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Sample records for 26s proteasome-dependent regulation

  1. COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis*S⃞

    PubMed Central

    Stuttmann, Johannes; Lechner, Esther; Guérois, Raphael; Parker, Jane E.; Nussaume, Laurent; Genschik, Pascal; Noël, Laurent D.

    2009-01-01

    Ubiquitination and proteasome-mediated degradation of proteins are crucial for eukaryotic physiology and development. The largest class of E3 ubiquitin ligases is made up of the cullin-RING ligases (CRLs), which themselves are positively regulated through conjugation of the ubiquitin-like peptide RUB/NEDD8 to cullins. RUB modification is antagonized by the COP9 signalosome (CSN), an evolutionarily conserved eight-subunit complex that is essential in most eukaryotes and cleaves RUB from cullins. The CSN behaves genetically as an activator of CRLs, although it abolishes CRL activity in vitro. This apparent paradox was recently reconciled in different organisms, as the CSN was shown to prevent autocatalytic degradation of several CRL substrate adaptors. We tested for such a mechanism in the model plant Arabidopsis by measuring the impact of a newly identified viable csn2 mutant on the activity and stability of SCFTIR1, a receptor to the phytohormone auxin and probably the best characterized plant CRL. Our analysis reveals that not only the F-box protein TIR1 but also relevant cullins are destabilized in csn2 and other Arabidopsis csn mutants. These results provide an explanation for the auxin resistance of csn mutants. We further observed in vivo a post-translational modification of TIR1 dependent on the proteasome inhibitor MG-132 and provide evidence for proteasome-mediated degradation of TIR1, CUL1, and ASK1 (Arabidopsis SKP1 homolog). These results are consistent with CSN-dependent protection of Arabidopsis CRLs from autocatalytic degradation, as observed in other eukaryotes, and provide evidence for antagonist roles of the CSN and 26S proteasome in modulating accumulation of the plant CRL SCFTIR1. PMID:19147500

  2. Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

    PubMed Central

    Cohen, Mickael M.J.; Leboucher, Guillaume P.; Livnat-Levanon, Nurit

    2008-01-01

    The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome–dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCFMdm30p ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion. PMID:18353967

  3. ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance

    PubMed Central

    McKerlie, Megan; Lin, Sichun; Zhu, Xu-Dong

    2012-01-01

    Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability. PMID:22266654

  4. Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway

    PubMed Central

    Lucas, Christopher D.; Allen, Keith C.; Dorward, David A.; Hoodless, Laura J.; Melrose, Lauren A.; Marwick, John A.; Tucker, Carl S.; Haslett, Christopher; Duffin, Rodger; Rossi, Adriano G.

    2013-01-01

    -1 via a proteasomal-dependent pathway. PMID:23195034

  5. Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

    PubMed Central

    Combaret, Lydie; Taillandier, Daniel; Dardevet, Dominique; Béchet, Daniel; Rallière, Cécile; Claustre, Agnès; Grizard, Jean; Attaix, Didier

    2004-01-01

    Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome. PMID:14636157

  6. Negative feedback regulation of NF-κB-inducing kinase is proteasome-dependent but does not require cellular inhibitors of apoptosis.

    PubMed

    Gray, Carolyn M; McCorkell, Kelly A; Chunduru, Srinivas K; McKinlay, Mark A; May, Michael J

    2014-07-18

    Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism. PMID:24942881

  7. Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

    PubMed Central

    Benharouga, Mohamed; Haardt, Martin; Kartner, Norbert; Lukacs, Gergely L.

    2001-01-01

    Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well. PMID:11381082

  8. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  9. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.

    PubMed

    Besche, Henrike C; Sha, Zhe; Kukushkin, Nikolay V; Peth, Andreas; Hock, Eva-Maria; Kim, Woong; Gygi, Steven; Gutierrez, Juan A; Liao, Hua; Dick, Lawrence; Goldberg, Alfred L

    2014-05-16

    Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly-ubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients. PMID:24811749

  10. The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death

    PubMed Central

    Livneh, Ido; Cohen-Kaplan, Victoria; Cohen-Rosenzweig, Chen; Avni, Noa; Ciechanover, Aaron

    2016-01-01

    The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms. PMID:27444871

  11. Regulation of Sperm Capacitation by the 26S Proteasome: An Emerging New Paradigm in Spermatology.

    PubMed

    Kerns, Karl; Morales, Patricio; Sutovsky, Peter

    2016-05-01

    The ubiquitin proteasome system (UPS) participates in many biological processes ranging from cell cycle and antigen processing to cellular defense and signaling. Work of the last decade has made it evident that the UPS is involved in many sperm-related processes leading up to and as part of fertilization. The current knowledge of UPS involvement and changes during sperm capacitation are reviewed together with a list of known proteasome-associated sperm proteins and a discussion of the relationships between these proteins and the proteasome. Proteasomal inhibitors such as MG-132 and epoxomicin significantly alter capacitation and prevent acrosome reaction. The 26S proteasome degrades AKAP3, an A-kinase anchoring protein, partially regulating the release of protein-kinase A (PKA), a vital component necessary for the steps leading up to capacitation. Further, changes occur in 20S core subunit localization and abundance throughout capacitation. Proteasome-interacting valosine-containing protein (VCP) undergoes tyrosine phosphorylation; however, its physiological roles in capacitation and fertilization remain unknown. The E1-type ubiquitin-activating enzyme (UBA1) inhibitor PYR-41 also alters acrosomal membrane remodeling during capacitation. Furthermore, after capacitation, the acrosomal proteasomes facilitate the degradation of zona pellucida glycoproteins leading up to fertilization. Methods to modulate the sperm proteasome activity during sperm storage and capacitation may translate to increased reproductive efficiency in livestock animals. Human male infertility diagnostics may benefit from incorporation of research outcomes built upon relationships between UPS and capacitation. Altogether, the studies reviewed here support the involvement of UPS in sperm capacitation and present opportunities for new discoveries. PMID:27053366

  12. HSP70 Mediates Dissociation and Reassociation of the 26S Proteasome During Adaptation to Oxidative Stress

    PubMed Central

    Grune, Tilman; Catalgol, Betül; Licht, Anke; Ermak, Gennady; Pickering, Andrew; Ngo, Jenny K.; Davies, Kelvin J. A.

    2011-01-01

    We report an entirely new role for the HSP70 chaperone in dissociating 26S proteasome complexes (into free 20S proteasomes and bound 19S regulators), preserving 19S regulators, and reconstituting 26S proteasomes in the first 1-3 hours following mild oxidative stress. These responses, coupled with direct 20S proteasome activation by poly-ADP ribose polymerase in the nucleus and by PA28αβ in the cytoplasm, instantly provides cells with increased capacity to degrade oxidatively damaged proteins and to survive the initial effects of stress exposure. Subsequent adaptive (hormetic) processes (3-24 hours following stress exposure), mediated by several signal transduction pathways and involving increased transcription/translation of 20S proteasomes, immunoproteasomes, and PA28αβ, abrogate the need for 26S proteasome dissociation. During this adaptive period, HSP70 releases its bound 19S regulators, 26S proteasomes are reconstituted, and ATP-stimulated proteolysis is restored. The 26S proteasome-dependent, and ATP-stimulated, turnover of ubiquitinylated proteins is essential for normal cell metabolism, and its restoration is required for successful stress-adaptation. PMID:21767633

  13. Hypo-osmotic shock induces nuclear export and proteasome-dependent decrease of UBL5

    SciTech Connect

    Hatanaka, Ken; Ikegami, Koji; Takagi, Hiroshi; Setou, Mitsutoshi . E-mail: setou@nips.ac.jp

    2006-11-24

    The osmolarity of body fluid is strictly controlled through the action of diuretic hormones, which are secreted in the hypothalamus. In the mammalian brain, ubiquitin-like 5 (UBL5) is expressed in oxytocin- and vasopressin-positive neurons in the hypothalamus, and these neurons play a role in regulating osmolarity. We examined the dynamics of UBL5 levels in response to hyper- or hypo-osmotic conditions. Hypo-osmotic conditions led to significantly reduced levels of UBL5 both in brain slices from the hypothalamus and in NIH-3T3 cells. This decrease in UBL5 was transcription-independent and proteasome-dependent. Time-course immunocytochemical studies using exogenous UBL5 revealed that the protein was exported from the nucleus under hypo-osmotic conditions and decreased in a proteasome-dependent manner. This report is the first to describe changes in the intracellular and subcellular localization of UBL5 in response to hypo-osmotic conditions. Our results imply osmoregulation of UBL5.

  14. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    SciTech Connect

    Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  15. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato

    PubMed Central

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  16. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.

    PubMed

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  17. Mouse homologue of yeast Prp19 interacts with mouse SUG1, the regulatory subunit of 26S proteasome

    SciTech Connect

    Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon . E-mail: shkim@khu.ac.kr

    2007-04-27

    Yeast Prp19 has been shown to involve in pre-mRNA splicing and DNA repair as well as being an ubiquitin ligase. Mammalian homologue of yeast Prp19 also plays on similar functional activities in cells. In the present study, we isolated mouse SUG1 (mSUG1) as binding partner of mouse Prp19 (mPrp19) by the yeast two-hybrid system. We confirmed the interaction of mPrp9 with mSUG1 by GST pull-down assay and co-immunoprecipitation assay. The N-terminus of mPrp19 including U-box domain was associated with the C-terminus of mSUG1. Although, mSUG1 is a regulatory subunit of 26S proteasome, mPrp19 was not degraded in the proteasome-dependent pathway. Interestingly, GFP-mPrp19 fusion protein was co-localized with mSUG1 protein in cytoplasm as the formation of the speckle-like structures in the presence of a proteasome inhibitor MG132. In addition, the activity of proteasome was increased in cells transfected with mPrp19. Taken together, these results suggest that mPrp19 involves the regulation of protein turnover and may transport its substrates to 26S proteasome through mSUG1 protein.

  18. Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon.

    PubMed Central

    Bose, Suchira; Stratford, Fiona L L; Broadfoot, Kerry I; Mason, Grant G F; Rivett, A Jennifer

    2004-01-01

    In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit. PMID:14583091

  19. 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.

  20. ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis.

    PubMed

    Sung, Dong-Yul; Kim, Tae-Houn; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2009-09-01

    A forward-genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 was the strongest arsenate- and arsenite-resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knock-out mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by the overexpression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild-type (WT) Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces the enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in the WT, and this arsenic induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5, compared with WT, suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background. PMID:19453443

  1. ARS5 is a component of the 26S proteasome complex and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis

    PubMed Central

    Sung, Dong-Yul; Kim, Tae-Houn; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2010-01-01

    Summary A forward genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 is the strongest arsenate and arsenite resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and the arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knockout mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by over expression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild type Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in wild type and this arsenic-induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5 compared to WT suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background. PMID:19453443

  2. Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin-proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of tra...

  3. Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

    PubMed

    Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

    2016-04-15

    We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. PMID:26789074

  4. 26S Proteasome: Hunter and Prey in Auxin Signaling.

