW)L(-1) for bacterial growth. A complete pathway for p-xylene degradation by strain WL1 was presented through identification of a major intermediate (p-toluic acid) and final products (2.193 gCO2 gp-xylene(-1) of CO? production and 0.215 g DCW gp-xylene(-1) of bacterial yield). Kinetics of bacterial growth and p-xylene degradation were evaluated using Haldane-Andrews model and pseudo first-order model, respectively. Furthermore, a biotrickling filter (BTF) was employed to evaluate the application of strain WL1 on the removal of gas-phase p-xylene under gas flow rates of 0.41?1.98 m(3)h(-1) for inlet loading rates of 5?248 gm(-3)h(-1). The BTF inoculated with strain WL1 proved to be robust against fluctuations of gas flow rates and inlet p-xylene concentrations. All the results obtained highlight the potential of strain WL1 for the treatment of p-xylene. PMID:25682514

  7. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    SciTech Connect

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-09-15

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

  8. Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes

    PubMed Central

    Paradkar, Prasad N.; Duchemin, Jean-Bernard; Rodriguez-Andres, Julio; Trinidad, Lee; Walker, Peter J.

    2015-01-01

    Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication. PMID:26325027

  9. Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Kapeli, Katannya; Hurlin, Peter J.

    2011-01-01

    Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-RasG12V) in very different ways. H-RasG12V promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-RasG12V-mediated destabilization of N-Myc. We show that H-RasG12V-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis. PMID:21908617

  10. Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-? protein turnover

    PubMed Central

    Zhang, P; Gao, K; Jin, X; Ma, J; Peng, J; Wumaier, R; Tang, Y; Zhang, Y; An, J; Yan, Q; Dong, Y; Huang, H; Yu, L; Wang, C

    2015-01-01

    Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7–10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-? (ER?), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ER?, and triggers ER? degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ER? degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ER? degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ER? protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer. PMID:25766326

  11. Potentiality of yeast Candida sp. SMN04 for degradation of cefdinir, a cephalosporin antibiotic: kinetics, enzyme analysis and biodegradation pathway.

    PubMed

    Selvi, A; Das, Devlina; Das, Nilanjana

    2015-12-01

    A new yeast strain isolated from the pharmaceutical wastewater was capable of utilizing cefdinir as a sole carbon source for their growth in mineral medium. The yeast was identified and named as Candida sp. SMN04 based on morphology and 18S-ITS-D1/D2/D3 rRNA sequence analysis. The interaction between factors pH (3.0-9.0), inoculum dosage (1-7%), time (1-11 day) and cefdinir concentration (50-450?mg/L) was studied using a Box-Behnken design. The factors were studied as a result of their effect on cell dry weight (R1; g/L), extended spectrum ?-lactamase (ESBL) assay (R2; mm), P450 activity (R3; U/mL) and degradation (R4; %). Maximum values of R1, R2, R3 and R4 were obtained at central values of all the parameters. The isolated yeast strain efficiently degraded 84% of 250?mg?L(-1) of cefdinir within 6 days with a half-life of 2.97 days and degradation rate constant of 0.2335 per day. Pseudo-first-order model efficiently described the process. Among the various enzymes tested, the order of activity at the end of Day 4 was noted to be: cytochrome P450 (1.76?±?0.03)?>?NADPH reductase (1.51?±?0.20)?>?manganese peroxidase and amylase (0.66?±?0.15; 0.66?±?0.70). Intermediates were successfully characterized by liquid chromatography-mass spectrometry. The opening of the ?-lactam ring involving ESBL activity was considered as one of the major steps in the cefdinir degradation process. Fourier transform-infrared spectroscopy analysis showed the absence of spectral vibrations between 1766 and 1519?cm(-1) confirming the complete removal of lactam ring during cefdinir degradation. The results of the present study are promising for the use of isolated yeast Candida sp. SMN04 as a potential bioremediation agent. PMID:26000889

  12. Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: new insights into the degradation pathway.

    PubMed

    Annamaria, Halasz; Manno, Dominic; Strand, Stuart E; Bruce, Neil C; Hawari, Jalal

    2010-12-15

    Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) aerobically via initial denitration. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using (18)O(2) and H(2)(18)O labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) with DN22 under similar conditions. DN22 degraded RDX and MNX giving NO(2)(-), NO(3)(-), NDAB (4-nitro-diazabutanal), NH(3), N(2)O, and HCHO with NO(2)(-)/NO(3)(-) molar ratio reaching 17 and ca. 2, respectively. In the presence of (18)O(2), DN22 degraded RDX and produced NO(2)(-) with m/z at 46 Da that subsequently oxidized to NO(3)(-) containing one (18)O atom, but in the presence of H(2)(18)O we detected NO(3)(-) without (18)O. A control containing NO(2)(-), DN22, and (18)O(2) gave NO(3)(-) with one (18)O, confirming biotic oxidation of NO(2)(-) to NO(3)(-). Treatment of MNX with DN22 and (18)O(2) produced NO(3)(-) with two mass ions, one (66 Da) incorporating two (18)O atoms and another (64 Da) incorporating only one (18)O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO(2)(-), respectively. In the presence of H(2)(18)O we detected NO(2)(-) with two different masses, one representing NO(2)(-) (46 Da) and another representing NO(2)(-) (48 Da) with the inclusion of one (18)O atom suggesting auto-oxidation of NO to NO(2)(-). Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO(2)(-). PMID:21105645

  13. TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

    PubMed Central

    Xu, W; Wang, S; Chen, Q; Zhang, Y; Ni, P; Wu, X; Zhang, J; Qiang, F; Li, A; Røe, O D; Xu, S; Wang, M; Zhang, R; Zhou, J

    2014-01-01

    Cisplatin is a cytotoxic platinum compound that triggers DNA crosslinking induced cell death, and is one of the reference drugs used in the treatment of several types of human cancers including gastric cancer. However, intrinsic or acquired drug resistance to cisplatin is very common, and leading to treatment failure. We have recently shown that reduced expression of base excision repair protein XRCC1 (X-ray repair cross complementing group1) in gastric cancerous tissues correlates with a significant survival benefit from adjuvant first-line platinum-based chemotherapy. In this study, we demonstrated the role of XRCC1 in repair of cisplatin-induced DNA lesions and acquired cisplatin resistance in gastric cancer by using cisplatin-sensitive gastric cancer cell lines BGC823 and the cisplatin-resistant gastric cancer cell lines BGC823/cis-diamminedichloridoplatinum(II) (DDP). Our results indicated that the protein expression of XRCC1 was significantly increased in cisplatin-resistant cells and independently contributed to cisplatin resistance. Irinotecan, another chemotherapeutic agent to induce DNA damaging used to treat patients with advanced gastric cancer that progressed on cisplatin, was found to inhibit the expression of XRCC1 effectively, and leading to an increase in the sensitivity of resistant cells to cisplatin. Our proteomic studies further identified a cofactor of 26S proteasome, the thioredoxin-like protein 1 (TXNL1) that downregulated XRCC1 in BGC823/DDP cells via the ubiquitin-proteasome pathway. In conclusion, the TXNL1-XRCC1 is a novel regulatory pathway that has an independent role in cisplatin resistance, indicating a putative drug target for reversing cisplatin resistance in gastric cancer. PMID:24525731

  14. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    SciTech Connect

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung; Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho; Lee, Gye Won; Yun, Mi-Young; Cuong, Nguyen Manh; Shin, Jae-Gook; Song, Gyu-Yong; Oh, Sangtaek

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  15. Regulating the ethylene response of a plant by modulation of F-box proteins

    DOEpatents

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2014-01-07

    The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

  16. [Translocation of MuRF isoforms to nuclei in postural muscle during gravitational unloading].

    PubMed

    Lomonosova, Iu N; Turtikova, O V; Shenkman, B S

    2015-01-01

    Ubiquitin-proteasomal proteolytic pathway is one of the key signaling pathways determining protein degradation in muscle fibers. Among the E3 ubiquitin ligases, rate limiting enzymes of the ubiquitin-proteasomal pathway, the most interesting ones are the MuRF isoforms: MuRF-1 and MuRF-2. There are some pieces of evidence that these enzymes are also involved in the regulation of gene expression in skeletal muscle under some specific conditions (i. e. muscle disuse). We supposed that it was disuse that brought about to altered localization of MuRFs in postural muscle fibers and their translocation to nuclei. In the study using the conventional simulation model of the gravitational unloading (rat hindlimb suspension according to Ilyin and Novikov modified by Morey-Holton) we found that from the 3rd day till 14th day of unloading the content of MuRF-1 and MuRF-2 in the nuclear fraction 4-5 fold increased in unloaded soleus as compared to the control values. These data obtained by means of electrophoresis and western blot of the nuclear fraction of rat soleus were confirmed in the immunohistochemical study of co-localization of MuRF-1 and MuRF-2 antibodies and DAPI nuclear stain on transverse frozen sections of soleus muscle. Thus in the present study we observed the phenomenon of MuRF isoforms accumulation in nuclei of soleus muscle fibers during simulated gravitational unloading. PMID:25868327

  17. The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

    PubMed Central

    David-Kadoch, Galit; Hazan, Rotem; Yu, Tzenlin; Glickman, Michael H.; Ben-Aroya, Shay

    2015-01-01

    Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments. PMID:25919710

  18. Myostatin Induces Insulin Resistance via Casitas B-Lineage Lymphoma b (Cblb)-mediated Degradation of Insulin Receptor Substrate 1 (IRS1) Protein in Response to High Calorie Diet Intake*

    PubMed Central

    Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

    2014-01-01

    To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat- mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner. PMID:24451368

  19. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro; Hayashi, Hidetoshi; Itoh, Saotomo; Takii, Takemasa; Onozaki, Kikuo

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  20. Induced Degradation of Tat by Nucleocapsid (NC) via the Proteasome Pathway and Its Effect on HIV Transcription

    PubMed Central

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-01-01

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation. PMID:23611845

  1. Miswiring the brain: ?9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway

    PubMed Central

    Tortoriello, Giuseppe; Morris, Claudia V; Alpar, Alan; Fuzik, Janos; Shirran, Sally L; Calvigioni, Daniela; Keimpema, Erik; Botting, Catherine H; Reinecke, Kirstin; Herdegen, Thomas; Courtney, Michael; Hurd, Yasmin L; Harkany, Tibor

    2014-01-01

    Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly ?9-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring. PMID:24469251

  2. A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway.

    PubMed

    Bahar, M; de Majnik, J; Wexler, M; Fry, J; Poole, P S; Murphy, P J

    1998-11-01

    Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway. PMID:9805393

  3. Two enzymes of a complete degradation pathway for linear alkylbenzenesulfonate (LAS) surfactants: 4-sulfoacetophenone Baeyer-Villiger monooxygenase and 4-sulfophenylacetate esterase in Comamonas testosteroni KF-1.

    PubMed

    Weiss, Michael; Denger, Karin; Huhn, Thomas; Schleheck, David

    2012-12-01

    Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C(4)-SPC). Second, these SPCs are mineralized. 3-C(4)-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C(4)-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria. PMID:23001656

  4. Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1

    PubMed Central

    Weiss, Michael; Denger, Karin; Huhn, Thomas

    2012-01-01

    Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria. PMID:23001656

  5. Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway.

    PubMed

    Duffner, F M; Kirchner, U; Bauer, M P; Müller, R

    2000-10-01

    Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400. PMID:11054550

  6. Diphenylarsinic acid contaminated soil remediation by titanium dioxide (P25) photocatalysis: Degradation pathway, optimization of operating parameters and effects of soil properties.