    PubMed

    Kong, Xiangpei; Zhang, Liangran; Ding, Zhaojun

    2016-07-01

    Auxin binds to TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALLING F-BOX proteins (TIR1/AFBs) and promotes the degradation of Aux/IAA transcriptional repressors. The proteasome regulator PROTEASOME REGULATOR1 (PTRE1) has now been shown to be required for auxin-mediated repression of 26S proteasome activity, thus providing new insights into the fine-tuning of the homoeostasis of Aux/IAA proteins and auxin signaling. PMID:27246455

  5. The 26S Proteasome and Initiation of Gene Transcription

    PubMed Central

    Durairaj, Geetha; Kaiser, Peter

    2014-01-01

    Transcription activation is the foremost step of gene expression and is modulated by various factors that act in synergy. Misregulation of this process and its associated factors has severe effects and hence requires strong regulatory control. In recent years, growing evidence has highlighted the 26S proteasome as an important contributor to the regulation of transcription initiation. Well known for its role in protein destruction, its contribution to protein synthesis was initially viewed with skepticism. However, studies over the past several years have established the proteasome as an important component of transcription initiation through proteolytic and non-proteolytic activities. In this review, we discuss findings made so far in understanding the connections between transcription initiation and the 26S proteasome complex. PMID:25211636

  6. The murine cardiac 26S proteasome: an organelle awaiting exploration.

    PubMed

    Gomes, Aldrin V; Zong, Chenggong; Edmondson, Ricky D; Berhane, Beniam T; Wang, Guang-Wu; Le, Steven; Young, Glen; Zhang, Jun; Vondriska, Thomas M; Whitelegge, Julian P; Jones, Richard C; Joshua, Irving G; Thyparambil, Sheeno; Pantaleon, Dawn; Qiao, Joe; Loo, Joseph; Ping, Peipei

    2005-06-01

    Multiprotein complexes have been increasingly recognized as essential functional units for a variety of cellular processes, including the protein degradation system. Selective degradation of proteins in eukaryotes is primarily conducted by the ubiquitin proteasome system. The current knowledge base, pertaining to the proteasome complexes in mammalian cells, relies largely upon information gained in the yeast system, where the 26S proteasome is hypothesized to contain a 20S multiprotein core complex and one or two 19S regulatory complexes. To date, the molecular structure of the proteasome system, the proteomic composition of the entire 26S multiprotein complexes, and the specific designated function of individual components within this essential protein degradation system in the heart remain virtually unknown. A functional proteomic approach, employing multidimensional chromatography purification combined with liquid chromatography tandem mass spectrometry and protein chemistry, was utilized to explore the murine cardiac 26S proteasome system. This article presents an overview on the subject of protein degradation in mammalian cells. In addition, this review shares the limited information that has been garnered thus far pertaining to the molecular composition, function, and regulation of this important organelle in the cardiac cells. PMID:16093497

  7. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

  8. Cdx2 represses Oct4 function via inducing its proteasome-dependent degradation in early porcine embryos.

    PubMed

    Bou, Gerelchimeg; Liu, Shichao; Guo, Jia; Zhao, Yueming; Sun, Mingju; Xue, Binghua; Wang, Jiaqiang; Wei, Yanchang; Kong, Qingran; Liu, Zhonghua

    2016-02-01

    Reciprocal repression of inner cell mass specific factor OCT4 and trophectoderm specific factor CDX2 promotes mouse first lineage segregation. Studies in mouse embryonic stem (ES) cells revealed that they bind to each other's regulatory regions to reciprocally suppress transcription, additionally they form protein complex for mutual antagonism. However, so far the molecular interaction of Oct4 and Cdx2 in other mammal's early embryo is not yet investigated. Here, over-expression of Cdx2 in early porcine embryo showed CDX2 represses Oct4 through neither the transcriptional repression nor forming repressive complex, but promoting OCT4 nuclear export and proteasomal degradation. The results showed novel molecular regulation of CDX2 on Oct4, and provided important clues for clarifying the mechanism of interaction between CDX2 and Oct4 in embryo of mammals other than mouse. PMID:26708097

  9. The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis

    PubMed Central

    Jang, Kiyoung; Gil Lee, Hong; Jung, Su-Jin; Paek, Nam-Chon; Joon Seo, Pil

    2015-01-01

    Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) was reduced because of its ambient temperature-dependent protein stability control, and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1, and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success. PMID:26159740

  10. Structural characterization of the interaction of Ubp6 with the 26S proteasome.

    PubMed

    Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, Andreas; Pfeifer, Günter; Goldberg, Alfred L; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich

    2015-07-14

    In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation. PMID:26130806

  11. Structural characterization of the interaction of Ubp6 with the 26S proteasome

    PubMed Central

    Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, Andreas; Pfeifer, Günter; Goldberg, Alfred L.; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich

    2015-01-01

    In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation. PMID:26130806

  12. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  13. [Structures and functions of the 26S proteasome Rpn10 family].

    PubMed

    Kawahara, Hiroyuki

    2002-09-01

    The ubiquitin-dependent proteolytic pathway is thought to be one of the vital systems for cellular regulations, including control of the cell cycle, differentiation and apoptosis. In this pathway, poly-ubiquitinated proteins are selectively degraded by the 26S proteasome, a multisubunit proteolytic machinery. Recognition of the poly-ubiquitin chain by the 26S proteasome should be a key step leading to the selective degradation of target proteins, and the Rpn10 subunit of the 26S proteasome has been shown to preferentially bind the poly-ubiquitin chain in vitro. We previously reported that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated, alternative splicing. To determine whether such alternative splicing mechanisms occur in organisms other than the mouse, we searched for Rpn10 isoforms in various species. Here we summarize the gene organization of the Rpn10 in lower species and provide evidence that the competence for generating all distinct forms of Rpn10 alternative splicing has expanded through evolution. Some of the Rpn10 family genes were found to be expressed in distinct developmental stages, suggesting that they have distinct functions during embryogenesis. For example, Rpn10c and Rpn10e were exclusively expressed at specific developmental stages and in specific tissues, while Rpn10a was expressed constitutively. Our experimental results indicate that the respective Rpn10 proteins possess distinct roles in the progression of development. Furthermore, some of the Rpn10 variants specifically interacted with important developmental regulators. PMID:12235853

  14. The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)...

  15. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome

    PubMed Central

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-01

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

  16. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome.

    PubMed

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-26

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

  17. Structure-Driven Developments of 26S Proteasome Inhibitors.

    PubMed

    Śledź, Paweł; Baumeister, Wolfgang

    2016-01-01

    The 26S proteasome is a 2.5-MDa complex, and it operates at the executive end of the ubiquitin-proteasome pathway. It is a proven target for therapeutic agents for the treatment of some cancers and autoimmune diseases, and moreover, it has potential as a target of antibacterial agents. Most inhibitors, including all molecules approved for clinical use, target the 20S proteolytic core complex; its structure was determined two decades ago. Hitherto, efforts to develop inhibitors targeting the 19S regulatory particle subunits have been less successful. This is, in part, because the molecular architecture of this subcomplex has been, until recently, poorly understood, and high-resolution structures have been available only for a few subunits. In this review, we describe, from a structural perspective, the development of inhibitory molecules that target both the 20S and 19S subunits of the proteasome. We highlight the recent progress achieved in structure-based drug-discovery approaches, and we discuss the prospects for further improvement. PMID:26738474

  18. DNA damage modulates interactions between microRNAs and the 26S proteasome

    PubMed Central

    Tsimokha, Anna S; Kulichkova, Valentina A.; Karpova, Elena V.; Zaykova, Julia J.; Aksenov, Nikolai D; Vasilishina, Anastasia A.; Kropotov, Andrei V.; Antonov, Alexey; Barlev, Nikolai A.

    2014-01-01

    26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs. PMID:25004448

  19. Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

    PubMed Central

    Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

    2015-01-01

    ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome

  20. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators. PMID:27155164

  1. Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13*

    PubMed Central

    Berko, Dikla; Herkon, Ora; Braunstein, Ilana; Isakov, Elada; David, Yael; Ziv, Tamar; Navon, Ami; Stanhill, Ariel

    2014-01-01

    The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps. PMID:24429290

  2. The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies.

    PubMed

    Nabhan, Joseph F; El-Shehabi, Fouad; Patocka, Nicholas; Ribeiro, Paula

    2007-11-01

    The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. Here, we used a bioinformatics approach to identify the proteasomal constituents of the parasitic trematode Schistosoma mansoni. A detailed search of the S. mansoni genome database identified a total of 31 putative proteasomal subunits, including 17 subunits of the regulatory (19S) complex and 14 predicted catalytic (20S) subunits. A quantitative real-time RT-PCR analysis of subunit expression levels revealed that the S. mansoni proteasome components are differentially expressed among cercaria, schistosomula, and adult worms. In particular, the data suggest that the proteasome may be downregulated during the early stages of schistosomula development and is subsequently upregulated as the parasite matures to the adult stage. To test for biological relevance, we developed a transfection-based RNA interference method to knockdown the expression of the proteasome subunit, SmRPN11/POH1. Transfection of in vitro transformed S. mansoni schistosomula with specific short-interfering RNAs (siRNAs) diminished SmRPN11/POH1 expression nearly 80%, as determined by quantitative RT-PCR analysis, and also decreased parasite viability 78%, whereas no significant effect could be seen after treatment with the same amount of an irrelevant siRNA. These results indicate that the subunit SmRPN11/POH1 is an essential gene in schistosomes and further suggest an important role for the proteasome in parasite development and survival. PMID:17892869

  3. Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    PubMed Central

    Li, Sheng; Yang, Li-juan; Wang, Ping; He, Yu-jiao; Huang, Jun-mei; Liu, Han-wei; Shen, Xiao-fei; Wang, Fei

    2016-01-01

    Background Type I interferons (IFN-α/β) have broad and potent immunoregulatory and antiproliferative activities. However, it is still known whether the dietary flavonoids exhibit their antiviral and anticancer properties by modulating the function of type I IFNs. Objective This study aimed at determining the role of apigenin, a dietary plant flavonoid abundant in common fruits and vegetables, on the type I IFN-mediated inhibition of cancer cell viability. Design Inhibitory effect of apigenin on human 26S proteasome, a known negative regulator of type I IFN signaling, was evaluated in vitro. Molecular docking was conducted to know the interaction between apigenin and subunits of 26S proteasome. Effects of apigenin on JAK/STAT pathway, 26S proteasome-mediated interferon receptor stability, and cancer cells viability were also investigated. Results Apigenin was identified to be a potent inhibitor of human 26S proteasome in a cell-based assay. Apigenin inhibited the chymotrypsin-like, caspase-like, and trypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Results from computational modeling of the potential interactions of apigenin with the chymotrypsin site (β5 subunit), caspase site (β1 subunit), and trypsin site (β2 subunit) of the proteasome were consistent with the observed proteasome inhibitory activity. Apigenin enhanced the phosphorylation of signal transducer and activator of transcription proteins (STAT1 and STAT2) and promoted the endogenous IFN-α-regulated gene expression. Apigenin inhibited the IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Apigenin also sensitized the inhibitory effect of IFN-α on viability of cervical carcinoma HeLa cells. Conclusion These results suggest that apigenin potentiates the inhibitory effect of IFN-α on cancer cell viability by activating JAK/STAT signaling pathway through inhibition of 26S proteasome

  4. Structure of the human 26S proteasome at a resolution of 3.9 Å

    PubMed Central

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M.; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-01-01

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  5. Structure of the human 26S proteasome at a resolution of 3.9 Å.

    PubMed

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-07-12

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  6. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

    PubMed Central

    Dahlmann, B; Kuehn, L; Reinauer, H

    1995-01-01

    The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7619056

  7. The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome*

    PubMed Central

    Jacobson, Andrew D.; Zhang, Nan-Yan; Xu, Ping; Han, Ke-Jun; Noone, Seth; Peng, Junmin; Liu, Chang-Wei

    2009-01-01

    The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates. PMID:19858201

  8. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

    PubMed Central

    Lasker, Keren; Förster, Friedrich; Bohn, Stefan; Walzthoeni, Thomas; Villa, Elizabeth; Unverdorben, Pia; Beck, Florian; Aebersold, Ruedi; Sali, Andrej; Baumeister, Wolfgang

    2012-01-01

    The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates. PMID:22307589

  9. Indirect inhibition of 26S proteasome activity in a cellular model of Huntington’s disease

    PubMed Central

    Hipp, Mark S.; Patel, Chetan N.; Bersuker, Kirill; Riley, Brigit E.; Kaiser, Stephen E.; Shaler, Thomas A.; Brandeis, Michael

    2012-01-01

    Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington’s disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt—whether aggregated or not—did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network. PMID:22371559

  10. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

    PubMed Central

    Hampton, R Y; Gardner, R G; Rine, J

    1996-01-01

    3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the

  11. Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii

    PubMed Central

    Gogliettino, Marta; Balestrieri, Marco; Riccio, Alessia; Facchiano, Angelo; Fusco, Carmela; Palazzo, Vincenzo Cecere; Rossi, Mosè; Cocca, Ennio; Palmieri, Gianna

    2016-01-01

    Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its ‘uncommon’ ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery. PMID:26933238

  12. The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway[C][W

    PubMed Central

    Yan, Jianbin; Li, Haiou; Li, Shuhua; Yao, Ruifeng; Deng, Haiteng; Xie, Qi; Xie, Daoxin

    2013-01-01

    Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCFCOI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCFCOI1 complexes, suggesting a function for SCFCOI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses. PMID:23386265

  13. Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

    PubMed

    Moulédous, Lionel; Neasta, Jérémie; Uttenweiler-Joseph, Sandrine; Stella, Alexandre; Matondo, Mariette; Corbani, Maïthé; Monsarrat, Bernard; Meunier, Jean-Claude

    2005-08-01

    The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role. PMID:15901846

  14. Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition

    PubMed Central

    Dambacher, Corey M; Worden, Evan J; Herzik, Mark A; Martin, Andreas; Lander, Gabriel C

    2016-01-01

    The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the ‘lid’ sub-complex. Prior to the lid’s incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation. DOI: http://dx.doi.org/10.7554/eLife.13027.001 PMID:26744777

  15. Near-atomic resolution structural model of the yeast 26S proteasome

    PubMed Central

    Beck, Florian; Unverdorben, Pia; Bohn, Stefan; Schweitzer, Andreas; Pfeifer, Günter; Sakata, Eri; Nickell, Stephan; Plitzko, Jürgen M.; Villa, Elizabeth; Baumeister, Wolfgang; Förster, Friedrich

    2012-01-01

    The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits. PMID:22927375

  16. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

    PubMed Central

    Eytan, E; Ganoth, D; Armon, T; Hershko, A

    1989-01-01

    Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway. Images PMID:2554287

  17. Structure of an endogenous yeast 26S proteasome reveals two major conformational states

    PubMed Central

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J.; Shi, Yigong; Wang, Feng

    2016-01-01

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function. PMID:26929360

  18. D1 dopamine receptor stimulation impairs striatal proteasome activity in Parkinsonism through 26S proteasome disassembly.

    PubMed

    Barroso-Chinea, Pedro; Thiolat, Marie-Laure; Bido, Simone; Martinez, Audrey; Doudnikoff, Evelyne; Baufreton, Jérôme; Bourdenx, Mathieu; Bloch, Bertrand; Bezard, Erwan; Martin-Negrier, Marie-Laure

    2015-06-01

    Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization. PMID:25766677

  19. Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.

    PubMed

    Concannon, Claire; Lahue, Robert S

    2014-01-01

    Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions. PMID:24359926

  20. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome.

    PubMed

    Unverdorben, Pia; Beck, Florian; Śledź, Paweł; Schweitzer, Andreas; Pfeifer, Günter; Plitzko, Jürgen M; Baumeister, Wolfgang; Förster, Friedrich

    2014-04-15

    The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle. PMID:24706844

  1. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

    PubMed Central

    Unverdorben, Pia; Beck, Florian; Śledź, Paweł; Schweitzer, Andreas; Pfeifer, Günter; Plitzko, Jürgen M.; Baumeister, Wolfgang; Förster, Friedrich

    2014-01-01

    The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin–proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle. PMID:24706844

  2. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

    PubMed

    Peth, Andreas; Nathan, James A; Goldberg, Alfred L

    2013-10-01

    The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost. PMID:23965995

  3. The ubiquitin–protein ligase Itch regulates p73 stability

    PubMed Central

    Rossi, Mario; De Laurenzi, Vincenzo; Munarriz, Eliana; Green, Douglas R; Liu, Yun-Cai; Vousden, Karen H; Cesareni, Gianni; Melino, Gerry

    2005-01-01

    p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post-transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal-dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome-dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions. PMID:15678106

  4. The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

    PubMed Central

    Grode, Kyle D.; Rogers, Stephen L.

    2015-01-01

    Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules. PMID:25886649

  5. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    PubMed

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. PMID:26945516

  6. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    SciTech Connect

    Kwak, Mi-Kyoung . E-mail: mkwak@yumail.ac.kr; Kensler, Thomas W.

    2006-07-14

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

  7. Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis.

    PubMed

    Marshall, Richard S; Li, Faqiang; Gemperline, David C; Book, Adam J; Vierstra, Richard D

    2015-06-18

    Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes. PMID:26004230

  8. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    SciTech Connect

    Foerster, Friedrich; Lasker, Keren; 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.

  9. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome.

    PubMed

    Förster, Friedrich; Lasker, Keren; 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. PMID:19653995

  10. An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

    PubMed

    Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing; Marko, John F; Matouschek, Andreas

    2016-09-15

    The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. PMID:27296635

  11. The ORC1 cycle in human cells: I. cell cycle-regulated oscillation of human ORC1.

    PubMed

    Tatsumi, Yasutoshi; Ohta, Satoshi; Kimura, Hiroshi; Tsurimoto, Toshiki; Obuse, Chikashi

    2003-10-17

    Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring. PMID:12909627

  12. Growth and developmental functions of a human immunodeficiency virus Tat-binding protein/26S protease subunit homolog from Dictyostelium discoideum.

    PubMed Central

    Cao, J G; Firtel, R A

    1995-01-01

    We have characterized a newly identified gene from Dictyostelium discoideum, DdTBP alpha, that encodes a member of the family of eukaryotic proteins. These proteins contain a conserved ATPase domain, include subunits of the 26S protease subunit, and are homologous to the mammalian human immunodeficiency virus Tat-binding protein TBP1. While information indicates that some family members are involved in the regulation of transcription in mammalian and yeast cells during growth, these proteins are also involved in other cellular functions, and nothing is known about their possible function in multicellular development. The Dictyostelium DdTBP alpha gene is developmentally regulated, with its expression at the highest levels occurring during growth and early development. The gene is present in two copies in the genome. Disruption of one copy by homologous recombination leads to aberrant morphogenesis, which lasts from the formation of the first finger until the onset of culmination. The gene appears to be essential for growth since we were unable to obtain a complete null phenotype and since expression of an inducible antisense construct in the partial null background resulted in cell death. Expression of the antisense construct during development accentuated the partial null phenotype and also resulted in very abnormal fruiting bodies. Overexpression of DdTBP alpha from its own promoter leads to very large multinucleated vegetative cells when the cells are grown in suspension culture. When the cells are plated onto petri dishes in growth medium, they rapidly split into multiple cells containing one to two nuclei, in a manner similar to that of wild-type cells. Overexpressing cells are significantly delayed in forming a multicellular aggregate, but development proceeds normally once the first finger stage is reached. The results indicate that DdTBP alpha plays an important role in regulating both growth and morphogenesis in D. discoideum. PMID:7862164

  13. Hyperfine structure of the 4{f}^{8}5{d}^{2}6s configuration in the Tb atom

    NASA Astrophysics Data System (ADS)

    Furmann, B.; Stefanska, D.; Krzykowski, A.

    2016-01-01

    Within this work new experimental results concerning the hyperfine structure (hfs) in the terbium atom are presented. Hfs constants A and B for eight levels belonging to the even-parity configuration 4{f}85{d}26s were determined, based on the results of measurements performed using the laser-induced fluorescence method in a hollow cathode discharge at 18 spectral lines. The configuration 4{f}85{d}26s in the terbium atom was hitherto very scarcely investigated; for seven of the levels examined within this work results concerning the hfs were obtained for the first time. As a by-product, hfs constants for 12 odd-parity levels, involved as upper levels in the transitions investigated, were also determined. A preliminary attempt at a semi-empirical analysis of Tb I hfs on a multi-configuration basis, based on the results of this work, yielded a value of the one-electron {a}6s10 parameter as well as the respective radial integral {< {r}-3> }6s10, which can be compared with the values along the lanthanide elements series reported in the literature. More conclusive results can certainly be obtained once the experimental database for Tb I becomes more extensive.

  14. Ca2O3Fe2.6S2: an antiferromagnetic Mott insulator at proximity to bad metal

    NASA Astrophysics Data System (ADS)

    Zhang, Han; Wu, Xiaozhi; Li, Dandan; Jin, Shifeng; Chen, Xiao; Zhang, Tao; Lin, Zhiping; Shen, Shijie; Yuan, Duanduan; Chen, Xiaolong

    2016-04-01

    We report here the first layered iron oxychalcogenide Ca2O3Fe2.6S2 that contains both planar [Ca2FeO2]2+ and [Fe2OS2]2- layers with the shortest Fe-Fe bond length. This compound is a narrow band gap (~0.073 eV) Mott insulator. The observed antiferromagnetic (AFM) transition at 77 K is due to the ordered Fe vacancies, which can be suppressed by partial substitution of Se for S. We show that the vacancy-free phase Ca2O3Fe3S2 may become a metal with moderate electron correlation comparable to the parent compound LaOFeAs of corresponding superconductors. Our results imply that iron oxychalcogenide can be converted from an AFM Mott insulator into a bad metal like iron pnictides through Fe-Fe bond length shrinking.

  15. Genomic organization and mapping of the mouse P26s4 ATPase gene: A member of the remarkably conserved AAA gene family

    SciTech Connect

    Hoyle, J.; Fisher, E.M.C.

    1996-01-01

    The eukaryotic genome contains a large family of ATPases in which each member has at least one highly conserved domain of approximately 200 amino acids with an ATP binding motif (the {open_quotes}AAA{close_quotes} domain). AAA ATPases play diverse roles in the cell and are of considerable interest to researchers investigating a number of different phenomena, including control of the cell cycle. We have characterized the mouse P26s4 AAA ATPase gene that encodes a subunit of the 26S protease, a multimeric complex that is responsible for the ubiquitin- and ATP-dependent degradation of specific proteins. The normal functioning of eukaryotic cells depends on this pathway to remove regulatory proteins such as cyclins or signal transduction molecules from the intracellular environment, with the appropriate timing to allow normal cell division and development. We have isolated mouse P26s4 cDNAs and mapped the P26s4 gene to chromosome 12. We have analyzed the intron-exon structure of the P26s4 genomic locus and have determined that the gene contains at least 10 introns, the first of which separates the start methionine from the rest of the coding sequence. 18 refs., 2 figs.

  16. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene

    PubMed Central

    Czub, Barbara; Shah, Amna Z.; Alfano, Giovanna; Kruczek, Przemysław M.; Chakarova, Christina F.; Bhattacharya, Shomi S.

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS. PMID:26872363

  17. Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

    PubMed Central

    Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. Objective We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. Methods Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. Results M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. Conclusion Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only. PMID:21747616

  18. Epidemiologic Study of Malassezia Yeasts in Acne Patients by Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Song, Young Chan; Hahn, Hyung Jin; Kim, Ji Young; Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background Although acne is a common follicular inflammatory dermatosis, studies of the relationship between Malassezia yeasts and acne have rarely been conducted. Objective We sought to identify Malassezia yeasts from acne patients and establish a relationship between specific types of species of Malassezia and acne. Methods Sixty acne patients were enrolled. Each strain obtained was identified as one of eleven species by 26S rDNA PCR-RFLP. We then compared these data with those of age- and sex-matched healthy subjects. Results Growth of Malassezia was evident in fewer patients with acne (50%) in comparison to controls (70.6%). M. restricta was dominant in patients with acne (23.9%), whereas M. globosa was most common (26.7%) in healthy controls. In the patients group, the rate was the highest (71.7%) in the twenties and, in terms of body site, the rate was the highest (60%) in the chest. In the control group, the rate was the highest (75.0%) in the thirties and in the forehead (85.0%). Conclusion The detection rate of Malassezia yeasts was conspicuously low in the acne patients group. Statistically significant differences were observed between the patient and the control groups in the twenties and thirties, and in terms of body site, in the forehead and chest. PMID:21909202

  19. Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

    SciTech Connect

    Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu; Kim, Ki-Jeong; Park, Chang-Jin; Kim, Young-Jin; Park, Ohkmae K.; Paek, Kyung-Hee . E-mail: khpaek95@korea.ac.kr

    2006-12-15

    Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P{sub 0} interaction, but not during compatible TMV-P{sub 1.2} interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.

  20. Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers.

    PubMed

    Suezawa, Yasuhiko; Suzuki, Motofumi; Mori, Haruhiko

    2008-09-01

    We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species. PMID:18776675

  1. Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae.

    PubMed Central

    Kominami, K; DeMartino, G N; Moomaw, C R; Slaughter, C A; Shimbara, N; Fujimuro, M; Yokosawa, H; Hisamatsu, H; Tanahashi, N; Shimizu, Y

    1995-01-01

    The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p. Images PMID:7621825

  2. The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

    PubMed

    Lee, Kwang-Hee; Minami, Atsushi; Marshall, Richard S; Book, Adam J; Farmer, Lisa M; Walker, Joseph M; Vierstra, Richard D

    2011-12-01

    The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly. PMID:22158466

  3. Expression of the 26S proteasome subunit RPN10 is upregulated by salt stress in Dunaliella viridis.

    PubMed

    Sun, Xiaobin; Meng, Xiangzong; Xu, Zhengkai; Song, Rentao

    2010-08-15

    Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis. PMID:20430475

  4. Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim.