    PubMed

    Wang, A-Nan; Teng, Ying; Hu, Xue-Feng; Wu, Long-Hua; Huang, Yu-Juan; Luo, Yong-Ming; Christie, Peter

    2016-01-15

    Diphenylarsinic acid (DPAA) is formed during the leakage of arsenic chemical weapons in sites and poses a high risk to biota. However, remediation methods for DPAA contaminated soils are rare. Here, the photocatalytic oxidation (PCO) process by nano-sized titanium dioxide (TiO2) was applied to degrade DPAA in soil. The degradation pathway was firstly studied, and arsenate was identified as the final product. Then, an orthogonal array experimental design of L9(3)(4), only 9 experiments were needed, instead of 81 experiments in a conventional one-factor-at-a-time, was used to optimize the operational parameters soil:water ratio, TiO2 dosage, irradiation time and light intensity to increase DPAA removal efficiency. Soil:water ratio was found to have a more significant effect on DPAA removal efficiency than other properties. The optimum conditions to treat 4g soil with a DPAA concentration of 20mgkg(-1) were found to be a 1:10 soil: water ratio, 40mWcm(-2) light intensity, 5% TiO2 in soil, and a 3-hour irradiation time, with a removal efficiency of up to 82.7%. Furthermore, this method (except for a change in irradiation time from 3 to 1.5h) was validated in nine different soils and the removal efficiencies ranged from 57.0 to 78.6%. Removal efficiencies were found to be negatively correlated with soil electrical conductivity, organic matter content, pH and total phosphorus content. Finally, coupled with electron spin resonance (ESR) measurement, these soil properties affected the generation of OH• by TiO2 in soil slurry. This study suggests that TiO2 photocatalytic oxidation is a promising treatment for removing DPAA from soil. PMID:26410709

  7. High quality draft genome sequence of Olivibacter sitiensis type strain (AW-6T), a diphenol degrader with genes involved in the catechol pathway

    PubMed Central

    Ntougias, Spyridon; Lapidus, Alla; Han, James; Mavromatis, Konstantinos; Pati, Amrita; Chen, Amy; Klenk, Hans-Peter; Woyke, Tanja; Fasseas, Constantinos; Kyrpides, Nikos C.; Zervakis, Georgios I.

    2014-01-01

    Olivibacter sitiensis Ntougias et al. 2007 is a member of the family Sphingobacteriaceae, phylum Bacteroidetes. Members of the genus Olivibacter are phylogenetically diverse and of significant interest. They occur in diverse habitats, such as rhizosphere and contaminated soils, viscous wastes, composts, biofilter clean-up facilities on contaminated sites and cave environments, and they are involved in the degradation of complex and toxic compounds. Here we describe the features of O. sitiensis AW-6T, together with the permanent-draft genome sequence and annotation. The organism was sequenced under the Genomic Encyclopedia for Bacteria and Archaea (GEBA) project at the DOE Joint Genome Institute and is the first genome sequence of a species within the genus Olivibacter. The genome is 5,053,571 bp long and is comprised of 110 scaffolds with an average GC content of 44.61%. Of the 4,565 genes predicted, 4,501 were protein-coding genes and 64 were RNA genes. Most protein-coding genes (68.52%) were assigned to a putative function. The identification of 2-keto-4-pentenoate hydratase/2-oxohepta-3-ene-1,7-dioic acid hydratase-coding genes indicates involvement of this organism in the catechol catabolic pathway. In addition, genes encoding for ?-1,4-xylanases and ?-1,4-xylosidases reveal the xylanolytic action of O. sitiensis. PMID:25197463

  8. The Drosophila insulin-degrading enzyme restricts growth by modulating the PI3K pathway in a cell-autonomous manner

    PubMed Central

    Galagovsky, Diego; Katz, Maximiliano J.; Acevedo, Julieta M.; Sorianello, Eleonora; Glavic, Alvaro; Wappner, Pablo

    2014-01-01

    Mammalian insulin-degrading enzyme (IDE) cleaves insulin, among other peptidic substrates, but its function in insulin signaling is elusive. We use the Drosophila system to define the function of IDE in the regulation of growth and metabolism. We find that either loss or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell-autonomous manner by affecting both cell size and cell number. dIDE can modulate Drosophila insulin-like peptide 2 levels, thereby restricting activation of the phosphatidylinositol-3-phosphate kinase pathway and promoting activation of Drosophila forkhead box, subgroup O transcription factor. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. We find that dIDE loss of function exacerbates this phenotype and that mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. We propose that dIDE is a modulator of insulin signaling and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II. PMID:24430872

  9. Epigallocatechin-3-gallate (EGCG) for Clinical Trials: More Pitfalls than Promises?

    PubMed Central

    Mereles, Derliz; Hunstein, Werner

    2011-01-01

    Epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, has shown numerous health promoting effects acting through different pathways, as antioxidant, anti-inflammatory and anti-atherogenic agent, showing gene expression activity, functioning through growth factor-mediated pathways, the mitogen-activated protein kinase-dependent pathway, the ubiquitin/proteasome degradation pathway, as well as eliciting an amyloid protein remodeling activity. However, epidemiological inferences are sometimes conflicting and in vitro and in vivo studies may seem discrepant. Current knowledge on how to enhance bioavailability could be the answer to some of these issues. Furthermore, dose levels, administration frequency and potential side effects remain to be examined. PMID:22016611

  10. Efficient Degradation of TCE in Groundwater Using Pd and Electro-generated H2 and O2: A Shift in Pathway from Hydrodechlorination to Oxidation in the Presence of Ferrous Ions

    PubMed Central

    Yuan, Songhu; Mao, Xuhui; Alshawabkeh, Akram N.

    2012-01-01

    Degradation of trichloroethylene (TCE) in simulated groundwater by Pd and electro-generated H2 and O2 is investigated in the absence and presence of Fe(II). In the absence of Fe(II), hydrodechlorination dominates TCE degradation, with accumulation of H2O2 up to 17 mg/L. Under weak acidity, low concentrations of oxidizing •OH radical are detected due to decomposition of H2O2, slightly contributing to TCE degradation via oxidation. In the presence of Fe(II), the degradation efficiency of TCE at 396 ?M improves to 94.9% within 80 min. The product distribution proves that the degradation pathway shifts from 79% hydrodechlorination in the absence of Fe(II) to 84% •OH oxidation in the presence of Fe(II). TCE degradation follows zeroth-order kinetics with rate constants increasing from 2.0 to 4.6 ?M/min with increasing initial Fe(II) concentration from 0 to 27.3 mg/L at pH 4. A good correlation between TCE degradation rate constants and •OH generation rate constants confirms that •OH is the predominant reactive species for TCE oxidation. Presence of 10 mM Na2SO4, NaCl, NaNO3, NaHCO3, K2SO4, CaSO4 and MgSO4 does not significantly influence degradation, but sulfite and sulfide greatly enhance and slightly suppresses degradation, respectively. A novel Pd-based electrochemical process is proposed for groundwater remediation. PMID:22315993

  11. Cathepsin D in a murine model of frontotemporal dementia with Parkinsonism-linked to chromosome 17.

    PubMed

    Fernández-Montoya, Julia; Pérez, Mar

    2015-01-01

    Tauopathies, such as Alzheimer's disease (AD) and Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), are characterized by tau accumulation. This accumulation could result from alterations in tau degradation by either the ubiquitin-proteasome system or the autophagy-lysosomal pathway. To analyze a possible alteration of the autophagy-lysosomal pathway in transgenic mice expressing human tau with three FTDP-17 missense mutations (TauVLW mice), we studied the lysosomal enzyme Cathepsin D. The hippocampi of TauVLW mice, where the human mutant tau accumulates, showed both increased Cathepsin D and partial colocalization of Cathepsin D with human mutant tau. At the ultrastructural level, some multivesicular bodies showed human mutant tau-immunopositive vesicles. This finding could provide insights into the molecular mechanisms of tau degradation in human tauopathies. PMID:25428250

  12. Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines.

    PubMed

    Jivan, Rupal; Damelin, Leonard Howard; Birkhead, Monica; Rousseau, Amanda Louise; Veale, Robin Bruce; Mavri-Damelin, Demetra

    2015-10-01

    Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC. PMID:25846272

  13. Patchwork Assembly of nag-Like Nitroarene Dioxygenase Genes and the 3-Chlorocatechol Degradation Cluster for Evolution of the 2-Chloronitrobenzene Catabolism Pathway in Pseudomonas stutzeri ZWLR2-1?

    PubMed Central

    Liu, Hong; Wang, Shu-Jun; Zhang, Jun-Jie; Dai, Hui; Tang, Huiru; Zhou, Ning-Yi

    2011-01-01

    Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective ? and ? subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals. PMID:21602392

  14. Degradation Kinetics of VX

    SciTech Connect

    Gary S. Groenewold

    2010-12-01

    O-ethyl S-(2-diisopropylaminoethyl)phosphonothiolate (VX) is the most toxic of the conventional chemical warfare agents. It is a persistent compound, an attribute derived from its relative involatility and slow rates of hydrolysis. These properties suggest that VX can linger in an exposed environment for extended periods of time long after the air has cleared. Concern over prolonged risk from VX exposure is exacerbated by the fact that it poses a dermal contact hazard. Hence a detailed understanding of volatilization rates, and degradation pathways and rates occurring in various environments is needed. Historically, volatilization has not been considered to be an important mechanism for VX depletion, but recent studies have shown that a significant fraction of VX may volatilize, depending on the matrix. A significant body of research has been conducted over the years to unravel VX degradation reaction pathways and to quantify the rates at which they proceed. Rigorous measurement of degradation rates is frequently difficult, and thus in many cases the degradation of VX has been described in terms of half lives, while in fewer instances rate constants have been measured. This variable approach to describing degradation kinetics reflects uncertainty regarding the exact nature of the degradation mechanisms. In this review, rates of VX degradation are compared on the basis of pseudo-first order rate constants, in order to provide a basis for assessing likelihood of VX persistence in a given environment. An issue of specific concern is that one VX degradation pathway produces S-2-(diisopropylaminoethyl) methylphosphonothioic acid (known as EA2192), which is a degradation product that retains much of the original toxicity of VX. Consequently degradation pathways and rates for EA2192 are also discussed.

  15. Alkaline hydrolysis of the cyclic nitramine explosives RDX, HMX, and CL-20: new insights into degradation pathways obtained by the observation of novel intermediates.