    PubMed

    Moujalled, Diane; Weston, Ross; Anderton, Holly; Ninnis, Robert; Goel, Pranay; Coley, Andrew; Huang, David C S; Wu, Li; Strasser, Andreas; Puthalakath, Hamsa

    2011-01-01

    The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy. PMID:21151042

  5. Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

    PubMed Central

    Haller, Martina; Hock, Andreas K; Giampazolias, Evangelos; Oberst, Andrew; Green, Douglas R; Debnath, Jayanta; Ryan, Kevin M; Vousden, Karen H; Tait, Stephen W G

    2015-01-01

    During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12. PMID:25629932

  6. CERAMIDE SYNTHASE 1 IS REGULATED BY PROTEASOMAL MEDIATED TURNOVER

    PubMed Central

    Sridevi, Priya; Alexander, Hannah; Laviad, Elad L.; Pewzner-Jung, Yael; Hannink, Mark; Futerman, Anthony H.; Alexander, Stephen

    2009-01-01

    Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis. PMID:19393694

  7. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  8. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

    PubMed Central

    Gardner, R C; Assinder, S J; Christie, G; Mason, G G; Markwell, R; Wadsworth, H; McLaughlin, M; King, R; Chabot-Fletcher, M C; Breton, J J; Allsop, D; Rivett, A J

    2000-01-01

    Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells. PMID:10677365

  9. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

  10. Autophagic Turnover of Inactive 26S Proteasomes in Yeast Is Directed by the Ubiquitin Receptor Cue5 and the Hsp42 Chaperone.

    PubMed

    Marshall, Richard S; McLoughlin, Fionn; Vierstra, Richard D

    2016-08-01

    The autophagic clearance of 26S proteasomes (proteaphagy) is an important homeostatic mechanism within the ubiquitin system that modulates proteolytic capacity and eliminates damaged particles. Here, we define two proteaphagy routes in yeast that respond to either nitrogen starvation or particle inactivation. Whereas the core autophagic machineries required for Atg8 lipidation and vesiculation are essential for both routes, the upstream Atg1 kinase participates only in starvation-induced proteaphagy. Following inactivation, 26S proteasomes become extensively modified with ubiquitin. Although prior studies with Arabidopsis implicated RPN10 in tethering ubiquitylated proteasomes to ATG8 lining the autophagic membranes, yeast proteaphagy employs the evolutionarily distinct receptor Cue5, which simultaneously binds ubiquitin and Atg8. Proteaphagy of inactivated proteasomes also requires the oligomeric Hsp42 chaperone, suggesting that ubiquitylated proteasomes are directed by Hsp42 to insoluble protein deposit (IPOD)-type structures before encapsulation. Together, Cue5 and Hsp42 provide a quality control checkpoint in yeast directed at recycling dysfunctional 26S proteasomes. PMID:27477278

  11. Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling

    PubMed Central

    Myeku, Natura; Clelland, Catherine L; Emrani, Sheina; Kukushkin, Nikolay V; Yu, Wai Haung; Goldberg, Alfred L; Duff, Karen E

    2016-01-01

    The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP–protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance. PMID:26692334

  12. MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells.

    PubMed

    Xie, Chen; Chen, Wei; Zhang, Mengdie; Cai, Qiuxian; Xu, Weiyi; Li, Xiaodi; Jiang, Songshan

    2015-07-01

    Despite extensive investigation into the role of let-7 miRNAs in pathological tumor processes, their involvement in the DNA damage response remains unclear. Here we show that most let-7 family members down-regulate MDM4 expression via binding to MDM4 mRNA at a conserved DNA sequence. Expression of exogenous let-7 miRNA mimics decreased MDM4 protein but not mRNA levels. Several DNA damage reagents increased let-7 expression, thereby decreasing MDM4 protein levels in glioma cells. Inhibition of endogenous let-7 with antisense RNAs rescued MDM4 protein levels with or without MG132, a proteasome-dependent degradation inhibitor. An MDM4 mutation identified in a glioma patient was associated with loss of the putative MDM4 target site. Therefore, let-7 binding to MDM4 is implicated in the DNA damage response. PMID:26028311

  13. Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: preliminary application to Physalis fruits from Egypt.

    PubMed

    El Sheikha, Aly Farag; Condur, Ana; Métayer, Isabelle; Nguyen, Doan Duy Le; Loiseau, Gérard; Montet, Didier

    2009-10-01

    The determination of geographical origin is a demand of the traceability system of import-export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR-DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location. PMID:19784935

  14. The porcine gene TBP10 encodes a protein homologous to the human tat-binding protein/26S protease subunit family.

    PubMed

    Leeb, T; Rettenberger, G; Breech, J; Hameister, H; Brenig, B

    1996-03-01

    We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region. PMID:8833236

  15. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    PubMed Central

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  16. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

    PubMed

    Maurer, Matthew J; Spear, Eric D; Yu, Allen T; Lee, Evan J; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  17. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  18. Gene Expression Analysis of the 26S Proteasome Subunit PSMB4 Reveals Significant Upregulation, Different Expression and Association with Proliferation in Human Pulmonary Neuroendocrine Tumours

    PubMed Central

    Mairinger, Fabian Dominik; Walter, Robert Fred Henry; Theegarten, Dirk; Hager, Thomas; Vollbrecht, Claudia; Christoph, Daniel Christian; Worm, Karl; Ting, Saskia; Werner, Robert; Stamatis, Georgios; Mairinger, Thomas; Baba, Hideo; Zarogoulidis, Konstantinos; Huang, Haidong; Li, Qiang; Tsakiridis, Kosmas; Zarogoulidis, Paul; Schmid, Kurt Werner; Wohlschlaeger, Jeremias

    2014-01-01

    Background: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. Material and methods: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. Results: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). Conclusion: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation. PMID:25157275

  19. Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

    PubMed Central

    Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

    2013-01-01

    The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

  20. Oxidative Stress-Mediated Regulation of Proteasome Complexes*

    PubMed Central

    Aiken, Charity T.; Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways. PMID:21543789

  1. CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

    PubMed

    Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

    2016-08-01

    Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1. PMID:27178152

  2. Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

    PubMed

    Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

    2015-06-01

    Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis

  3. The E3 ubiquitin ligase WWP1 regulates {Delta}Np63-dependent transcription through Lys63 linkages

    SciTech Connect

    Peschiaroli, Angelo; Scialpi, Flavia; Bernassola, Francesca; Sherbini, El Said El; Melino, Gerry

    2010-11-12

    Research highlights: {yields} WWP1 ubiquitylates {Delta}Np63 through conjugation of Lys63-linked poly-ubiquitin chains. {yields} WWP1 does not control {Delta}Np63 protein stability. {yields} WWP1 regulates {Delta}Np63-dependent transcription. -- Abstract: The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and tumorigenesis through the regulation of epithelial progenitor cell proliferation, differentiation and apoptosis. Similarly to p53, p63 activity is regulated by post-translational modifications, including ubiquitylation. Here, we report that the WWP1 E3 ubiquitin ligase binds specifically to {Delta}Np63 isoform but it does not trigger {Delta}Np63 proteasome-dependent degradation. Accordingly, we found that WWP1-dependent ubiquitylation of {Delta}Np63 occurs through the formation of Lys63-linked poly-ubiquitin chains. Importantly, we found that WWP1 is able to increase {Delta}Np63-dependent transcription and depletion of WWP1 in human primary keratinocytes induces cell cycle arrest. All together these results indicate that WWP1 regulates {Delta}Np63 transcriptional activity, acting thus as a potential regulator of the proliferation and survival of epithelial-derived cells.

  4. Physical mapping of 5S and 18S-5.8S-26S RNA gene families in polyploid series of Cenchrus ciliaris Linnaeus, 1771 (Poaceae)

    PubMed Central

    Kharrat-Souissi, Amina; Siljak-Yakovlev, Sonja; Pustahija, Fatima; Chaieb, Mohamed

    2012-01-01

    Abstract The Buffelgrass (Cenchrus ciliaris L., Poaceae) is one of the most important pasturage grasses due to its high productivity and good forage qualities. This species possess a high adaptability to bioclimatic constraints of arid zones and may be used for the restoration of degraded arid ecosystems. Tunisian populations present three ploidy levels (4x, 5x and 6x) with a basic chromosome number x=9. This study reported for the first time the distribution of the ribosomal genes (rRNA) for pentaploid and hexaploid cytotypes of Cenchrus ciliaris. Molecular cytogenetic study using double fluorescence in situ hybridization has shown that the two rDNA families, 5S and 18S-5.8S-26S (18S), displayed intraspecific variation in number of loci among different ploidy levels. Each ploidy level was characterized by specific number of both 5S and 18S rDNA loci (two loci in tetraploid, five in pentaploid and six in hexaploid level). For three studied cytotypes (4x, 5x and 6x) all 5S rDNA loci were localized on the subcentromeric region of chromosomes, while 18S loci were situated on the telomeric region of short chromosome arms. Data of the FISH experiments show proportional increase of ribosomal loci number during polyploidization processes. PMID:24260668

  5. In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

    PubMed Central

    Inácio, João; Behrens, Sebastian; Fuchs, Bernhard M.; Fonseca, Álvaro; Spencer-Martins, Isabel; Amann, Rudolf

    2003-01-01

    Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring. PMID:12732564

  6. Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

    2010-01-01

    Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

  7. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

    PubMed

    Schlax, Peter E; Zhang, Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T Glen

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination. PMID:17150238

  8. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    SciTech Connect

    Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen . E-mail: tlawson@bates.edu

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

  9. The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes.

    PubMed

    Żabka, Aneta; Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

    2015-11-01

    DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities. PMID:26233708

  10. Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

    PubMed

    Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

    2013-01-01

    Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

  11. Heat shock protein 70 regulates Tcl1 expression in leukemia and lymphomas

    PubMed Central

    Gaudio, Eugenio; Paduano, Francesco; Ngankeu, Apollinaire; Lovat, Francesca; Fabbri, Muller; Sun, Hui-Lung; Gasparini, Pierluigi; Efanov, Alexey; Peng, Yong; Zanesi, Nicola; Shuaib, Mohammed A.; Rassenti, Laura Z.; Kipps, Thomas J.; Li, Chenglong; Aqeilan, Rami I.; Lesinski, Gregory B.; Trapasso, Francesco

    2013-01-01

    T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The inhibition of Hsp70 significantly reduced the growth of lymphoma xenografts in vivo and down-regulated the expression of Tcl1 protein. Our findings reveal a functional interaction between Tcl1 and Hsp70 and identify Tcl1 as a novel Hsp70 client protein. These findings suggest that inhibition of Hsp70 may represent an alternative effective therapy for chronic lymphocytic leukemia and lymphomas via its ability to inhibit the oncogenic functions of Tcl1. PMID:23160471

  12. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1.

    PubMed

    Brady, Owen A; Meng, Peter; Zheng, Yanqiu; Mao, Yuxin; Hu, Fenghua

    2011-01-01

    TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, such as frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Phosphorylated and ubiquitinated TDP-43 C-terminal fragments have been found in cytoplasmic inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis patients. However, the factors and pathways that regulate TDP-43 aggregation are still not clear. We found that the C-terminal 15 kDa fragment of TDP-43 is sufficient to induce aggregation but the aggregation phenotype is modified by additional sequences. Aggregation is accompanied by phosphorylation at serine residues 409/410. Mutation of 409/410 to phosphomimetic aspartic acid residues significantly reduces aggregation. Inhibition of either proteasome or autophagy dramatically increases TDP-43 aggregation. Furthermore, TDP-43 aggregates colocalize with markers of autophagy and the adaptor protein p62/SQSTM1. Over-expression of p62/SQSTM1 reduces TDP-43 aggregation in an autophagy and proteasome-dependent manner. These studies suggest that aggregation of TDP-43 C-terminal fragments is regulated by phosphorylation events and both the autophagy and proteasome-mediated degradation pathways. PMID:21062285

  13. Zyxin is a critical regulator of the apoptotic HIPK2-p53 signaling axis.

    PubMed

    Crone, Johanna; Glas, Carolina; Schultheiss, Kathrin; Moehlenbrink, Jutta; Krieghoff-Henning, Eva; Hofmann, Thomas G

    2011-03-15

    HIPK2 activates the apoptotic arm of the DNA damage response by phosphorylating tumor suppressor p53 at serine 46. Unstressed cells keep HIPK2 levels low through targeted polyubiquitination and subsequent proteasomal degradation. Here we identify the LIM domain protein Zyxin as a novel regulator of the HIPK2-p53 signaling axis in response to DNA damage. Remarkably, depletion of endogenous Zyxin, which colocalizes with HIPK2 at the cytoskeleton and in the cell nucleus, stimulates proteasome-dependent HIPK2 degradation. In contrast, ectopic expression of Zyxin stabilizes HIPK2, even upon enforced expression of its ubiquitin ligase Siah-1. Consistently, Zyxin physically interacts with Siah-1, and knock-down of Siah-1 rescues HIPK2 expression in Zyxin-depleted cancer cells. Mechanistically, our data suggest that Zyxin regulates Siah-1 activity through interference with Siah-1 dimerization. Furthermore, we show that endogenous Zyxin coaccumulates with HIPK2 in response to DNA damage in cancer cells, and that depletion of endogenous Zyxin results in reduced HIPK2 protein levels and compromises DNA damage-induced p53 Ser46 phosphorylation and caspase activation. These findings suggest an unforeseen role for Zyxin in DNA damage-induced cell fate control through modulating the HIPK2-p53 signaling axis. PMID:21248071

  14. CUEDC2 modulates cardiomyocyte oxidative capacity by regulating GPX1 stability.

    PubMed

    Jian, Zhao; Liang, Bing; Pan, Xin; Xu, Guang; Guo, Sai-Sai; Li, Ting; Zhou, Tao; Xiao, Ying-Bin; Li, Ai-Ling

    2016-01-01

    The irreversible loss of cardiomyocytes due to oxidative stress is the main cause of heart dysfunction following ischemia/reperfusion (I/R) injury and ageing-induced cardiomyopathy. Here, we report that CUEDC2, a CUE domain-containing protein, plays a critical role in oxidative stress-induced cardiac injury. Cuedc2(-/-) cardiomyocytes exhibited a greater resistance to oxidative stress-induced cell death. Loss of CUEDC2 enhanced the antioxidant capacity of cardiomyocytes, promoted reactive oxygen species (ROS) scavenging, and subsequently inhibited the redox-dependent activation of signaling pathways. Notably, CUEDC2 promoted E3 ubiquitin ligases tripartite motif-containing 33 (TRIM33)-mediated the antioxidant enzyme, glutathione peroxidase 1 (GPX1) ubiquitination, and proteasome-dependent degradation. Ablation of CUEDC2 upregulated the protein level of GPX1 in the heart significantly. Strikingly, in vivo, the infarct size of Cuedc2(-/-) heart was significantly decreased after I/R injury, and aged Cuedc2(-/-) mice preserved better heart function as the overall ROS levels in their hearts were significantly lower. Our results demonstrated a novel role of CUEDC2 in cardiomyocyte death regulation. Manipulating CUEDC2 level might be an attractive therapeutic strategy for promoting cardiomyocyte survival following oxidative stress-induced cardiac injury. PMID:27286733

  15. Identification and Regulation of Plasma Membrane Sulfate Transporters in Chlamydomonas1[W][OA

    PubMed Central

    Pootakham, Wirulda; Gonzalez-Ballester, David; Grossman, Arthur R.

    2010-01-01

    Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO42−). Aspects of SO42− transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO42− transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO42− transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO42− transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO42− into S-deprived cells. PMID:20498339

  16. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    PubMed Central

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

  17. Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome†

    PubMed Central

    Wilson, Heather L.; Ou, Mark S.; Aldrich, Henry C.; Maupin-Furlow, Julie

    2000-01-01

    The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high Vmax for ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Pi per min per mg of protein as well as a relatively low affinity for CTP and ATP with Km values of 307 and 497

  18. Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7)

    PubMed Central

    Ding, Xiaodan; Jiang, Wei; Zhou, Peipei; Liu, Lulu; Wan, Xiaoling; Yuan, Xiujie; Wang, Xizi; Chen, Miao; Chen, Jun; Yang, Jing; Kong, Chao; Li, Bin; Peng, Chao; Wong, Catherine C. L.; Hou, Fajian; Zhang, Yan

    2015-01-01

    Mixed lineage leukemia 5 (MLL5) protein is a trithorax family histone 3 lysine 4 (H3K4) methyltransferase that regulates diverse biological processes, including cell cycle progression, hematopoiesis and cancer. The mechanisms by which MLL5 protein stability is regulated have remained unclear to date. Here, we showed that MLL5 protein stability is cooperatively regulated by O-GlcNAc transferase (OGT) and ubiquitin-specific protease 7 (USP7). Depletion of OGT in cells led to a decrease in the MLL5 protein level through ubiquitin/proteasome-dependent proteolytic degradation, whereas ectopic expression of OGT protein suppressed MLL5 ubiquitylation. We further identified deubiquitinase USP7 as a novel MLL5-associated protein using mass spectrometry. USP7 stabilized the MLL5 protein through direct binding and deubiquitylation. Loss of USP7 induced degradation of MLL5 protein. Conversely, overexpression of USP7, but not a catalytically inactive USP7 mutant, led to decreased ubiquitylation and increased MLL5 stability. Co-immunoprecipitation and co-immunostaining assays revealed that MLL5, OGT and USP7 interact with each other to form a stable ternary complex that is predominantly located in the nucleus. In addition, upregulation of MLL5 expression was correlated with increased expression of OGT and USP7 in human primary cervical adenocarcinomas. Our results collectively reveal a novel molecular mechanism underlying regulation of MLL5 protein stability and provide new insights into the functional interplay among O-GlcNAc transferase, deubiquitinase and histone methyltransferase. PMID:26678539

  19. Essential Phosphatases and a Phospho-Degron Are Critical for Regulation of SRC-3/AIB1 Coactivator Function and Turnover

    PubMed Central

    Li, Chao; Liang, Yao-Yun; Feng, Xin-Hua; Tsai, Sophia Y.; Tsai, Ming-Jer; O’Malley, Bert W.

    2008-01-01

    SRC-3/AIB1 is a master growth coactivator and oncogene, and phosphorylation activates it into a powerful coregulator. Dephosphorylation is a potential regulatory mechanism for SRC-3 function but the identity of such phosphatases remains unexplored. Herein, we report that using functional genomic screening of human Ser/Thr phosphatases targeting SRC-3’s known phosphorylation sites, the phosphatases PDXP, PP1 and PP2A were identified to be key negative regulators of SRC-3 transcriptional coregulatory activity in steroid receptor signalings. PDXP and PP2A dephosphorylate SRC-3 and inhibit its ligand-dependent association with estrogen receptor. PP1 stabilizes SRC-3 protein by blocking its proteasome-dependent turnover through dephosphorylation of two previously unidentified phosphorylation sites (Ser101 and S102) required for activity. These two sites are located within a degron of SRC-3, and are primary determinants of SRC-3 turnover. Moreover, PP1 regulates the oncogenic cell proliferation and invasion functions of SRC-3 in breast cancer cells. PMID:18922467

  20. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response.

    PubMed

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-01-01

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage. PMID:26238070

  1. 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

  2. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    SciTech Connect

    Yamagata, Kazutsune; Kitabayashi, Issay

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  3. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response

    PubMed Central

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-01-01

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage. PMID:26238070

  4. Emerging Mechanistic Insights into AAA Complexes Regulating Proteasomal Degradation

    PubMed Central

    Förster, Friedrich; Schuller, Jan M.; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system (UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells. It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates. The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases, shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship. Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  5. Emerging mechanistic insights into AAA complexes regulating proteasomal degradation.

    PubMed

    Förster, Friedrich; Schuller, Jan M; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system(UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells.It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates.The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases,shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship.Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  6. TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

    PubMed Central

    Yang, Bo; Yan, Shanshan; Zhou, Haiyan; He, Lan; Lin, Guomei; Lian, Zhexiong; Jiang, Zhengfan; Sun, Bing

    2015-01-01

    Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING. PMID:26114947

  7. Glycogen synthase kinase-3β antagonizes ROS-induced hepatocellular carcinoma cell death through suppression of the apoptosis signal-regulating kinase 1.

    PubMed

    Zhang, Na; Liu, Lu; Dou, Yueying; Song, Danqing; Deng, Hongbin

    2016-07-01

    Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is an important regulator of cancer cell survival. Apoptosis signal-regulating kinase 1 (ASK1) is also a key factor for controlling several cellular events including the cell cycle, senescence, and apoptosis, in response to reactive oxygen species (ROS). The role of GSK-3β regulating the activity and protein level of ASK1 in the cancer cells remains largely unexplored. In this study, we showed that GSK-3β inhibits ROS-induced hepatocellular carcinoma cell death by suppressing ASK1. We first found that ectopic expression of GSK-3β suppressed hydrogen peroxide (H2O2)-induced cell death in HepG2 cells and knockdown of endogenous GSK-3β expression exhibited opposite effects. Moreover, GSK-3β expression clearly inhibited H2O2-induced phosphorylation of ASK1 in HepG2 cells, in association with a decrease in ASK1 protein level. Further exploration revealed that GSK-3β induced ubiquitination and proteasome-dependent degradation of ASK1 via inhibition of ubiquitin-specific protease USP9X. Our results thus suggest that GSK-3β is a key factor involved in ASK1 activation and ROS-induced cell death. PMID:27221474

  8. SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation.

    PubMed

    Wang, Ming; Sang, Jing; Ren, Yanhua; Liu, Kejia; Liu, Xinyi; Zhang, Jian; Wang, Haolu; Wang, Jian; Orian, Amir; Yang, Jie; Yi, Jing

    2016-01-01

    SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4. PMID:26511642

  9. The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression

    PubMed Central

    He, Chunbo; Mao, Dagan; Hua, Guohua; Lv, Xiangmin; Chen, Xingcheng; Angeletti, Peter C; Dong, Jixin; Remmenga, Steven W; Rodabaugh, Kerry J; Zhou, Jin; Lambert, Paul F; Yang, Peixin; Davis, John S; Wang, Cheng

    2015-01-01

    The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes-associated protein (YAP). Here, we show that YAP plays a central role in controlling the progression of cervical cancer. Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer. TGF-α and amphiregulin (AREG), via EGFR, inhibit the Hippo signaling pathway and activate YAP to induce cervical cancer cell proliferation and migration. Activated YAP allows for up-regulation of TGF-α, AREG, and EGFR, forming a positive signaling loop to drive cervical cancer cell proliferation. HPV E6 protein, a major etiological molecule of cervical cancer, maintains high YAP protein levels in cervical cancer cells by preventing proteasome-dependent YAP degradation to drive cervical cancer cell proliferation. Results from human cervical cancer genomic databases and an accepted transgenic mouse model strongly support the clinical relevance of the discovered feed-forward signaling loop. Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer. PMID:26417066

  10. PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation.

    PubMed

    Ito, Koichi; Park, Sun Hee; Nayak, Anupma; Byerly, Jessica H; Irie, Hanna Y

    2016-08-01

    Patients with triple-negative breast cancers (TNBC) are at high risk for recurrent or metastatic disease despite standard treatment, underscoring the need for novel therapeutic targets and strategies. Here we report that protein tyrosine kinase 6 (PTK6) is expressed in approximately 70% of TNBCs where it acts to promote survival and metastatic lung colonization. PTK6 downregulation in mesenchymal TNBC cells suppressed migration and three-dimensional culture growth, and enhanced anoikis, resistance to which is considered a prerequisite for metastasis. PTK6 downregulation restored E-cadherin levels via proteasome-dependent degradation of the E-cadherin repressor SNAIL. Beyond being functionally required in TNBC cells, kinase-active PTK6 also suppressed E-cadherin expression, promoted cell migration, and increased levels of mesenchymal markers in nontransformed MCF10A breast epithelial cells, consistent with a role in promoting an epithelial-mesenchymal transition (EMT). SNAIL downregulation and E-cadherin upregulation mediated by PTK6 inhibition induced anoikis, leading to impaired metastatic lung colonization in vivo Finally, effects of PTK6 downregulation were phenocopied by treatment with a recently developed PTK6 kinase inhibitor, further implicating kinase activity in regulation of EMT and metastases. Our findings illustrate the clinical potential for PTK6 inhibition to improve treatment of patients with high-risk TNBC. Cancer Res; 76(15); 4406-17. ©2016 AACR. PMID:27302163

  11. Selective Restriction of Nef-Defective Human Immunodeficiency Virus Type 1 by a Proteasome-Dependent Mechanism▿

    PubMed Central

    Qi, Mingli; Aiken, Christopher

    2007-01-01

    The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nef-defective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells. PMID:17108041

  12. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    PubMed

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. PMID:25687693

  13. Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

    PubMed Central

    Hachmeister, Matthias; Bobowski, Karolina D.; Hogl, Sebastian; Dislich, Bastian; Fukumori, Akio; Eggert, Carola; Mack, Brigitte; Kremling, Heidi; Sarrach, Sannia; Coscia, Fabian; Zimmermann, Wolfgang; Steiner, Harald; Lichtenthaler, Stefan F.; Gires, Olivier