    PubMed

    Balakrishnan, Vimal K; Halasz, Annamaria; Hawari, Jalal

    2003-05-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, I) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) hydrolyze at pH > 10 to form end products including NO2-, HCHO, HCOOH, NH3, and N2O, but little information is available on intermediates, apart from the tentatively identified pentahydro-3,5-dinitro-1,3,5-triazacyclohex-1-ene (II). Despite suggestions that RDX and HMX contaminated groundwater could be economically treated via alkaline hydrolysis, the optimization of such a process requires more detailed knowledge of intermediates and degradation pathways. In this study, we hydrolyzed the monocyclic nitramines RDX, MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), and HMX in aqueous solution (pH 10-12.3) and found that nitramine removal was accompanied by formation of 1 molar equiv of nitrite and the accumulation of the key ring cleavage product 4-nitro-2,4-diazabutanal (4-NDAB, O2NNHCH2NHCHO). Most of the remaining C and N content of RDX, MNX, and HMX was found in HCHO, N2O, HCOOH, and NH3. Consequently, we selected RDX as a model compound and hydrolyzed it in aqueous acetonitrile solutions (pH 12.3) in the presence and absence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to explore other early intermediates in more detail. We observed a transient LC-MS peak with a [M-H] at 192 Da that was tentatively identified as 4,6-dinitro-2,4,6-triaza-hexanal (O2NNHCH2NNO2CH2NHCHO, III) considered as the hydrolyzed product of II. In addition, we detected another novel intermediate with a [M-H] at 148 Da that was tentatively identified as a hydrolyzed product of III, namely, 5-hydroxy-4-nitro-2,4-diaza-pentanal (HOCH2NNO2CH2NHCHO, IV). Both III and IV can act as precursors to 4-NDAB. In the case of the polycyclic nitramine 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), denitration (two NO2-) also led to the formation of HCOOH, NH3, and N2O, but neither HCHO nor 4-NDAB were detected. The results provide strong evidence that initial denitration of cyclic nitramines in water is sufficient to cause ring cleavage followed by spontaneous decomposition to form the final products. PMID:12775055

  16. Ataxin-3 like (ATXN3L), a member of the Josephin family of deubiquitinating enzymes, promotes breast cancer proliferation by deubiquitinating Krüppel-like factor 5 (KLF5)

    PubMed Central

    Zhou, Zhongmei; Liu, Rong; Liu, Linlin; Tan, Jing; Zou, Tianning; Li, Hongyuan; Ren, Guosheng; Chen, Ceshi

    2015-01-01

    The Krüppel-like factor 5 (KLF5) has been suggested to promote breast cell proliferation, survival and tumorigenesis. KLF5 protein degradation is increased by several E3 ubiquitin ligases, including WWP1 and SCFFbw7, through the ubiquitin-proteasome pathway. However, the deubiquitinase (DUB) of KLF5 has not been demonstrated. In this study, we identified ATXN3L as a KLF5 DUB by genome-wide siRNA screening. ATXN3L directly binds to KLF5, decreasing its ubiquitination and thus degradation. Functionally, knockdown of ATXN3L inhibits breast cancer cell proliferation partially through KLF5. These findings reveal a previously unrecognized role of ATXN3L in the regulation of KLF5 stability in breast cancer. ATXN3L might be a therapeutic target for breast cancer. PMID:26079537

  17. Integration of multiple ubiquitin signals in proteasome regulation.

    PubMed

    Isasa, Marta; Zuin, Alice; Crosas, Bernat

    2012-01-01

    The ubiquitin-proteasome system has emerged in the last decades as a new paradigm in cell physiology. Ubiquitin is found in fundamental levels of cell regulation, as a target for degradation to the proteasome or as a signal that controls protein function in a complex manner. Even though many aspects of the ubiquitin system remain unexplored, the contributions on the field uncover that ubiquitin represents one of the most sophisticated codes in cellular biology. The proteasome is an ATP-dependent protease that degrades a large number of protein substrates in the cell. The proteasome recruits substrates by a number of receptors that interact with polyubiquitin. Recently, it has been shown that one of these receptors, Rpn10, is regulated by monoubiquitination. In this chapter, we show an overview of the central aspects of the pathway and describe the methodology to characterize in vitro the monoubiquitination of proteasome subunits. PMID:22821603

  18. Proteasome regulates the mediators of cytoplasmic polyadenylation signaling during late-phase long-term potentiation.

    PubMed

    Dong, Chenghai; Vashisht, Anirudh; Hegde, Ashok N

    2014-11-01

    The ubiquitin-proteasome pathway is essential for long-term synaptic plasticity, but its exact roles remain unclear. Previously we established that proteasome inhibition increased the early, induction part of late-phase long-term potentiation (L-LTP) but blocks the late, maintenance part. Our prior work also showed that the proteasome modulates components of the mammalian target of rapamycin pathway for translation. In this study, we tested the possible role of the proteasome in regulating the cytoplasmic polyadenylation signaling required for translation during L-LTP. We found that a polyadenylation inhibitor cordycepin diminishes the enhancement of early L-LTP mediated by proteasome inhibition. Furthermore, blocking Aurora-A kinase and calcium-calmodulin-dependent kinase II reduces the increase in early L-LTP brought about by proteasome inhibition. Our results suggest a link between polyadenylation-mediated translational control and protein degradation during induction of long-term synaptic plasticity. PMID:25263789

  19. Composition, Roles, and Regulation of Cullin-Based Ubiquitin E3 Ligases

    PubMed Central

    Choi, Christina M.; Gray, William M.; Mooney, Sutton; Hellmann, Hanjo

    2014-01-01

    Due to their sessile nature, plants depend on flexible regulatory systems that allow them to adequately regulate developmental and physiological processes in context with environmental cues. The ubiquitin proteasome pathway, which targets a great number of proteins for degradation, is cellular tool that provides the necessary flexibility to accomplish this task. Ubiquitin E3 ligases provide the needed specificity to the pathway by selectively binding to particular substrates and facilitating their ubiquitylation. The largest group of E3 ligases known in plants is represented by CULLIN-REALLY INTERESTING NEW GENE (RING) E3 ligases (CRLs). In recent years, a great amount of knowledge has been generated to reveal the critical roles of these enzymes across all aspects of plant life. This review provides an overview of the different classes of CRLs in plants, their specific complex compositions, the variety of biological processes they control, and the regulatory steps that can affect their activities. PMID:25505853

  20. The N-end rule degradation pathway : substrate recognition and staged delivery to the CIpAP protease by the CIpS adaptor protein

    E-print Network

    Román Hernández, Giselle

    2011-01-01

    Regulated protein degradation is crucial in the regulation of many physiological processes as well as in protein quality control. In all organisms, ranging from bacteria to mammals, ATP-dependent proteases carry out regulated ...

  1. Eukaryotic Cells Producing Ribosomes Deficient in Rpl1 Are Hypersensitive to Defects in the Ubiquitin-Proteasome System

    E-print Network

    Bhattacharya, Arpita

    It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal ...

  2. The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

    ERIC Educational Resources Information Center

    Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

    2014-01-01

    Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

  3. Control of DAPK-1 degradation 

    E-print Network

    Lin, Yao

    2009-01-01

    DAPK-1 is calcium-calmodulin regulated protein kinase involved in multiple cellular pathways including apoptosis, autophagy, cell survival and motility. The cytokine TNF-? has been reported to induce the degradation of DAPK-1. Here I identified...

  4. NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

    PubMed Central

    Craxton, A; Butterworth, M; Harper, N; Fairall, L; Schwabe, J; Ciechanover, A; Cohen, G M

    2012-01-01

    Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity. PMID:22361683

  5. PHENOXYACETIC ACID DEGRADATION BY THE 2,4-DICHLOROPHENOXYACETIC ACID (TFD) PATHWAY OF PLASMID PJP4: MAPPING AND CHARACTERIZATION OF THE TFD REGULATORY GENE

    EPA Science Inventory

    Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD), Plasmid pROl0 is a derivative of pJP4 obtained by insertion of TN1721 into a nonessential region of pJP4. lasmid pROl0l was transferred by conjugation to severa...

  6. Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes.

    PubMed

    Peters, Franziska; Shinoda, Yoshifumi; McInerney, Michael J; Boll, Matthias

    2007-02-01

    In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes. PMID:17122342

  7. Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

    PubMed Central

    Harker, A R; Olsen, R H; Seidler, R J

    1989-01-01

    Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols. Images PMID:2914848

  8. Physiology of the Alzheimer's disease.

    PubMed

    Hahr, Jung Y

    2015-12-01

    Alzheimer's disease is a disease that is resulted from increased plasma osmolality both the excessive consumption of animal-based proteins and reduction of sodium intake, that resulted to increase plasma osmolality. When we are exposed to high animal-based protein diets throughout life, we gradually lose extracellular sodium and the body cannot retain water, resulting in a gradual rise of plasma osmolality. When the neuronal cells of the central nervous system are exposed to high osmolality stress, they produce of phosphorylated tau, APP, and pathologic beta amyloid protein peptides. The BACE 1 protein which influences the cleavage of amyloid precursor proteins (APP) and affects the production of beta amyloid protein peptides, is also increased in a hyperosmotic stress. When pathologic beta amyloid protein peptides are produced, they are degraded by the ubiquitin proteasome proteolytic pathway, and only then are the neurotoxic effects on the central nervous system manifested, leading to Alzheimer's disease. PMID:26386488

  9. Natural Product Inspired N-Terminal Hsp90 Inhibitors: From Bench to Bedside?

    PubMed

    Khandelwal, Anuj; Crowley, Vincent M; Blagg, Brian S J

    2016-01-01

    The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Proteins dependent upon Hsp90 are associated with all six hallmarks of cancer. Upon Hsp90 inhibition, protein substrates are degraded via the ubiquitin-proteasome pathway. Consequentially, inhibition of Hsp90 offers a therapeutic opportunity for the treatment of cancer. Natural product inhibitors of Hsp90 have been identified in vitro, which have served as leads for the development of more efficacious inhibitors and analogs that have entered clinical trials. This review highlights the development of natural product analogs, as well as the development of clinically important inhibitors that arose from natural products. PMID:26010985

  10. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    SciTech Connect

    Foerster, Friedrich; Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences , University of California at San Francisco, San Francisco ; Lasker, Keren; Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv ; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  11. Targeting ubiquitination for cancer therapies.

    PubMed

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

    2015-11-01

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

  12. Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde.

    PubMed Central

    He, Z; Spain, J C

    1997-01-01

    Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism. PMID:9471964

  13. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK?)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness.

    PubMed

    Chacon-Cabrera, Alba; Fermoselle, Clara; Urtreger, Alejandro J; Mateu-Jimenez, Mercè; Diament, Miriam J; de Kier Joffé, Elisa D Bal; Sandri, Marco; Barreiro, Esther

    2014-11-01

    Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-?B, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-?B, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-?B (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-?B and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-?B and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-?B signaling pathway effects on muscles is beneficial in cancer-induced cachexia. PMID:24615622

  17. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins.

    PubMed

    Braun, Ralf J

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

  18. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

    PubMed Central

    Braun, Ralf J.

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

  19. Expression of K6W-ubiquitin inhibits proliferation of human lens epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway plays an important role in controlling the cell cycle. The purpose of this study was to examine if expression of a dominant negative form of ubiquitin can inhibit the proliferation of lens epithelial cells. Dominant negative K6W-ubiquitin was expressed in cultured hu...

  20. Lifting DELLA repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DELLA repression of Arabidopsis seed germination can be lifted through the ubiquitin-proteasome pathway and proteolysis-independent GA signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiq...

  1. THE PROTEASOME IS A TARGET OF OXIDATIVE DAMAGE IN HUMAN RETINA PIGMENT EPITHELIAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study is to investigate the effect of oxidative stress on the UPP in retina pigment epithelial cells. Methods: To mimic physiological oxidative stress...

  2. Analysis of the Durability of PEM FC Membrane Electrode Assemblies in Automotive Applications through the Fundamental Understanding of Membrane and MEA Degradation Pathways

    SciTech Connect

    Perry, Randal L.

    2013-10-31

    The Project focused on mitigation of degradation processes on membrane electrode assemblies. The approach was to develop a model to improve understanding of the mechanisms, and to use it to focus mitigation strategies. The detailed effects of various accelerated stress tests (ASTs) were evaluated to determine the best subset to use in model development. A combination of ASTs developed by the Fuel Cell Commercialization Conference of Japan and the Fuel Cell Tech Team were selected for use. The ASTs were compared by measuring effects on performance, running in-situ diagnostics, and performing microscopic analyses of the membrane electrode assemblies after the stress tests were complete. Nissan ran FCCJ AST protocols and performed in situ and ex-situ electrochemical testing. DuPont ran FCTT and USFCC AST protocols, performed scanning and transmission electron microscopy and ran in-situ electrochemical tests. Other ex-situ testing was performed by IIT, along with much of the data analysis and model development. These tests were then modified to generate time-dependent data of the degradation mechanisms. Three different catalyst types and four membrane variants were then used to generate data for a theoretically-based degradation model. An important part of the approach was to use commercially available materials in the electrodes and membranes made in scalable semiworks processes rather than lab-based materials. This constraint ensured all materials would be practicable for full-scale testing. The initial model for the electrode layer was tested for internal consistency and agreement with the data. A Java-based computer application was developed to analyze the time-dependent AST data using polarization curves with four different cathode gas feeds and generate model parameters. Data showed very good reproducibility and good consistency as cathode catalyst loadings were varied. At the point of termination of the project, a basic electrode model was in hand with several areas identified for improvement. Time dependence and the membrane portion of the model were not addressed due to cancellation of Phase 2 of the Project.