    2013-01-01

    Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation. PMID:24009667

  14. Stabilization of ATF5 by TAK1–Nemo-Like Kinase Critically Regulates the Interleukin-1β-Stimulated C/EBP Signaling Pathway

    PubMed Central

    Zhang, Ze-Yan; Li, Shang-Ze; Zhang, Hui-Hui; Wu, Qu-Ran; Gong, Jun; Liang, Tong; Gao, Lu; Xing, Na-Na; Liu, Wen-Bin

    2014-01-01

    Interleukin-1β (IL-1β) is a key proinflammatory cytokine that initiates several signaling cascades, including those involving CCAAT/enhancer binding proteins (C/EBPs). The mechanism by which IL-1β propagates a signal that activates C/EBP has remained elusive. Nemo-like kinase (NLK) is a mitogen-activated protein kinase (MAPK)-like kinase associated with many pathways and phenotypes that are not yet well understood. Using a luciferase reporter screen, we found that IL-1β-induced C/EBP activation was positively regulated by NLK. Overexpression of NLK activated C/EBP and potentiated IL-1β-triggered C/EBP activation, whereas knockdown or knockout of NLK had the opposite effect. NLK interacted with activating transcription factor 5 (ATF5) and inhibited the proteasome-dependent degradation of ATF5 in a kinase-independent manner. Consistently, NLK deficiency resulted in decreased levels of ATF5. NLK cooperated with ATF5 to activate C/EBP, whereas NLK could not activate C/EBP upon knockdown of ATF5. Moreover, TAK1, a downstream effector of IL-1β that acts upstream of NLK, mimicked the ability of NLK to stabilize ATF5 and activate C/EBP. Thus, our findings reveal the TAK1-NLK pathway as a novel regulator of basal or IL-1β-triggered C/EBP activation though stabilization of ATF5. PMID:25512613

  15. Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

    PubMed Central

    Xu, Shan; Cherok, Edward; Das, Shweta; Li, Sunan; Roelofs, Brian A.; Ge, Shealinna X.; Polster, Brian M.; Boyman, Liron; Lederer, W. Jonathan; Wang, Chunxin; Karbowski, Mariusz

    2016-01-01

    Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress. PMID:26564796

  16. C-terminus of heat shock protein 70– interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1– dependent apoptosis

    PubMed Central

    Hwang, Jae Ryoung; Zhang, Chunlian; Patterson, Cam

    2005-01-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress. PMID:16038411

  17. The phylogenetic relationships of Rhodosporidium dacryoidum Fell, Hunter et Tallman based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Sakaguchia gen. nov., a heterobasidiomycetous yeast genus.

    PubMed

    Yamada, Y; Maeda, K; Mikata, K

    1994-01-01

    The partial base sequences of 18S and 26S rRNAs of Rhodosporidium fluviale, R. lusitaniae, and Erythrobasidium hasegawianum were analyzed. In the 26S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had 81-82 and 77 percent similarities compared with R. toruloides (type species of genus Rhodosporidium) IFO 0559 and IFO 0880. Erythrobasidium hasegawianum IFO 1058 showed 69-71, 59, 63, and 61 percent similarities with R. toruloides IFO 0559 and IFO 0880, L. scottii (type species of genus Leucosporidium) IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and Kondoa malvinella IFO 1936, respectively. In the 18S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had zero and two base differences with R. toruloides. Erythrobasidium hasegawianum IFO 1058 showed ten, sixteen, three, and twenty base differences with R. toruloides IFO 0559 and IFO 0880, L. scottii IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and K. malvinella IFO 1936, respectively. Based on the sequence data obtained, a new genus, Sakaguchia was proposed for R. dacryoidum with a new combination, Sakaguchia dacryoides. PMID:7765151

  18. Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity

    PubMed Central

    Niemann, Michael C. E.; Bartrina, Isabel; Ashikov, Angel; Weber, Henriette; Spíchal, Lukáš; Strnad, Miroslav; Strasser, Richard; Bakker, Hans; Schmülling, Thomas; Werner, Tomáš

    2015-01-01

    The formation of glycoconjugates depends on nucleotide sugars, which serve as donor substrates for glycosyltransferases in the lumen of Golgi vesicles and the endoplasmic reticulum (ER). Import of nucleotide sugars from the cytosol is an important prerequisite for these reactions and is mediated by nucleotide sugar transporters. Here, we report the identification of REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1, At5g65000) as an ER-localized facilitator of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transport in Arabidopsis thaliana. Mutant alleles of ROCK1 suppress phenotypes inferred by a reduced concentration of the plant hormone cytokinin. This suppression is caused by the loss of activity of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKXs). Cytokinin plays an essential role in regulating shoot apical meristem (SAM) activity and shoot architecture. We show that rock1 enhances SAM activity and organ formation rate, demonstrating an important role of ROCK1 in regulating the cytokinin signal in the meristematic cells through modulating activity of CKX proteins. Intriguingly, genetic and molecular analysis indicated that N-glycosylation of CKX1 was not affected by the lack of ROCK1-mediated supply of UDP-GlcNAc. In contrast, we show that CKX1 stability is regulated in a proteasome-dependent manner and that ROCK1 regulates the CKX1 level. The increased unfolded protein response in rock1 plants and suppression of phenotypes caused by the defective brassinosteroid receptor bri1-9 strongly suggest that the ROCK1 activity is an important part of the ER quality control system, which determines the fate of aberrant proteins in the secretory pathway. PMID:25535363

  19. RelB is differentially regulated by IkappaB Kinase-alpha in B cells and mouse lung by cigarette smoke.

    PubMed

    Yang, Se-Ran; Yao, Hongwei; Rajendrasozhan, Saravanan; Chung, Sangwoon; Edirisinghe, Indika; Valvo, Samantha; Fromm, George; McCabe, Michael J; Sime, Patricia J; Phipps, Richard P; Li, Jian-Dong; Bulger, Michael; Rahman, Irfan

    2009-02-01

    The activation of transcription factor NF-kappaB is controlled by two main pathways: the classical canonical (RelA/p65-p50)- and the alternative noncanonical (RelB/p52)-NF-kappaB pathways. RelB has been shown to play a protective role in RelA/p65-mediated proinflammatory cytokine release in immune-inflammatory lymphoid cells. Increased infiltration of macrophages and lymphoid cells occurs in lungs of patients with chronic obstructive pulmonary disease, leading to abnormal inflammation. We hypothesized that RelB, and its signaling pathway, is differentially regulated in macrophages and B cells and in lung cells, leading to differential regulation of proinflammatory cytokines in response to cigarette smoke (CS). CS exposure increased the levels of RelB and NF-kappaB-inducing kinase associated with recruitment of RelB on promoters of the IL-6 and macrophage inflammatory protein-2 genes in mouse lung. Treatment of macrophage cell line, MonoMac6, with CS extract showed activation of RelB. In contrast, RelB was degraded by a proteasome-dependent mechanism in B lymphocytes (human Ramos, mouse WEHI-231, and primary mouse spleen B cells), suggesting that RelB is differentially regulated in lung inflammatory and lymphoid cells in response to CS exposure. Transient transfection of dominant negative IkappaB-kinase-alpha and double mutants of NF-kappaB-inducing kinase partially attenuated the CS extract-mediated loss of RelB in B cells and normalized the increased RelB level in macrophages. Taken together, these data suggest that RelB is differentially regulated in response to CS exposure in macrophages, B cells, and in lung cells by IkappaB-kinase-alpha-dependent mechanism. Rapid degradation of RelB signals for RelA/p65 activation and loss of its protective ability to suppress the proinflammatory cytokine release in lymphoid B cells. PMID:18688039

  20. CHIP has a protective role against oxidative stress-induced cell death through specific regulation of endonuclease G.

    PubMed

    Lee, J S; Seo, T W; Yi, J H; Shin, K S; Yoo, S J

    2013-01-01

    Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging. PMID:23764847

  1. Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Yasuda, Shigetaka; Domeki, Yukie; Sueyoshi, Kuni; Fujiwara, Masayuki; Fukao, Yoichiro; Goto, Derek B; Yamaguchi, Junji

    2011-10-01

    The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3χ, was selected for detailed characterization. The interaction between ATL31 and 14-3-3χ was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3χ is catalyzed by ATL31. Degradation of 14-3-3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3χ leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status. PMID:21668537

  2. Expression of the kinetochore protein Hec1 during the cell cycle in normal and cancer cells and its regulation by the pRb pathway.

    PubMed

    Ferretti, Chiara; Totta, Pierangela; Fiore, Mario; Mattiuzzo, Marta; Schillaci, Tiziana; Ricordy, Ruggero; Di Leonardo, Aldo; Degrassi, Francesca

    2010-10-15

    Highly Expressed in Cancer protein 1 (Hec1) is a subunit of the Ndc80 complex, a constituent of the mitotic kinetochore. HEC1 has been shown to be overexpressed in many cancers, suggesting that HEC1 upregulation is involved in the generation and/or maintenance of the tumour phenotype. However, the regulation of Hec1 expression in normal and tumour cells and the molecular alterations promoting accumulation of this protein in cancer cells are still unknown. Here we show that elevated Hec1 protein levels are characteristic of transformed cell lines of different origins and that kinetochore recruitment of this protein is also increased in cancer cell lines in comparison with normal human cells. Using different cell synchronization strategies, Hec1 expression was found to be tightly regulated during the cell cycle in both normal and cancer cells. A limited proteasome-dependent degradation of Hec1 cellular content was observed at mitotic exit, with no evident differences between normal and cancer cells. Interestingly, increased expression of HEC1 mRNA and Hec1 protein was observed after transient silencing of the retinoblastoma gene by siRNA or following microRNA-mediated permanent depletion of the retinoblastoma protein in HCT116 cells. Our data provide evidence for a functional link between Hec1 expression and the pRb pathway. These observations suggest that disruption of pRb function may lead to chromosome segregation errors and mitotic defects through Hec1 overexpression. This may importantly contribute to aneuploidy and chromosomal instability in RB-defective cancer cells. PMID:20948316

  3. VOLTAGE REGULATOR

    DOEpatents

    Von Eschen, R.L.; Scheele, P.F.

    1962-04-24

    A transistorized voltage regulator which provides very close voitage regulation up to about 180 deg F is described. A diode in the positive line provides a constant voltage drop from the input to a regulating transistor emitter. An amplifier is coupled to the positive line through a resistor and is connected between a difference circuit and the regulating transistor base which is negative due to the difference in voltage drop across thc diode and the resistor so that a change in the regulator output causes the amplifier to increase or decrease the base voltage and current and incrcase or decrease the transistor impedance to return the regulator output to normal. (AEC)

  4. Preschool Regulations.

    ERIC Educational Resources Information Center

    Nebraska State Dept. of Health and Human Services, Lincoln.

    Published by the Department of Health and Human Services, as required by Nebraska law, this guide details regulations for the physical well-being, safety, and protection of children and defines the minimum levels of acceptable services to be provided in Nebraska preschools. The first section of the guide lists specific preschool regulations,…

  5. Acceleration of Gastric Tumorigenesis Through MKRN1-Mediated Posttranslational Regulation of p14ARF

    PubMed Central

    2012-01-01

    Background We investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis. Methods A link between MKRN1 and ARF was examined in MKRN1 null mouse embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA (shRNA). Ubiquitination and proteasomal degradation assays were used to assess p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels were analyzed with immunohistochemistry in malignant and normal tissues from gastric cancer patients and with χ2 tests. The tumor growth of gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was examined in mouse xenograft models (n = 4–6) and analyzed with unpaired t tests. All statistical tests were two-sided. Results MKRN1 knockout MEFs exhibited premature senescence and growth retardation with increased p19ARF protein expression. Similar results were obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown. Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and subsequent proteasome-dependent degradation. A statistically significant association was shown between MKRN1 overexpression and p14ARF underexpression (P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional SNU601 cells displayed statistically significant tumor growth retardation by silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1 knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs 464.8mm3, difference = 300.2mm3, 95% CI = 189.1 to 411.3mm3, P < .001). Conclusions We demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer. PMID:23104211

  6. Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner, pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation ▿

    PubMed Central

    Bolin, Lisa L.; Hanson, Laura K.; Slater, Jacquelyn S.; Kerry, Julie A.; Campbell, Ann E.