  3. Rho iso-alpha acids from hops inhibit the GSK-3/NF-?B pathway and reduce inflammatory markers associated with bone and cartilage degradation

    PubMed Central

    Konda, Veera Reddy; Desai, Anuradha; Darland, Gary; Bland, Jeffrey S; Tripp, Matthew L

    2009-01-01

    Background Rho iso-alpha acids (RIAA) from hops have been shown to have anti-inflammatory properties. To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models. We also investigated their therapeutic effect in mice with collagen-induced arthritis. Methods The LPS-stimulated RAW 264.7 macrophages were used to evaluate the effect of RIAA on the NF-?B and MAPK signaling pathways; phosphorylation of ERK1/2, p38 and JNK was assessed by western blotting and NF-?B binding by electrophoretic mobility shift assays. Effect on the NF-?B activity was evaluated by the luciferase reporter assays in LPS-stimulated RAW 264.7 cells. GSK-3?/? kinase activity was measured in cell-free assays. The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-?/IL-1?-mediated MMP-13 expression in SW1353 cells. Mice with collagen-induced arthritis were fed with RIAA for 2 weeks. Symptoms of joint swelling, arthritic index and joint damage were assessed. Results RIAA selectively inhibited the NF-?B pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells. RIAA also inhibited GSK-3?/? kinase activity and GSK-3? dependent phosphorylation of ?-catenin in RAW 264.7 cells. In addition, RIAA inhibited NF-?B-mediated inflammatory markers in various cell models, including nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-?/IL-1?-mediated MMP-13 expression in SW1353 human chondrosarcoma cells. Finally, in a mouse model of collagen-induced arthritis, RIAA ameliorated joint damage as evidenced by significant reduction of the arthritis index and histology score; at 250 mg/kg-body weight, RIAA had efficacy similar to that of 20 mg/kg-body weight of celecoxib. Conclusion RIAA may have potential as an anti-inflammatory therapeutic. PMID:19712471

  4. Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover.

    PubMed

    Kalapis, Dorottya; Bezerra, Ana R; Farkas, Zoltán; Horvath, Peter; Bódi, Zoltán; Daraba, Andreea; Szamecz, Béla; Gut, Ivo; Bayes, Mónica; Santos, Manuel A S; Pál, Csaba

    2015-11-01

    Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature. PMID:26544557

  5. Cardiac Nav1.5 is modulated by ubiquitin protein ligase E3 component n-recognin UBR3 and 6

    PubMed Central

    Zhao, Chunxia; Wang, Lijie; Ma, Xiue; Zhu, Weidong; Yao, Lei; Cui, Yingyu; Liu, Yi; Li, Jun; Liang, Xingqun; Sun, Yunfu; Li, Li; Chen, Yi-Han

    2015-01-01

    The voltage-gated Na+ channel Nav1.5 is essential for action potential (AP) formation and electrophysiological homoeostasis in the heart. The ubiquitin–proteasome system (UPS) is a major degradative system for intracellular proteins including ion channels. The ubiquitin protein ligase E3 component N-recognin (UBR) family is a part of the UPS; however, their roles in regulating cardiac Nav1.5 channels remain elusive. Here, we found that all of the UBR members were expressed in cardiomyocytes. Individual knockdown of UBR3 or UBR6, but not of other UBR members, significantly increased Nav1.5 protein levels in neonatal rat ventricular myocytes, and this effect was verified in HEK293T cells expressing Nav1.5 channels. The UBR3/6-dependent regulation of Nav1.5 channels was not transcriptionally mediated, and pharmacological inhibition of protein biosynthesis failed to counteract the increase in Nav1.5 protein caused by UBR3/6 reduction, suggesting a degradative modulation of UBR3/6 on Nav1.5. Furthermore, the effects of UBR3/6 knockdown on Nav1.5 proteins were abolished under the inhibition of proteasome activity, and UBR3/6 knockdown reduced Nav1.5 ubiquitylation. The double UBR3–UBR6 knockdown resulted in comparable increases in Nav1.5 proteins to that observed for single knockdown of either UBR3 or UBR6. Electrophysiological recordings showed that UBR3/6 reduction-mediated increase in Nav1.5 protein enhanced the opening of Nav1.5 channels and thereby the amplitude of the AP. Thus, our findings indicate that UBR3/6 regulate cardiomyocyte Nav1.5 channel protein levels via the ubiquitin–proteasome pathway. It is likely that UBR3/6 have the potential to be a therapeutic target for cardiac arrhythmias. PMID:26059563

  6. Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

    PubMed Central

    Farkas, Zoltán; Horvath, Peter; Bódi, Zoltán; Daraba, Andreea; Szamecz, Béla; Gut, Ivo; Bayes, Mónica; Santos, Manuel A. S.; Pál, Csaba

    2015-01-01

    Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature. PMID:26544557

  7. Chemical Modulation of Endocytic Sorting Augments Adeno-associated Viral Transduction.

    PubMed

    Berry, Garrett E; Asokan, Aravind

    2016-01-01

    Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, and autophagy-based or endoplasmic reticulum-associated machinery. In the case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well understood. In the current study, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles toward large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors toward more productive trafficking pathways by deregulating cellular degradation mechanisms. PMID:26527686

  8. Free radical reaction pathway, thermochemistry of peracetic acid homolysis, and its application for phenol degradation: spectroscopic study and quantum chemistry calculations.

    PubMed

    Rokhina, Ekaterina V; Makarova, Katerina; Golovina, Elena A; Van As, Henk; Virkutyte, Jurate

    2010-09-01

    The homolysis of peracetic acid (PAA) as a relevant source of free radicals (e.g., *OH) was studied in detail. Radicals formed as a result of chain radical reactions were detected with electron spin resonance and nuclear magnetic resonance spin trapping techniques and subsequently identified by means of the simulation-based fitting approach. The reaction mechanism, where a hydroxyl radical was a primary product of O-O bond rupture of PAA, was established with a complete assessment of relevant reaction thermochemistry. Total energy analysis of the reaction pathway was performed by electronic structure calculations (ab initio and semiempirical methods) at different levels and basis sets [e.g., HF/6-311G(d), B3LYP/6-31G(d)]. Furthermore, the heterogeneous MnO2/PAA system was tested for the elimination of a model aromatic compound, phenol from aqueous solution. An artificial neural network (ANN) was designed to associate the removal efficiency of phenol with relevant process parameters such as concentrations of both the catalyst and PAA and the reaction time. Results were used to train and test ANN to identify an optimized network structure, which represented the correlations between the operational parameters and removal efficiency of phenol. PMID:20669941

  9. Oxidized low-density lipoprotein impairs the anti-coagulant function of tissue-factor-pathway inhibitor through oxidative modification by its high association and accelerated degradation in cultured human endothelial cells.

    PubMed Central

    Horie, S; Hiraishi, S; Hirata, Y; Kazama, M; Matsuda, J

    2000-01-01

    We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixed with 10 microg of ox-LDL for 30 min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time-dependent manner. It did not affect the mRNA level, but ox-LDL-dependent reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively. PMID:11085919

  10. Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate.

    PubMed

    Hesslinger, C; Fairhurst, S A; Sawers, G

    1998-01-01

    An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD. PMID:9484901

  11. Microbial degradation of herbicides.

    PubMed

    Singh, Baljinder; Singh, Kashmir

    2014-08-27

    Abstract Herbicides remain the most effective, efficient and economical way to control weeds; and its market continues to grow even with the plethora of generic products. With the development of herbicide-tolerant crops, use of herbicides is increasing around the world that has resulted in severe contamination of the environment. The strategies are now being developed to clean these substances in an economical and eco-friendly manner. In this review, an attempt has been made to pool all the available literature on the biodegradation of key herbicides, clodinafop propargyl, 2,4-dichlorophenoxyacetic acid, atrazine, metolachlor, diuron, glyphosate, imazapyr, pendimethalin and paraquat under the following objectives: (1) to highlight the general characteristic and mode of action, (2) to enlist toxicity in animals, (3) to pool microorganisms capable of degrading herbicides, (4) to discuss the assessment of herbicides degradation by efficient microbes, (5) to highlight biodegradation pathways, (6) to discuss the molecular basis of degradation, (7) to enlist the products of herbicides under degradation process, (8) to highlight the factors effecting biodegradation of herbicides and (9) to discuss the future aspects of herbicides degradation. This review may be useful in developing safer and economic microbiological methods for cleanup of soil and water contaminated with such compounds. PMID:25159042

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

    PubMed Central

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

    2014-01-01

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

  13. DDE remediation and degradation.

    PubMed

    Thomas, John E; Ou, Li-Tse; All-Agely, Abid

    2008-01-01

    DDT and its metabolites, DDD and DDE, have been shown to be recalcitrant to degradation. The parent compound, DDT, was used extensively worldwide starting in 1939 and was banned in the United States in 1973. The daughter compound, DDE, may result from aerobic degradation, abiotic dehydrochlorination, or photochemical decomposition. DDE has also occurred as a contaminant in commercial-grade DDT. The p,p'-DDE isomer is more biologically active than the o,p-DDE, with a reported half-life of -5.7 years. However, when DDT was repeatedly applied to the soil, the DDE concentration may remain unchanged for more than 20 yr. Remediation of DDE-contaminated soil and water may be done by several techniques. Phytoremediation involves translocating DDT, DDD, and DDE from the soil into the plant, although some aquatic species (duckweed > elodea > parrot feather) can transform DDT into predominantly DDD with some DDE being formed. Of all the plants that can uptake DDE, Cucurbita pepo has been the most extensively studied, with translocation values approaching "hyperaccumulation" levels. Soil moisture, temperature, and plant density have all been documented as important factors in the uptake of DDE by Cucurbita pepo. Uptake may also be influenced positively by amendments such as biosurfactants, mycorrhizal inoculants, and low molecular weight organic acids (e.g., citric and oxalic acids). DDE microbial degradation by dehalogenases, dioxygenases, and hydrolases occurs under the proper conditions. Although several aerobic degradation pathways have been proposed, none has been fully verified. Very few aerobic pure cultures are capable of fully degrading DDE to CO2. Cometabolism of DDE by Pseudomonas sp., Alicaligens sp., and Terrabacter sp. grown on biphenyl has been reported; however, not all bacterial species that produce biphenyl dioxygenase degraded DDE. Arsenic and copper inhibit DDE degradation by aerobic microorganisms. Similarly, metal chelates such as EDTA inhibit the breakdown of DDE by the extracellular lignolytic enzymes produced by white rot fungi. The addition of adjutants such as sodium ion, surfactants, and cellulose increased the rate of DDT aerobic or anaerobic degradation but did little to enhance the rate of DDE disappearance under anaerobic conditions. Only in the past decade has it been demonstrated that DDE can undergo reductive dechlorination under methanogenic and sulfidogenic conditions to form the degradation product DDMU, 1-chloro-2,2'-bis-(4'-chlorophenyl)ethane. The only pure culture reported to degrade DDE under anaerobic conditions was the denitrifier Alcaligens denitrificans. The degradation of DDE by this bacterium was enhanced by glucose, whereas biphenyl fumes had no effect. Abiotic remediation by DDE volatilization was enhanced by flooding and irrigation and deepplowing inhibited the volatilization. The use of zero-valent iron and surfactants in flooded soils enhanced DDT degradation but did not significantly alter the rate of DDE removal. Other catalysts (palladized magnesium, palladium on carbon, and nickel/aluminum alloys) degraded DDT and its metabolites, including DDE. However, these systems are often biphasic or involve explosive gases or both. Safer abiotic alternatives use UV light with titanium oxide or visible light with methylene green to degrade DDT, DDD, and DDE in aqueous or mixed solvent systems. Remediation and degradation of DDE in soil and water by phytoextraction, aerobic and anaerobic microorganisms, or abiotic methods can be accomplished. However, success has been limited, and great care must be taken that the method does not transfer the contaminants to another locale (by volatilization, deep plowing, erosion, or runoff) or to another species (by ingestion of accumulating plants or contaminated water). Although the remediation of DDT-, DDD-, and DDE-contaminated soil and water is beset with myriad problems, there remain many open avenues of research. PMID:18069646

  14. Metabolic Pathway Involved in 2-Methyl-6-Ethylaniline Degradation by Sphingobium sp. Strain MEA3-1 and Cloning of the Novel Flavin-Dependent Monooxygenase System meaBA.