    2010-01-01

    Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages. PMID:19955315

  7. A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway.

    PubMed

    Betting, J; Seufert, W

    1996-10-18

    The UBC9 gene of the yeast Saccharomyces cerevisiae is essential for cell viability and encodes a soluble protein of the nucleus that is metabolically stable. Products of mutant alleles selected to confer temperature-sensitive in vivo function were found to be extremely short-lived at the restrictive but long-lived at the permissive condition. An extragenic suppressor mutation was isolated which increased thermoresistance of a ubc9-1 strain. This suppressor turned out to stabilize the mutated gene product, indicating that the physiological activity of ubc9-1 protein is primarily controlled by conditional proteolysis. The labile ubc9-1 protein appears to be a substrate for ubiquitination, and its turnover was substantially reduced by expression of a ubiquitin derivative that interferes with formation of multi-ubiquitin chains. Stabilization resulted also from competitive inhibition of Ubc4-related ubiquitin-conjugating enzymes. Activity of the proteasome complex was crucial to rapid breakdown, whereas vacuolar proteases were dispensable. Thus, the heat-denatured ubc9-1 protein is targeted for proteolysis by the ubiquitin-proteasome pathway and may serve as a useful tool to further define the process by which a misfolded polypeptide is recognized. PMID:8824207

  8. Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

    PubMed

    Becker, Hans Jiro; Kondo, Eisei; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; von Bergwelt-Baildon, Michael S

    2016-08-01

    Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway. PMID:26561366

  9. Characterization of the ORF YBR264c in Saccharomyces cerevisiae, which encodes a new yeast Ypt that is degraded by a proteasome-dependent mechanism.

    PubMed

    Louvet, O; Roumanie, O; Barthe, C; Peypouquet, M F; Schaeffer, J; Doignon, F; Crouzet, M

    1999-06-01

    We identified the ORF YBR264c during the systematic sequencing of the Saccharomyces cerevisiae genome. It encodes a putative protein of 218 amino acids. We demonstrate here that the gene is indeed expressed and encodes a new Ypt in yeast. This protein specifically binds guanine nucleotides and interacts via its C-terminal end with the unique Rab GDP Dissociation Inhibitor (RabGDI). In accordance with a recent proposal, the gene is now designated YPT10. No mutant phenotype could be associated with inactivation of the gene. However, overexpression of YPT10 resulted in defects in growth; microscopic examination of such cells revealed an overabundance of vesicular and tubular structures, suggesting some alteration in the function of the Golgi apparatus. In addition, degradation of the Ypt10 protein, which possesses a PEST sequence, is shown to be dependent on proteasome activity. PMID:10394895

  10. Evolution of Proteasome Regulators in Eukaryotes

    PubMed Central

    Fort, Philippe; Kajava, Andrey V.; Delsuc, Fredéric; Coux, Olivier

    2015-01-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles. PMID:25943340

  11. Voltage regulator

    SciTech Connect

    Rossetti, N.

    1986-12-09

    This patent describes a prior art integrated circuit voltage regulator having an unregulated voltage input terminal and a regulated voltage output terminal, and further comprising: a first transistor having an emitter, a collector and a base, the first transistor having a first base-emitter voltage characteristic, the collector of the first transistor being connected through a first resistor to a current source. The current source is derived from the unregulated voltage, the emitter of the first transistor being connected through a second resistor to a reference voltage; and a second transistor having an emitter, a collector and a base, the second transistor having a second base-emitter voltage characteristic, the base of the second transistor being connected to the collector of the first transistor. The collector of the second transistor is connected to the current source, the emitter of the second transistor being connected to the reference voltage. The regulated output of the voltage regulator is provided at the collector of the second transistor and the regulated voltage output is a function of the first base-emitter voltage characteristic of the first transistor plus the quantity comprising the difference between the first base-emitter voltage characteristic of the first transistor and the second base-emitter voltage characteristic of the second transistor, times the ratio of the value of resistance of the first resistor and the value of resistance of the second resistor. The improvement described here comprises: a third transistor having a collector, an emitter and a base.

  12. NORM regulations

    SciTech Connect

    Gray, P.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  13. 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

  14. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  15. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  16. Regulation of BRAF protein stability by a negative feedback loop involving the MEK-ERK pathway but not the FBXW7 tumour suppressor.

    PubMed

    Hernandez, Maria Aguilar; Patel, Bipin; Hey, Fiona; Giblett, Susan; Davis, Hayley; Pritchard, Catrin

    2016-06-01

    The (V600E)BRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK-ERK signalling cascade. Although output of the BRAF-MEK-ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous (V600E)BRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and (V600E)BRAF has a reduced half-life compared to (WT)BRAF in HEK293(T) cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400-405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in (V600E)BRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either (WT)BRAF or (V600E)BRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of (WT)BRAF and (V600E)BRAF were not altered in MEFs derived from mice with the homozygous knockin (R482Q)FBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation. PMID:26898828

  17. Charge regulation circuit

    DOEpatents

    Ball, Don G.

    1992-01-01

    A charge regulation circuit provides regulation of an unregulated voltage supply in the range of 0.01%. The charge regulation circuit is utilized in a preferred embodiment in providing regulated voltage for controlling the operation of a laser.

  18. The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice[OPEN

    PubMed Central

    Yang, Ying; Fu, Debao; Zhu, Chunmei; He, Yizhou; Zhang, Huijun; Liu, Tao; Li, Xianghua; Wu, Changyin

    2015-01-01

    The photoperiodic response is one of the most important factors determining heading date in rice (Oryza sativa). Although rhythmic expression patterns of flowering time genes have been reported to fine-tune the photoperiodic response, posttranslational regulation of key flowering regulators has seldom been elucidated in rice. Heading date 1 (Hd1) encodes a zinc finger transcription factor that plays a crucial role in the photoperiodic response, which determines rice regional adaptability. However, little is known about the molecular mechanisms of Hd1 accumulation during the photoperiod response. Here, we identify a C3HC4 RING domain-containing E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), which physically interacts with Hd1. HAF1 mediates ubiquitination and targets Hd1 for degradation via the 26S proteasome-dependent pathway. The haf1 mutant exhibits a later flowering heading date under both short-day and long-day conditions. In addition, the haf1 hd1 double mutant headed as late as hd1 plants under short-day conditions but exhibited a heading date similar to haf1 under long-day conditions, thus indicating that HAF1 may determine heading date mainly through Hd1 under short-day conditions. Moreover, high levels of Hd1 accumulate in haf1. Our results suggest that HAF1 is essential to precise modulation of the timing of Hd1 accumulation during the photoperiod response in rice. PMID:26296966

  19. Proteasome, but Not Autophagy, Disruption Results in Severe Eye and Wing Dysmorphia: A Subunit- and Regulator-Dependent Process in Drosophila

    PubMed Central

    Pantazi, Asimina D.; Mpakou, Vassiliki E.; Zervas, Christos G.; Papassideri, Issidora S.; Stravopodis, Dimitrios J.

    2013-01-01

    Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}BxMS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly’s eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing’s, but not eye’s, morphogenetic organization and architecture. However, Atg9 proved indispensable

  20. Multimedia regulated chemicals

    SciTech Connect

    Lee, C.C.; Huffman, G.L.; Mao, Y.L.

    1999-10-01

    This article examines those chemicals that are listed in either environmental laws or regulations. Its objective is to help readers determine which laws regulate what types of chemicals and which types of chemicals are regulated by what laws. It is multimedia in scope, describing the various chemicals that are regulated in the different media (i.e., air, water, or land).

  1. Regulating Rho GTPases and their regulators.

    PubMed

    Hodge, Richard G; Ridley, Anne J

    2016-08-01

    Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases. PMID:27301673

  2. Structural basis of antizyme-mediated regulation of polyamine homeostasis

    PubMed Central

    Wu, Hsiang-Yi; Chen, Shin-Fu; Hsieh, Ju-Yi; Chou, Fang; Wang, Yu-Hsuan; Lin, Wan-Ting; Lee, Pei-Ying; Yu, Yu-Jen; Lin, Li-Ying; Lin, Te-Sheng; Lin, Chieh-Liang; Liu, Guang-Yaw; Tzeng, Shiou-Ru; Hung, Hui-Chih; Chan, Nei-Li

    2015-01-01

    Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation. PMID:26305948

  3. Proteasome-mediated turnover of the transcriptional activator FIT is required for plant iron-deficiency responses.

    PubMed

    Sivitz, Alicia; Grinvalds, Claudia; Barberon, Marie; Curie, Catherine; Vert, Grégory

    2011-06-01

    Plants display a number of responses to low iron availability in order to increase iron uptake from the soil. In the model plant Arabidopsis thaliana, the ferric-chelate reductase FRO2 and the ferrous iron transporter IRT1 control iron entry from the soil into the root epidermis. To maintain iron homeostasis, the expression of FRO2 and IRT1 is tightly controlled by iron deficiency at the transcriptional level. The basic helix-loop-helix (bHLH) transcription factor FIT represents the most upstream actor known in the iron-deficiency signaling pathway, and directly regulates the expression of the root iron uptake machinery genes FRO2 and IRT1. However, how FIT is controlled by iron and acts to activate transcription of its targets remains obscure. Here we show that FIT mRNA and endogenous FIT protein accumulate in Arabidopsis roots upon iron deficiency. However, using plants constitutively expressing FIT, we observed that FIT protein accumulation is reduced in iron-limited conditions. This post-transcriptional regulation of FIT is perfectly synchronized with the accumulation of endogenous FIT and IRT1 proteins, and therefore is part of the early responses to low iron. We demonstrated that such regulation affects FIT protein stability under iron deficiency as a result of 26S proteasome-dependent degradation. In addition, we showed that FIT post-translational regulation by iron is required for FRO2 and IRT1 gene expression. Taken together our results indicate that FIT transcriptional and post-translational regulations are integrated in plant roots to ensure that the positive regulator FIT accumulates as a short-lived protein following iron shortage, and to allow proper iron-deficiency responses. PMID:21426424

  4. Activity and regulation of the centrosome-associated proteasome.

    PubMed

    Fabunmi, R P; Wigley, W C; Thomas, P J; DeMartino, G N

    2000-01-01

    Regulated proteolysis is important for maintaining appropriate cellular levels of many proteins. The bulk of intracellular protein degradation is catalyzed by the proteasome. Recently, the centrosome was identified as a novel site for concentration of the proteasome and associated regulatory proteins (Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) J. Cell Biol. 145, 481-490). Here we provide evidence that centrosomes contain the active 26 S proteasome that degrades ubiquitinated-protein and proteasome-specific peptide substrates. Moreover, the centrosomes contain an ubiquitin isopeptidase activity. The proteolytic activity is ATP-dependent and is inhibited by proteasome inhibitors. Notably, treatment of cells with inhibitors of proteasome activity promotes redistribution of the proteasome and associated regulatory proteins to the centrosome independent of an intact microtubule system. These data provide biochemical evidence for active proteasomal complexes at the centrosome, highlighting a novel function for this organizing structure. PMID:10617632

  5. Load regulating expansion fixture

    DOEpatents

    Wagner, Lawrence M.; Strum, Michael J.

    1998-01-01

    A free standing self contained device for bonding ultra thin metallic films, such as 0.001 inch beryllium foils. The device will regulate to a predetermined load for solid state bonding when heated to a bonding temperature. The device includes a load regulating feature, whereby the expansion stresses generated for bonding are regulated and self adjusting. The load regulator comprises a pair of friction isolators with a plurality of annealed copper members located therebetween. The device, with the load regulator, will adjust to and maintain a stress level needed to successfully and economically complete a leak tight bond without damaging thin foils or other delicate components.

  6. Load regulating expansion fixture

    DOEpatents

    Wagner, L.M.; Strum, M.J.

    1998-12-15

    A free standing self contained device for bonding ultra thin metallic films, such as 0.001 inch beryllium foils is disclosed. The device will regulate to a predetermined load for solid state bonding when heated to a bonding temperature. The device includes a load regulating feature, whereby the expansion stresses generated for bonding are regulated and self adjusting. The load regulator comprises a pair of friction isolators with a plurality of annealed copper members located therebetween. The device, with the load regulator, will adjust to and maintain a stress level needed to successfully and economically complete a leak tight bond without damaging thin foils or other delicate components. 1 fig.

  7. Emotion Regulation in Parenthood

    PubMed Central

    Rutherford, Helena J.V.; Wallace, Norah S.; Laurent, Heidemarie K.; Mayes, Linda C.