    PubMed

    Dong, Weiliang; Chen, Qiongzhen; Hou, Ying; Li, Shuhuan; Zhuang, Kai; Huang, Fei; Zhou, Jie; Li, Zhoukun; Wang, Jue; Fu, Lei; Zhang, Zhengguang; Huang, Yan; Wang, Fei; Cui, Zhongli

    2015-12-15

    2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02' in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase. PMID:26386060

  15. TALSPEAK Solvent Degradation

    SciTech Connect

    Leigh R. Martin; Bruce J. Mincher

    2009-09-01

    Understanding the radiolytic degradation behavior of organic molecules involved in new or existing schemes for the recycle of used nuclear fuels is of significant interest for sustaining a closed nuclear fuel cycle. Here we have conducted several lines of investigation to begin understanding the effects of radiolysis on the aqueous phase of the TALSPEAK process for the separation of the trivalent lanthanides from the trivalent actinides. Using the 60-Co irradiator at the INL, we have begun to quantify the effects of radiation on the aqueous phase complexants used in this separation technique, and how this will affect the actinide lanthanide separation factor. In addition we have started to develop methodologies for stable product identification, a key element in determining the degradation pathways. We have also introduced a methodology to investigate the effects of alpha radiolysis that has previously received limited attention.

  16. The Ku70/80 ring in Non-Homologous End-Joining: easy to slip on, hard to remove.

    PubMed

    Kragelund, Birthe B; Weterings, Eric; Hartmann-Petersen, Rasmus; Keijzers, Guido

    2016-01-01

    Non-homologous end-joining (NHEJ) is an essential DNA double strand break repair pathway during all cell cycle stages. Deficiency in NHEJ factors can lead to accumulation of unrepaired DNA breaks or faulty DNA repair, which may ultimately result in cell death, senescence or carcinogenesis. The Ku70/80 heterodimer is a key-player in the NHEJ pathway and binds to DNA termini with high affinity, where it helps to protect DNA ends from degradation and to recruit other NHEJ factors required for repair. The mechanism of Ku70/80 detachment from the DNA helix after completion of DNA repair is incompletely understood. Some data suggest that certain DNA repair factors are ubiquitylated and targeted for proteasomal degradation after repair. Recent studies suggest that Ku80 is conjugated to lysine48-linked ubiquitin chains by the Skp1-Cullin-F-box (SCF) complex and/or the RING finger protein 8 (RNF8) ubiquitin-protein ligases, followed by rapid proteasomal degradation. In this review we address the structure and function of the Ku70/80 heterodimer and how ubiquitylation may affect the release of Ku70/80 from chromatin and its subsequent degradation via the ubiquitin-proteasome system. PMID:26709791

  17. Isoform-Specific SCFFbw7 Ubiquitination Mediates Differential Regulation of PGC-1?

    PubMed Central

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

    2015-01-01

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

  18. A data science approach to understanding photovoltaic module degradation

    NASA Astrophysics Data System (ADS)

    Wheeler, Nicholas R.; Gok, Abdulkerim; Peshek, Timothy J.; Bruckman, Laura S.; Goel, Nikhil; Zabiyaka, Davis; Fagerholm, Cara L.; Dang, Thomas; Alcantara, Christopher; Terry, Mason L.; French, Roger H.

    2015-09-01

    The expected lifetime performance and degradation of photovoltaic (PV) modules is a major issue facing the levelized cost of electricity of PV as a competitive energy source. Studies that quantify the rates and mechanisms of performance degradation are needed not only for bankability and adoption of these promising technologies, but also for the diagnosis and improvement of their mechanistic degradation pathways. Towards this goal, a generalizable approach to degradation science studies utilizing data science principles has been developed and applied to c-Si PV modules. By combining domain knowledge and data derived insights, mechanistic degradation pathways are indicated that link environmental stressors to the degradation of PV module performance characteristics. Targeted studies guided by these results have yielded predictive equations describing rates of degradation, and further studies are underway to achieve this for additional mechanistic pathways of interest.

  19. Microbial degradation of hydrazine

    SciTech Connect

    Ou, L.T.

    1987-07-01

    Hydrazine is toxic to many forms of bacteria. The activities of the autotrophic nitrifiers Nitrosomonas and Nitrobacter, denitrifying bacteria, and anaerobic methanogens were inhibited by the chemical. In addition, hydrazine prolonged the lag phase of growth as well as inhibited growth of the soil bacterium Enterobacter cloacae. Due to rapid disappearance in soil, hydrazine at 100 ..mu..g/g exhibited only temporary inhibition on soil bacterial populations. However, hydrazine at 500 ..mu..g/g caused a significant reduction of soil bacterial populations, although fungal populations in soil were enhanced by the presence of hydrazine. Despite its toxicity to soil bacteria, hydrazine in small quantities was cometabolically degraded to nitrogen gas by Nitrosomonas. An enzyme system of nitrogen-fixing heterotrophic bacteria was able to convert hydrazine to ammonia. However, it was not clear that the bacteria could utilize hydrazine as a sole source of nitrogen for growth. In the present study, the authors describe the isolation of an Achromobacter sp. from soil that had a high capacity to degrade hydrazine. Attempts to establish degradation pathways are also reported.

  20. Ubiquitin, Hormones and Biotic Stress in Plants

    PubMed Central

    Dreher, Kate; Callis, Judy

    2007-01-01

    Background The covalent attachment of ubiquitin to a substrate protein changes its fate. Notably, proteins typically tagged with a lysine48-linked polyubiquitin chain become substrates for degradation by the 26S proteasome. In recent years many experiments have been performed to characterize the proteins involved in the ubiquitylation process and to identify their substrates, in order to understand better the mechanisms that link specific protein degradation events to regulation of plant growth and development. Scope This review focuses on the role that ubiquitin plays in hormone synthesis, hormonal signalling cascades and plant defence mechanisms. Several examples are given of how targeted degradation of proteins affects downstream transcriptional regulation of hormone-responsive genes in the auxin, gibberellin, abscisic acid, ethylene and jasmonate signalling pathways. Additional experiments suggest that ubiquitin-mediated proteolysis may also act upstream of the hormonal signalling cascades by regulating hormone biosynthesis, transport and perception. Moreover, several experiments demonstrate that hormonal cross-talk can occur at the level of proteolysis. The more recently established role of the ubiquitin/proteasome system (UPS) in defence against biotic threats is also reviewed. Conclusions The UPS has been implicated in the regulation of almost every developmental process in plants, from embryogenesis to floral organ production probably through its central role in many hormone pathways. More recent evidence provides molecular mechanisms for hormonal cross-talk and links the UPS system to biotic defence responses. PMID:17220175

  1. Molecular pathogenesis of Spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins

    PubMed Central

    Bin, Bum-Ho; Hojyo, Shintaro; Hosaka, Toshiaki; Bhin, Jinhyuk; Kano, Hiroki; Miyai, Tomohiro; Ikeda, Mariko; Kimura-Someya, Tomomi; Shirouzu, Mikako; Cho, Eun-Gyung; Fukue, Kazuhisa; Kambe, Taiho; Ohashi, Wakana; Kim, Kyu-Han; Seo, Juyeon; Choi, Dong-Hwa; Nam, Yeon-Ju; Hwang, Daehee; Fukunaka, Ayako; Fujitani, Yoshio; Yokoyama, Shigeyuki; Superti-Furga, Andrea; Ikegawa, Shiro; Lee, Tae Ryong; Fukada, Toshiyuki

    2014-01-01

    The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13G64D, in which Gly at amino acid position 64 is replaced by Asp, and ZIP13?FLA, which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13G64D and ZIP13?FLA protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS. PMID:25007800

  2. Nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated protein quality control in cardiomyocytes.

    PubMed

    Cui, Taixing; Lai, Yimu; Janicki, Jospeh S; Wang, Xuejun

    2016-01-01

    Protein quality control (PQC) acts to minimize the level and toxicity of malfolded proteins in the cell. It is performed by an elaborate network of molecular chaperones and targeted protein degradation pathways. PQC monitors and maintains protein homeostasis or proteostasis in the cells. Whilst chaperones may actively promote refolding of malfolded proteins, the malfolded proteins which cannot be correctly refolded are degraded by the ubiquitin proteasome system (UPS) and the autophagic-lysosome pathway (ALP). The UPS degrades individual misfolded protein molecules, whereas the ALP removes large and less soluble protein aggregates and organelles. Emerging evidence indicates that dysregulated and inadequate PQC play an important role in the pathogenesis of not only classic conformational disease but more common forms of cardiac pathology such as cardiac pathological hypertrophy and heart failure. Nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor of cellular defense, appears to regulate the USP and the ALP by directly controlling the expression of UPS- and ALP- related genes. This article highlights an emerging role of Nrf2 in the regulation of intracellular PQC as well as its potential involvement in cardiac pathology. PMID:26709769

  3. In: The Ubiquitin Proteasome System... ISBN 978-1-60021-749-4 Eds: Mario Di Napoli and Cezary Wojcik, pp. 553-577 2007 Nova Science Publishers, Inc.

    E-print Network

    Li, Lian

    of Hsc70-interacting protein; CMT, Charcot-Marie-Tooth disease; DUB, deubiquitinating enzyme; E1¥ Department of Pharmacology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322-3090, USA. ABSTRACT The causes of various neurodegenerative diseases, particularly

  4. Polysaccharide Degradation

    NASA Astrophysics Data System (ADS)

    Stone, Bruce A.; Svensson, Birte; Collins, Michelle E.; Rastall, Robert A.

    An overview of current and potential enzymes used to degrade polysaccharides is presented. Such depolymerases are comprised of glycoside hydrolases, glycosyl transferases, phosphorylases and lyases, and their classification, active sites and action patterns are discussed. Additionally, the mechanisms that these enzymes use to cleave glycosidic linkages is reviewed as are inhibitors of depolymerase activity; reagents which react with amino acid residues, glycoside derivatives, transition state inhibitors and proteinaceous inhibitors. The characterization of various enzymes of microbial, animal or plant origin has led to their widespread use in the production of important oligosaccharides which can be incorporated into food stuffs. Sources of polysaccharides of particular interest in this chapter are those from plants and include inulin, dextran, xylan and pectin, as their hydrolysis products are purported to be functional foods in the context of gastrointestinal health. An alternative use of degraded polysaccharides is in the treatment of disease. The possibility exists to treat bacterial exopolysaccharide with lyases from bacteriophage to produce oligosaccharides exhibiting bioactive sequences. Although this area is currently in its infancy the knowledge is available to investigate further.

  5. Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates.

    PubMed

    Sandu, Cristinel; Chandramouli, Nagaranjan; Glickman, Joseph Fraser; Molina, Henrik; Kuo, Chueh-Ling; Kukushkin, Nikolay; Goldberg, Alfred L; Steller, Hermann

    2015-09-01

    Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based I?B? degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition. PMID:26033448

  6. Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

    PubMed Central

    Sandu, Cristinel; Chandramouli, Nagaranjan; Glickman, Joseph Fraser; Molina, Henrik; Kuo, Chueh-Ling; Kukushkin, Nikolay; Goldberg, Alfred L; Steller, Hermann

    2015-01-01

    Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based I?B? degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition. PMID:26033448

  7. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei; Volta, Viviana; Dipartimento di Scienze dell'Ambiente e della Vita , University of Eastern Piedmont ; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong ; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-09-04

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  8. Degradation of triclocarban by a triclosan-degrading Sphingomonas sp. strain YL-JM2C.

    PubMed

    Mulla, Sikandar I; Hu, Anyi; Wang, Yuwen; Sun, Qian; Huang, Shir-Ly; Wang, Han; Yu, Chang-Ping

    2016-02-01

    Bacterial degradation plays a vital role in determining the environmental fate of micropollutants like triclocarban. The mechanism of triclocarban degradation by pure bacterium is not yet explored. The purpose of this study was to identify metabolic pathway that might be involved in bacterial degradation of triclocarban. Triclosan-degrading Sphingomonas sp. strain YL-JM2C was first found to degrade up to 35% of triclocarban (4 mg L(-1)) within 5 d. Gas chromatography-mass spectrometry detected 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol as the major metabolites of the triclocarban degradation. Furthermore, total organic carbon results confirmed that the intermediates, 3,4-dichloroaniline (4 mg L(-1)) and 4-chloroaniline (4 mg L(-1)) could be degraded up to 77% and 80% by strain YL-JM2C within 5 d. PMID:26364219

  9. Thermal battery degradation mechanisms

    SciTech Connect

    Missert, Nancy A.; Brunke, Lyle Brent

    2015-09-01

    Diffuse reflectance IR spectroscopy (DRIFTS) was used to investigate the effect of accelerated aging on LiSi based anodes in simulated MC3816 batteries. DRIFTS spectra showed that the oxygen, carbonate, hydroxide and sulfur content of the anodes changes with aging times and temperatures, but not in a monotonic fashion that could be correlated to phase evolution. Bands associated with sulfur species were only observed in anodes taken from batteries aged in wet environments, providing further evidence for a reaction pathway facilitated by H2S transport from the cathode, through the separator, to the anode. Loss of battery capacity with accelerated aging in wet environments was correlated to loss of FeS2 in the catholyte pellets, suggesting that the major contribution to battery performance degradation results from loss of active cathode material.

  10. Gene Expression Profiles of Lens Regeneration and Development in Xenopus laevis

    PubMed Central

    Malloch, Erica L.; Perry, Kimberly J.; Fukui, Lisa; Johnson, Verity R.; Wever, Jason; Beck, Caroline W.; King, Michael W.; Henry, Jonathan J.

    2009-01-01

    734 unique genes were recovered from a cDNA library enriched for genes up-regulated during the process of lens regeneration in the frog X. laevis. The sequences represent transcription factors, proteins involved in RNA synthesis/processing, components of prominent cell signaling pathways, genes involved in protein processing, transport and degradation (e.g., the ubiquitin/proteasome pathway), matrix metalloproteases (MMPs), as well as many other proteins. The findings implicate specific signal transduction pathways in the process of lens regeneration, including the FGF, TGF-beta, MAPK, Retinoic acid, Wnt, and hedgehog signaling pathways, which are known to play important roles in eye/lens development and regeneration in various systems. In situ hybridization revealed that the majority of genes recovered are expressed during embryogenesis, including in eye tissues. Several novel genes specifically expressed in lenses were identified. The suite of genes was compared to those up-regulated in other regenerating tissues/organisms, and a small degree of overlap was detected. PMID:19681139

  11. Huntington's disease: degradation of mutant huntingtin by autophagy.

    PubMed

    Sarkar, Sovan; Rubinsztein, David C

    2008-09-01

    Autophagy is a nonspecific bulk degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. This process is also a major degradation pathway for many aggregate-prone, disease-causing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington's disease. In this review, we discuss factors regulating the degradation of mutant huntingtin by autophagy. We also report the growing list of new drugs/pathways that upregulate autophagy to enhance the clearance of this mutant protein, as autophagy upregulation may be a tractable strategy for the treatment of Huntington's disease. PMID:18637946

  12. Lignin-degrading enzymes.

    PubMed

    Pollegioni, Loredano; Tonin, Fabio; Rosini, Elena

    2015-04-01

    A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by ?-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies. PMID:25649492

  13. Muscle wasting in cancer.

    PubMed

    Johns, N; Stephens, N A; Fearon, K C H

    2013-10-01

    Skeletal muscle loss appears to be the most significant clinical event in cancer cachexia and is associated with a poor outcome. With regard to such muscle loss, despite extensive study in a range of models, there is ongoing debate as to whether a reduction in protein synthesis, an increase in degradation or a combination of both is the more relevant. Each model differs in terms of key mediators and the pathways activated in skeletal muscle. Certain models do suggest that decreased synthesis accompanied by enhanced protein degradation via the ubiquitin proteasome pathway (UPP) is important. Murine models tend to involve rapid development of cachexia and may represent more acute muscle atrophy rather than the chronic wasting observed in humans. There is a paucity of human data both at a basic descriptive level and at a molecular/mechanism level. Progress in treating the human form of cancer cachexia can only move forwards through carefully designed large randomised controlled clinical trials of specific therapies with validated biomarkers of relevance to underlying mechanisms. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. PMID:23770121

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

    PubMed Central

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

    2012-01-01

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

  15. Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant ?-synuclein.

    PubMed

    Lan, Dan-Mei; Liu, Feng-Tao; Zhao, Jian; Chen, Yan; Wu, Jian-Jun; Ding, Zheng-Tong; Yue, Zhen-Yu; Ren, Hui-Min; Jiang, Yu-Ping; Wang, Jian

    2012-09-01

    Accumulation of ?-synuclein (?-Syn) is a common pathology for both familiar and sporadic Parkinson's disease (PD), enhancing its clearance might be a promising strategy for treating PD. To assess the potential of trehalose in this regard, we investigated its effect on the PC12 cells overexpressing wild type (WT) or A53T mutant ?-Syn and the implicated pathway it might mediated. We observed that trehalose promoted the clearance of A53T ?-Syn but not WT ?-Syn in PC12 cells, and confirmed the increased LC3 and Lysotracker RED positive autolysosomes by using lysotracker and LC3 staining, the enhanced expression of LC3-II in Western blot, and more autophagosomes under Transmission Electron Microscope in a dose dependent manner after the trehalose treatment. The activation of autophagy can be alleviated by applying macroautophagy inhibitor 3-methyladenine (3-MA). In addition, degradation of A53T and WT ?-Syn was blocked after Ubiquitin Proteasome System (UPS) inhibitor (MG132) was applied in those PC12 cells overexpressing A53T or WT ?-Syn, suggesting that A53T ?-Syn could be degraded by both UPS and macroautophagy. But the effect of trehalose on A53T ?-Syn is mainly mediated through the macroautophagy pathway, which is not a dominant way for WT ?-Syn clearance. Further in vivo research will be needed to verify the effectiveness of trehalose in treating PD. PMID:22707286

  16. Resveratrol Inhibits Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Pathological Neovascularization

    PubMed Central

    Lee, Christopher Seungkyu; Choi, Eun Young; Lee, Sung Chul; Koh, Hyoung Jun; Lee, Joon Haeng

    2015-01-01

    Purpose To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1? (HIF-1?) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. Materials and Methods ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1?, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1? were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. Results In ARPE-19 cells, resveratrol significantly inhibited HIF-1? and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1? degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. Conclusion Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization. PMID:26446654

  17. Skeletal muscle wasting in cachexia and sarcopenia: molecular pathophysiology and impact of exercise training

    PubMed Central

    Bowen, T Scott; Schuler, Gerhard; Adams, Volker

    2015-01-01

    Skeletal muscle provides a fundamental basis for human function, enabling locomotion and respiration. Transmission of external stimuli to intracellular effector proteins via signalling pathways is a highly regulated and controlled process that determines muscle mass by balancing protein synthesis and protein degradation. An impaired balance between protein synthesis and breakdown leads to the development of specific myopathies. Sarcopenia and cachexia represent two distinct muscle wasting diseases characterized by inflammation and oxidative stress, where specific regulating molecules associated with wasting are either activated (e.g. members of the ubiquitin-proteasome system and myostatin) or repressed (e.g. insulin-like growth factor 1 and PGC-1?). At present, no therapeutic interventions are established to successfully treat muscle wasting in sarcopenia and cachexia. Exercise training, however, represents an intervention that can attenuate or even reverse the process of muscle wasting, by exerting anti-inflammatory and anti-oxidative effects that are able to attenuate signalling pathways associated with protein degradation and activate molecules associated with protein synthesis. This review will therefore discuss the molecular mechanisms associated with the pathology of muscle wasting in both sarcopenia and cachexia, as well as highlighting the intracellular effects of exercise training in attenuating the debilitating loss of muscle mass in these specific conditions. PMID:26401465

  18. E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif

    PubMed Central

    Ferreira, Rita T.; Menezes, Regina A.; Rodrigues-Pousada, Claudina

    2015-01-01

    ABSTRACT Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation of arsenic detoxification machinery by the Yap8 transcription factor. Yap8 is targeted by the ubiquitin proteasome system for degradation under physiological conditions, yet it escapes proteolysis in arsenic-injured cells by a mechanism that remains to be elucidated. Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels. Under these conditions, Ufd2 interacts with Yap8 mediating its stabilization, thereby controlling expression of ACR3 and capacity of cells to adapt to arsenic injury. We also show that Ufd2 U-box domain, which is associated to the ubiquitination activity of specific ubiquitin ligases, is dispensable for Yap8 stability and has no role in cell tolerance to arsenic stress. Thus, our data disclose a novel Ufd2 role beyond degradation. This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation. PMID:26276098

  19. Role of E2-Ub-conjugating enzymes during skeletal muscle atrophy

    PubMed Central

    Polge, Cecile; Attaix, Didier; Taillandier, Daniel

    2015-01-01

    The Ubiquitin Proteasome System (UPS) is a major actor of muscle wasting during various physio-pathological situations. In the past 15 years, increasing amounts of data have depicted a picture, although incomplete, of the mechanisms implicated in myofibrillar protein degradation, from the discovery of muscle-specific E3 ligases to the identification of the signaling pathways involved. The targeting specificity of the UPS relies on the capacity of the system to first recognize and then label the proteins to be degraded with a poly-ubiquitin (Ub) chain. It is fairly assumed that the recognition of the substrate is accomplished by the numerous E3 ligases present in mammalian cells. However, most E3s do not possess any catalytic activity and E2 enzymes may be more than simple Ub-providers for E3s since they are probably important actors in the ubiquitination machinery. Surprisingly, most authors have tried to characterize E3 substrates, but the exact role of E2s in muscle protein degradation is largely unknown. A very limited number of the 35 E2s described in humans have been studied in muscle protein breakdown experiments and the vast majority of studies were only descriptive. We review here the role of E2 enzymes in skeletal muscle and the difficulties linked to their study and provide future directions for the identification of muscle E2s responsible for the ubiquitination of contractile proteins. PMID:25805999

  20. Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    PubMed Central

    Gerö, Domokos; Szoleczky, Petra; Chatzianastasiou, Athanasia; Papapetropoulos, Andreas; Szabo, Csaba

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress–induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD+) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1–mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury. PMID:24842055

  1. TRICHLOROETHYLENE METABOLISM BY MICROORGANISMS THAT DEGRADE AROMATIC COMPOUNDS

    EPA Science Inventory

    Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol. Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxyge...