    2015-01-01

    Emotion regulation, defined as the capacity to influence one’s experience and expression of emotion, is a complex skill now recognized to evolve throughout the lifetime. Here we examine the role of emotion regulation in parenthood, and propose that regulatory function during this period is distinct from the emotion regulation skills acquired and implemented during other periods of life. In this review, we consider the unique demands of caring for a child and recognize that parents have to maintain a regulated state as well as facilitate regulation in their child, especially early in development. We examine neurobiological, hormonal and behavioral shifts during the transition to parenthood that may facilitate parental regulation in response to infant cues. Furthermore, we consider how parents shape emotion regulation in their child, and the clinical implications of regulatory functioning within the parent-child relationship. PMID:26085709

  8. Pressure reducing regulator

    DOEpatents

    Whitehead, John C.; Dilgard, Lemoyne W.

    1995-01-01

    A pressure reducing regulator that controls its downstream or outlet pressure to a fixed fraction of its upstream or inlet pressure. The regulator includes a housing which may be of a titanium alloy, within which is located a seal or gasket at the outlet end which may be made of annealed copper, a rod, and piston, each of which may be made of high density graphite. The regulator is insensitive to temperature by virtue of being without a spring or gas sealed behind a diaphragm, and provides a reference for a system in which it is being used. The rod and piston of the regulator are constructed, for example, to have a 1/20 ratio such that when the downstream pressure is less than 1/20 of the upstream pressure the regulator opens and when the downstream pressure exceeds 1/20 of the upstream pressure the regulator closes.

  9. Pressure reducing regulator

    DOEpatents

    Whitehead, J.C.; Dilgard, L.W.

    1995-10-10

    A pressure reducing regulator that controls its downstream or outlet pressure to a fixed fraction of its upstream or inlet pressure is disclosed. The regulator includes a housing which may be of a titanium alloy, within which is located a seal or gasket at the outlet end which may be made of annealed copper, a rod, and piston, each of which may be made of high density graphite. The regulator is insensitive to temperature by virtue of being without a spring or gas sealed behind a diaphragm, and provides a reference for a system in which it is being used. The rod and piston of the regulator are constructed, for example, to have a 1/20 ratio such that when the downstream pressure is less than 1/20 of the upstream pressure the regulator opens and when the downstream pressure exceeds 1/20 of the upstream pressure the regulator closes. 10 figs.

  10. TOWARD MORE EFFECTIVE REGULATION

    SciTech Connect

    J. GRAF

    2000-06-01

    This paper proposes a model relationship between the operator engaged in a hazardous activity, the regulator of that activity, and the general public. The roles and responsibilities of each entity are described in a way that allows effective communication flow. The role of the regulator is developed using the steam boiler as an example of a hazard subject to regulation; however, the model applies to any regulated activity. In this model the safety analyst has the extremely important role of communicating sometimes difficult technical information to the regulator in a way that the regulator can provide credible assurance to the general public as to the adequacy of the control of the hazardous activity. The conclusion asserts that acceptance of the model, understanding of the roles and responsibilities and definition of who communicates what information to whom will mitigate frustration on the part of each of the three entities.

  11. [Epigenetic regulation in spermatogenesis].

    PubMed

    Xu, Chen; Song, Ning

    2014-05-01

    Spermatogenesis is a process consisting of spermatogonial proliferation, spermatocytic meiosis, and spermiogenesis, and is also considered to be a process in which heterochromatins gradually aggregate and finally reach a highly condensed formation in the sperm head. Recent studies show that epigenetic regulation plays a key role in spermatogenesis. This review discusses the mechanisms of epigenetic regulation in spermatogenesis in three aspects, DNA methylation, histone modification, and noncoding RNAs. These factors are essential for spermatogenesis, fertilization, and embryogenesis by mutual regulation as well as by gene expression regulation, transposon activation, sex chromosome inactivation, and genome imprinting. PMID:24908726

  12. Federal Powers of Regulation.

    ERIC Educational Resources Information Center

    Rhyne, William S.

    1979-01-01

    Reflects the special considerations in affecting, rather than merely evaluating, the outcome of constitutional litigation over federal regulation of state and local government labor relations. (Author/IRT)

  13. The poxvirus encoded ubiquitin ligase, p28, is regulated by proteasomal degradation and autoubiquitination.

    PubMed

    Mottet, Kelly; Bareiss, Bettina; Milne, Craig D; Barry, Michele

    2014-11-01

    Virus manipulation of the ubiquitin-proteasome system has become increasingly apparent. Ubiquitin is a 76 amino acid protein that is post-translationally conjugated to target proteins, while poly-ubiquitination subsequently leads to degradation via the 26S proteasome. Target specificity is determined by a large family of ubiquitin ligases. Poxviruses encode p28, a highly conserved ubiquitin ligase expressed in a wide range of poxviruses (J. Virol. 79:597). Here we investigate the relationship between p28 and ubiquitination. Confocal microscopy indicated that orthologs of p28 co-localized with ubiquitin at the virus factory. Flow cytometry assays further demonstrated that p28 was regulated by proteasomal degradation. Moreover, when the ubiquitin ligase activity of p28 was disrupted by mutating the RING domain conjugated ubiquitin still localized to the viral factories, indicating that an unknown ubiquitin ligase(s) was responsible for regulating p28. Our observations indicate that p28 is a ubiquitin ligase that is regulated by ubiquitination and proteasomal degradation. PMID:25240226

  14. Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

    PubMed Central

    Leichner, Gil S.; Avner, Rachel; Harats, Dror; Roitelman, Joseph

    2011-01-01

    In mammalian cells, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between β-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR. PMID:21778231

  15. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. PMID:27241217

  16. ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance.

    PubMed

    Miura, Kenji; Ohta, Masaru; Nakazawa, Machiko; Ono, Michiyuki; Hasegawa, Paul M

    2011-07-01

    ICE1, a MYC-type transcription factor, has an important role in the induction of CBF3/DREB1A for regulation of cold signaling and tolerance. Here we reveal that serine 403 of ICE1 is involved in regulating the transactivation and stability of the ICE1 protein. Substitution of serine 403 by alanine enhanced the transactivational activity of ICE1 in Arabidopsis protoplasts. Over-expression of ICE1(S403A) conferred more freezing tolerance than ICE1(WT) in Arabidopsis, and the expression of cold-regulated genes such as CBF3/DREB1A, COR47 and KIN1 was enhanced in plants over-expressing ICE1(S403A). Furthermore, the ICE1(S403A) protein level was not changed after cold treatment, whereas the ICE1(WT) protein level was reduced. Interestingly, polyubiquitylation of the ICE1(S403A) protein in vivo was apparently blocked. These results demonstrate that serine 403 of ICE1 has roles in both transactivation and cold-induced degradation of ICE1 via the ubiquitin/26S proteasome pathway, suggesting that serine 403 is a key residue for the attenuation of cold-stress responses by HOS1-mediated degradation of ICE1. PMID:21447070

  17. The Right to Regulate

    NASA Technical Reports Server (NTRS)

    Vittek, J. F.

    1972-01-01

    An introduction to the historical and constitutional framework of industry regulation by local and Federal Governments is presented. Problems of the confiscation of private property without due process, government control and the rights and duties of the regulated industry are discussed.

  18. Regulation of University Teaching

    ERIC Educational Resources Information Center

    Lindblom-Ylanne, Sari; Nevgi, Anne; Trigwell, Keith

    2011-01-01

    The aims of the present study are twofold: firstly, to explore dimensions in the regulation of teaching in a multidisciplinary sample of university teachers, and secondly, to analyse factors related to the regulation of university teaching. Seventy-three university teachers representing several disciplines participated in the study. These teachers…

  19. Plant Growth Regulators.

    ERIC Educational Resources Information Center

    Nickell, Louis G.

    1978-01-01

    Describes the effect of "plant growth regulators" on plants, such as controlling the flowering, fruit development, plant size, and increasing crop yields. Provides a list of plant growth regulators which includes their chemical, common, and trade names, as well as their different use(s). (GA)

  20. Regulation of megakaryocytopoiesis.

    PubMed

    Caen, J P; Han, Z C; Bellucci, S; Alemany, M

    1999-09-01

    After 35 years of research, a physiological regulator of platelet production has been identified and the recombinant protein is available. With the discovery of thrombopoietin (TPO), its potential use in a wide variety of clinical megakaryocytic and platelet disorders has been expected and clinical trials have been undertaken. To date, the reported encouraging pre-clinical studies indicate that, as with erythropoietin or G-CSF, minimal toxicity can be expected. A potential limiting side-effect of TPO could be the induction of thrombosis. Nevertheless, it is too early to know whether this cytokine will be of major therapeutic importance for patients with life-threatening thrombocytopenia, such as patients undergoing bone marrow transplantation or subjected to a high dose of chemotherapy. Several experimental and clinical studies are still needed to determine the efficacy of TPO in the prevention or the amelioration of bleeding, which is the ultimate goal for the appropriate use of cytokines with haemostatic benefit. Basic and clinical studies on regulators of megakaryocytopoiesis have rapidly progressed. Now, there is no doubt that some of these regulators are effective in correcting haematopoietic disorders of various aetiologies. Studies on negative regulators not only are important to understand the regulation of megakaryocytopoiesis in normal and pathological states but also have a potential clinical application. Some of these regulators have been shown to be effective in the treatment of essential thrombocythaemia and other myeloproliferative disorders. Platelet factor 4 (PF4) and some other chemokines are also capable of protecting progenitor cells from the cytotoxicity of chemotherapeutic drugs. However, detailed investigations are still required to determine the precise mechanism(s) of action of these regulators and to establish the optimal clinical protocols of negative regulators alone or in association with positive regulators for the treatment of various

  1. Phosphoinositides regulate ion channels

    PubMed Central

    Hille, Bertil; Dickson, Eamonn J.; Kruse, Martin; Vivas, Oscar; Suh, Byung-Chang

    2014-01-01

    Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. PMID:25241941

  2. HIGH PRESSURE GAS REGULATOR

    DOEpatents

    Ramage, R.W.

    1962-05-01

    A gas regulator operating on the piston and feedback principle is described. The device is particularly suitable for the delicate regulation of high pressure, i.e., 10,000 psi and above, gas sources, as well as being perfectly adaptable for use on gas supplies as low as 50 psi. The piston is adjustably connected to a needle valve and the movement of the piston regulates the flow of gas from the needle valve. The gas output is obtained from the needle valve. Output pressure is sampled by a piston feedback means which, in turn, regulates the movement of the main piston. When the output is other than the desired value, the feedback system initiates movement of the main piston to allow the output pressure to be corrected or to remain constant. (AEC)

  3. R2 REGULATED FACILITIES

    EPA Science Inventory

    The Facility Registry System (FRS) is a centrally managed database that identifies facilities, sites or places subject to environmental regulations or of environmental interest. FRS creates high-quality, accurate, and authoritative facility identification records through rigorous...

  4. Proposed EEOC Regulations.

    ERIC Educational Resources Information Center

    Farrell, Michael

    1978-01-01

    This article explains how proposed Equal Employment Opportunity Commission (EEOC) regulations attempt to circumvent the case of Weber vs Kaiser Aluminum Corp. by providing employers with backpay immunity in reverse discrimination suits. (Author)

  5. SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through Its Interaction with PHR2 in Rice[W][OPEN

    PubMed Central

    Lv, Qundan; Zhong, Yongjia; Wang, Yuguang; Wang, Zhiye; Zhang, Li; Shi, Jing; Wu, Zhongchang; Liu, Yu; Mao, Chuanzao; Yi, Keke; Wu, Ping

    2014-01-01

    PHR2, a central regulator of phosphate signaling in rice, enhanced the expression of phosphate starvation-induced (PSI) genes and resulted in the enhancement of Pi acquisition under Pi deficiency stress. This occurred via PHR2 binding to a cis-element named the PHR1 binding sequences. However, the transcription level of PHR2 was not responsive to Pi starvation. So how is activity of transcription factor PHR2 adjusted to adapt diverse Pi status? Here, we identify an SPX family protein, Os-SPX4 (SPX4 hereafter), involving in Pi starvation signaling and acting as a negative regulator of PHR2. SPX4 is shown to be a fast turnover protein. When Pi is sufficient, through its interaction with PHR2, SPX4 inhibits the binding of PHR2 to its cis-element and reduces the targeting of PHR2 to the nucleus. However, when plants grow under Pi deficiency, the degradation of SPX4 is accelerated through the 26S proteasome pathway, thereby releasing PHR2 into the nucleus and activating the expression of PSI genes. Because the level of SPX4 is responsive to Pi concentration and SPX4 interacts with PHR2 and