  2. Delineating role of ubiquitination on nuclear factor-kappa B pathway by a computational modeling approach

    SciTech Connect

    Lee, Jungsul; Choi, Kyungsun; Choi, Chulhee; Graduate School of Medical Science and Engineering, KAIST, Daejeon 305-701; KI for Bio Century, KAIST, Daejeon 305-701

    2010-01-01

    Mutant ubiquitin found in neurodegenerative diseases has been thought to hamper activation of transcription factor nuclear factor-kappa B (NF-{kappa}B) by inhibiting ubiquitin-proteasome system (UPS). It has been reported that ubiquitin also is involved in signal transduction in an UPS-independent manner. We used a modeling and simulation approach to delineate the roles of ubiquitin on NF-{kappa}B activation. Inhibition of proteasome complex increased maximal activation of IKK mainly by decreasing the UPS efficiency. On the contrary, mutant ubiquitin decreased maximal activity of IKK. Computational modeling showed that the inhibition effect of mutant ubiquitin is mainly attributed to decreased activity of UPS-independent function of ubiquitin. Collectively, our results suggest that mutant ubiquitin affects NF-{kappa}B activation in an UPS-independent manner.

  3. Microbial degradation and toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine.

    PubMed

    Khan, Muhammad Imran; Lee, Jaejin; Park, Joonhong

    2012-10-01

    In the present work, current knowledge on the potential fate, microbial degradation, and toxicity of hexahydro- 1,3,5-trinitro-1,3,5-triazine (RDX) was thoroughly reviewed, focusing on the toxicological assessment of a variety of potential RDX degradation pathways in bacteria and fungi. The present review on microbial degradation pathways and toxicities of degradation intermediates suggests that, among aerobic RDX degradation pathways, the one via denitration may be preferred in a toxicological perspective, and that among anaerobic pathways, those forming 4- nitro-2,4-diazabutanal (NDAB) via ring cleavage of 1-nitroso- 3,5-dinitro-1,3,5-triazinane (MNX) may be toxicologically advantageous owing to its potential mineralization under partial or complete anoxic conditions. These findings provide important information on RDX-degrading microbial pathways, toxicologically most suitable to be stimulated in contaminated fields. PMID:23075780

  4. Investigating Degradation Enzymes for Pentachlorophenols (PCPs)

    E-print Network

    Collins, Gary S.

    Investigating Degradation Enzymes for Pentachlorophenols (PCPs) Emily White1, Mark Nissen2, Chul chlorophenolicum that breaks down PCP has been studied extensively but a complete picture of how the enzymes work is still lacking. This study aims to solve the crystal structure of PcpC, which is an enzyme in the pathway

  5. Bacterial degradation of natural and synthetic rubber.

    PubMed

    Bode, H B; Kerkhoff, K; Jendrossek, D

    2001-01-01

    The degradation of natural rubber (NR), synthetic poly(cis-1,4-isoprene) (SR), and cross-linked NR (latex gloves) by Gram-positive and Gram-negative bacteria was analyzed by weight loss, gel permeation chromatography, and determination of the protein content. Weight losses of 11-18% and an increase in protein up to 850 microg/mL after incubation of Nocardia sp. DSMZ43191, Streptomyces coelicolor, Streptomyces griseus, bacterial isolate 18a, Acinetobacter calcoaceticus, and Xanthomonas sp. with latex gloves as a carbon source indicated degradation of the polymer. An increase of protein up to 1250 microg/mL was obtained upon incubation of the bacteria with SR. No or only little weight losses and no increase in the protein content were found for nondegrading control strains such as Streptomyces lividans and Streptomyces exfoliatus and for mutants of degrading strains of S. coelicolor and S. griseus, which have been identified by their inability to produce clearing zones on opaque latex agar. Measurement of the average molecular weight of synthetic rubber before and after degradation showed a time-dependent shift to lower values for the degrading strains. Diketone derivates of oligo(cis-1,4-isoprene) were identified as metabolites of rubber degradation. An oxidative degradation pathway of poly(cis-1,4-isoprene) to acetyl-coenzymeA and propionyl-coenzymeA by beta-oxidation is suggested for bacterial degradation of isoprene rubber. PMID:11749186

  6. Bacterial Degradation of Aromatic Compounds

    PubMed Central

    Seo, Jong-Su; Keum, Young-Soo; Li, Qing X.

    2009-01-01

    Aromatic compounds are among the most prevalent and persistent pollutants in the environment. Petroleum-contaminated soil and sediment commonly contain a mixture of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatics. Aromatics derived from industrial activities often have functional groups such as alkyls, halogens and nitro groups. Biodegradation is a major mechanism of removal of organic pollutants from a contaminated site. This review focuses on bacterial degradation pathways of selected aromatic compounds. Catabolic pathways of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, and benzo[a]pyrene are described in detail. Bacterial catabolism of the heterocycles dibenzofuran, carbazole, dibenzothiophene, and dibenzodioxin is discussed. Bacterial catabolism of alkylated PAHs is summarized, followed by a brief discussion of proteomics and metabolomics as powerful tools for elucidation of biodegradation mechanisms. PMID:19440284

  7. Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration.

    PubMed

    Wakatsuki, Shuji; Furuno, Akiko; Ohshima, Makiko; Araki, Toshiyuki

    2015-11-23

    Oxidative stress is a well-known inducer of neuronal apoptosis and axonal degeneration. We previously showed that the E3 ubiquitin ligase ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B activation. We now demonstrate that oxidative stress serves as an activator of the ubiquitin ligase activity of ZNRF1 by inducing epidermal growth factor receptor (EGFR)-mediated phosphorylation at the 103rd tyrosine residue and that the up-regulation of ZNRF1 activity by oxidative stress leads to neuronal apoptosis and Wallerian degeneration. We also show that nicotinamide adenine dinucleotide phosphate-reduced oxidase activity is required for the EGFR-dependent phosphorylation-induced activation of ZNRF1 and resultant AKT degradation via the ubiquitin proteasome system to induce Wallerian degeneration. These results indicate the pathophysiological significance of the EGFR-ZNRF1 pathway induced by oxidative stress in the regulation of neuronal apoptosis and Wallerian degeneration. A deeper understanding of the regulatory mechanism for ZNRF1 catalytic activity via phosphorylation will provide a potential therapeutic avenue for neurodegeneration. PMID:26572622

  8. Heat shock protein 90? stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    SciTech Connect

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90? (HSP90?) interacts with FAK and the middle domain (amino acids 233–620) of HSP90? is mainly responsible for this interaction. Furthermore, we found that HSP90? regulates FAK stability since HSP90? inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90? interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90? regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90? protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90? or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90? or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90? or FAK interferes cell invasion and cytoskeleton.

  9. KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models.

    PubMed

    Riz, Irene; Hawley, Teresa S; Hawley, Robert G

    2015-06-20

    Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease. However, MM cells still develop resistance to proteasome inhibitors such as carfilzomib. Toward this end, we have established carfilzomib-resistant derivatives of MM cell lines. We found that resistance to carfilzomib was associated with elevated levels of prosurvival autophagy, and Kruppel-like factor 4 (KLF4) was identified as a contributing factor. Expression levels as well as nuclear localization of KLF4 protein were elevated in MM cells with acquired carfilzomib resistance. Chromatin immunoprecipitations indicated that endogenous KLF4 bound to the promoter regions of the SQSTM1 gene encoding the ubiquitin-binding adaptor protein sequestosome/p62 that links the proteasomal and autophagic protein degradation pathways. Ectopic expression of KLF4 induced upregulation of SQSTM1. On the other hand, inhibitors of autophagy sensitized MM cells to carfilzomib, even in carfilzomib-resistant derivatives having increased expression of the multidrug resistance protein P-glycoprotein. Thus, we report here a novel function for KLF4, one of the Yamanaka reprogramming factors, as being a contributor to autophagy gene expression which moderates preclinical proteasome inhibitor efficacy in MM. PMID:26109433

  10. KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

    PubMed Central

    Riz, Irene; Hawley, Teresa S.; Hawley, Robert G.

    2015-01-01

    Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease. However, MM cells still develop resistance to proteasome inhibitors such as carfilzomib. Toward this end, we have established carfilzomib-resistant derivatives of MM cell lines. We found that resistance to carfilzomib was associated with elevated levels of prosurvival autophagy, and Kruppel-like factor 4 (KLF4) was identified as a contributing factor. Expression levels as well as nuclear localization of KLF4 protein were elevated in MM cells with acquired carfilzomib resistance. Chromatin immunoprecipitations indicated that endogenous KLF4 bound to the promoter regions of the SQSTM1 gene encoding the ubiquitin-binding adaptor protein sequestosome/p62 that links the proteasomal and autophagic protein degradation pathways. Ectopic expression of KLF4 induced upregulation of SQSTM1. On the other hand, inhibitors of autophagy sensitized MM cells to carfilzomib, even in carfilzomib-resistant derivatives having increased expression of the multidrug resistance protein P-glycoprotein. Thus, we report here a novel function for KLF4, one of the Yamanaka reprogramming factors, as being a contributor to autophagy gene expression which moderates preclinical proteasome inhibitor efficacy in MM. PMID:26109433

  11. Proteasome malfunction activates macroautophagy in the heart.

    PubMed

    Zheng, Qingwen; Su, Huabo; Tian, Zongwen; Wang, Xuejun

    2011-01-01

    Protein quality control (PQC) senses and repairs misfolded/unfolded proteins and, if the repair fails, degrades the terminally misfolded polypeptides through an intricate collaboration between molecular chaperones and targeted proteolysis. Proteolysis of damaged proteins is performed primarily by the ubiquitin-proteasome system (UPS). Macroautophagy (commonly known as autophagy) may also play a role in PQC-associated proteolysis, especially when UPS function becomes inadequate. The development of a range of heart diseases, including bona fide cardiac proteinopathies and various forms of cardiac dysfunction has been linked to proteasome functional insufficiency (PFI). Both PFI and activation of autophagy have been observed in the heart of well-established mouse models of cardiac proteinopathy. A causal relationship between PFI and autophagic activation was suggested by a study using cultured cardiomyocytes but has not been established in the heart of intact animals. Taking advantage of an autophagy reporter, we demonstrated here that pharmacologically induced proteasome inhibition is sufficient to activate autophagy in cardiomyocytes in both intact animals and cell cultures, unveiling a potential cross-talk between the two major degradation pathways in cardiac PQC. PMID:22081794

  12. Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia

    PubMed Central

    Carlucci, Annalisa; Adornetto, Annagrazia; Scorziello, Antonella; Viggiano, Davide; Foca, Mariapaola; Cuomo, Ornella; Annunziato, Lucio; Gottesman, Max; Feliciello, Antonio

    2008-01-01

    A-kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post-translationally by the ubiquitin/proteasome pathway. Seven In-Absentia Homolog 2 (Siah2), an E3–ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2-mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2-dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121-signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability. PMID:18323779

  13. Use of proteome arrays to globally identify substrates for E3 ubiquitin ligases.

    PubMed

    Persaud, Avinash; Rotin, Daniela

    2011-01-01

    Ubiquitin-protein ligases (E3s) are responsible for target recognition and subsequent modification of selected substrates within the ubiquitin proteasomal system (UPS). Substrates of this pathway are covalently modified by the attachment of ubiquitin usually onto Lys residues. As a result, these modified proteins can be targeted for degradation, endocytosis, protein sorting, subnuclear trafficking, or other fates. Despite the advancements in understanding the underlying mechanisms of the ubiquitin system, the substrates of most E3 enzymes remain largely unknown. Here, we describe the development of a high-throughput method to identify in vitro substrates for E3 ligases on a global proteomic scale. The enzymatic activity (ubiquitylation) and binding of ubiquitin ligases to their substrates are performed using protein (proteome) microarrays as the experimental platform, and using Nedd4/Rsp5 family members as examples of HECT E3 ligases. The in vitro ubiquitylation and binding substrates identified in these screens can provide invaluable insight into the cellular pathways in which E3 ligases participate. PMID:21863490

  14. STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Wu, Chengsheng; Molavi, Ommoleila; Zhang, Haifeng; Gupta, Nidhi; Alshareef, Abdulraheem; Bone, Kathleen M; Gopal, Keshav; Wu, Fang; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Lai, Raymond

    2015-07-16

    The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-?, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis. PMID:25921060

  15. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

    PubMed

    Bañuelos, Adriana; Reyes, Elba; Ocadiz, Rodolfo; Alvarez, Elizabeth; Moreno, Martha; Monroy, Alberto; Gariglio, Patricio

    2003-08-01

    Human papillomavirus (HPV) E6 viral oncoprotein plays an important role during cervical carcinogenesis. This oncoprotein binds the tumor suppressor protein p53, leading to its degradation via the ubiquitin-proteasome pathway. Therefore, it is generally assumed that in HPV-positive cancer cells p53 function is completely abolished. Nevertheless, recent findings suggest that p53 activity can be recovered in cells expressing endogenous E6 protein. To investigate whether p53-dependent functions controlling genome integrity, cell proliferation, and apoptosis can be reactivated in cervical cancer cells, we examined the capacity of HeLa, INBL, CaSki, C33A, and ViBo cell lines to respond to neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA. We found that NCS treatment inhibits cellular proliferation through G2 cell cycle arrest and apoptosis induction. This effect was preceded by nuclear accumulation of p53 protein and by an increase of p21 transcripts. Although apoptosis was blocked in ViBo cells (HPV-negative), nuclear accumulation of transcriptionally active p53 and inhibition of cell proliferation are observed after NCS treatment. These results suggest that HPV-positive cervical cancer cells are capable of responding efficiently to DNA damage provoked by NCS treatment through a p53-dependent pathway in spite of the presence of E6 protein. PMID:12750435

  16. Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies

    PubMed Central

    Gazzerro, Elisabetta; Assereto, Stefania; Bonetto, Andrea; Sotgia, Federica; Scarfì, Sonia; Pistorio, Angela; Bonuccelli, Gloria; Cilli, Michele; Bruno, Claudio; Zara, Federico; Lisanti, Michael P.; Minetti, Carlo

    2010-01-01

    Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, ?-sarcoglycan, and ?-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases. PMID:20304949

  17. Increased ubiquitination and the crosstalk of G protein signaling in cardiac myocytes: involvement of Ric-8B in Gs suppression by Gq signal.

    PubMed

    Jenie, Riris I; Nishimura, Motoki; Fujino, Mika; Nakaya, Michio; Mizuno, Norikazu; Tago, Kenji; Kurose, Hitoshi; Itoh, Hiroshi

    2013-12-01

    Hyperactivation of Gq signaling causes cardiac hypertrophy, and ?-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of G?s in neonatal rat cardiomyocytes, reduced G?s expression, and suppressed cAMP response to ?-adrenergic receptor stimulation. Ectopic expression of G?q induced a similar suppression, which is due to the degradation of G?s by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of G?s, effectively canceled the G?q-induced ubiquitination of G?s and recovered the cAMP accumulation. In vitro, G?q competes for the binding of G?s to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure. PMID:24134321

  18. HAT3-mediated acetylation of PCNA precedes PCNA monoubiquitination following exposure to UV radiation in Leishmania donovani

    PubMed Central

    Kumar, Devanand; Saha, Swati

    2015-01-01

    Histone modifications impact various processes. In examining histone acetyltranferase HAT3 of Leishmania donovani, we find elimination of HAT3 causes decreased cell viability due to defects in histone deposition, and aberrant cell cycle progression pattern. HAT3 associates with proliferating cell nuclear antigen (PCNA), helping load PCNA onto chromatin in proliferating cells. HAT3-nulls show heightened sensitivity to UV radiation. Following UV exposure, PCNA cycles off/on chromatin only in cells expressing HAT3. Inhibition of the ubiquitin-proteasome pathway prior to UV exposure allows accumulation of chromatin-bound PCNA, and reveals that HAT3-nulls are deficient in PCNA monoubiquitination as well as polyubiquitination. While poor monoubiquitination of PCNA may adversely affect translesion DNA synthesis-based repair processes, polyubiquitination deficiencies may result in continued retention of chromatin-bound PCNA, leading to genomic instability. On suppressing the proteasome pathway we also find that HAT3 mediates PCNA acetylation in response to UV. HAT3-mediated PCNA acetylation may serve as a flag for PCNA ubiquitination, thus aiding DNA repair. While PCNA acetylation has previously been linked to its degradation following UV exposure, this is the first report linking a HAT-mediated PCNA acetylation to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes. PMID:25948582

  19. Bacterial degradation of monocyclic aromatic amines

    PubMed Central

    Arora, Pankaj K.

    2015-01-01

    Aromatic amines are an important group of industrial chemicals, which are widely used for manufacturing of dyes, pesticides, drugs, pigments, and other industrial products. These compounds have been considered highly toxic to human beings due to their carcinogenic nature. Three groups of aromatic amines have been recognized: monocyclic, polycyclic, and heterocyclic aromatic amines. Bacterial degradation of several monocyclic aromatic amines has been studied in a variety of bacteria, which utilizes monocyclic aromatic amines as their sole source of carbon and energy. Several degradation pathways have been proposed and the related enzymes and genes have also been characterized. Many reviews have been reviewed toxicity of monocyclic aromatic amines; however, there is lack of review on biodegradation of monocyclic aromatic amines. The aim of this review is to summarize bacterial degradation of monocyclic aromatic amines. This review will increase our current understanding of biochemical and molecular basis of bacterial degradation of monocyclic aromatic amines. PMID:26347719

  20. Development of disease-resistant rice using regulatory components of induced disease resistance

    PubMed Central

    Takatsuji, Hiroshi

    2014-01-01

    Infectious diseases cause huge crop losses annually. In response to pathogen attacks, plants activate defense systems that are mediated through various signaling pathways. The salicylic acid (SA) signaling pathway is the most powerful of these pathways. Several regulatory components of the SA signaling pathway have been identified, and are potential targets for genetic manipulation of plants’ disease resistance. However, the resistance associated with these regulatory components is often accompanied by fitness costs; that is, negative effects on plant growth and crop yield. Chemical defense inducers, such as benzothiadiazole and probenazole, act on the SA pathway and induce strong resistance to various pathogens without major fitness costs, owing to their ‘priming effect.’ Studies on how benzothiadiazole induces disease resistance in rice have identified WRKY45, a key transcription factor in the branched SA pathway, and OsNPR1/NH1. Rice plants overexpressing WRKY45 were extremely resistant to rice blast disease caused by the fungus Magnaporthe oryzae and bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo), the two major rice diseases. Disease resistance is often accompanied by fitness costs; however, WRKY45 overexpression imposed relatively small fitness costs on rice because of its priming effect. This priming effect was similar to that of chemical defense inducers, although the fitness costs were amplified by some environmental factors. WRKY45 is degraded by the ubiquitin–proteasome system, and the dual role of this degradation partly explains the priming effect. The synergistic interaction between SA and cytokinin signaling that activates WRKY45 also likely contributes to the priming effect. With a main focus on these studies, I review the current knowledge of SA-pathway-dependent defense in rice by comparing it with that in Arabidopsis, and discuss potential strategies to develop disease-resistant rice using signaling components. PMID:25431577

  1. Chemical Warfare Agent Degradation and Decontamination

    SciTech Connect

    Talmage, Sylvia Smith; Watson, Annetta Paule; Hauschild, Veronique; Munro, Nancy B; King, J.

    2007-02-01

    The decontamination of chemical warfare agents (CWA) from structures, environmental media, and even personnel has become an area of particular interest in recent years due to increased homeland security concerns. In addition to terrorist attacks, scenarios such as accidental releases of CWA from U.S. stockpile sites or from historic, buried munitions are also subjects for response planning. To facilitate rapid identification of practical and effective decontamination approaches, this paper reviews pathways of CWA degradation by natural means as well as those resulting from deliberately applied solutions and technologies; these pathways and technologies are compared and contrasted. We then review various technologies, both traditional and recent, with some emphasis on decontamination materials used for surfaces that are difficult to clean. Discussion is limited to the major threat CWA, namely sulfur mustard (HD, bis(2-chloroethyl)sulfide), VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate), and the G-series nerve agents. The principal G-agents are GA (tabun, ethyl N,N-dimethylphosphoramidocyanidate), GB (sarin, isopropyl methylphosphonofluoridate), and GD (soman, pinacolyl methylphosphonofluoridate). The chemical decontamination pathways of each agent are outlined, with some discussion of intermediate and final degradation product toxicity. In all cases, and regardless of the CWA degradation pathway chosen for decontamination, it will be necessary to collect and analyze pertinent environmental samples during the treatment phase to confirm attainment of clearance levels.

  2. mRNA Degradation Machinery in Plants Yukako Chiba & Pamela J. Green

    E-print Network

    Green, Pamela

    be degraded in a 3 to 5 direction by the exosome following deadenylation. These two pathways comprise the bulk, transcripts lacking translation termination codons are degraded rapidly from 3 to 5 direction by exosome ribosome leading to exonucleolytic degradation of the mRNA by Xrn1p and exosome. In addition to an active

  3. Proteolysis in the secretory pathway

    SciTech Connect

    Guzowski, D.E.; Bienkowski, R.S.

    1987-05-01

    Many secretory proteins are degraded intracellularly rather than secreted, however the location of this catabolic process is not known. The authors have tested the hypothesis that the degradation occurs in the organelles of the secretory pathway. Slices of rat liver were incubated with (/sup 14/C)leucine for 3 h and then incubated under chase conditions for 30 min. The tissue was homogenized and the Golgi apparatus, smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER) were isolated by ultracentrifugation on a discontinuous sucrose gradient. The organelles were incubated in 0.3M sucrose-50 mM citrate (pH 4) for 8-12 h at 37 C; control samples were incubated at 4 C. Percent degradation was calculated as the amount of acid soluble radioactivity released relative to total radioactivity in the sample. Proteolysis in the organelles incubated at 37 C was as follows: Golgi: 15-25%; sER: 10-20%; rER: 10-20%. Proteolysis at 4 C was negligible in all cases. These results support the hypothesis that the compartments of the secretory pathway are capable of degrading newly synthesized secretory proteins.

  4. Current Status on Biochemistry and Molecular Biology of Microbial Degradation of Nicotine

    PubMed Central

    Gurusamy, Raman; Natarajan, Sakthivel

    2013-01-01

    Bioremediation is one of the most promising methods to clean up polluted environments using highly efficient potent microbes. Microbes with specific enzymes and biochemical pathways are capable of degrading the tobacco alkaloids including highly toxic heterocyclic compound, nicotine. After the metabolic conversion, these nicotinophilic microbes use nicotine as the sole carbon, nitrogen, and energy source for their growth. Various nicotine degradation pathways such as demethylation pathway in fungi, pyridine pathway in Gram-positive bacteria, pyrrolidine pathway, and variant of pyridine and pyrrolidine pathways in Gram-negative bacteria have been reported. In this review, we discussed the nicotine-degrading pathways of microbes and their enzymes and biotechnological applications of nicotine intermediate metabolites. PMID:24470788

  5. Targeted polypeptide degradation

    DOEpatents

    Church, George M. (Brookline, MA); Janse, Daniel M. (Brookline, MA)

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides m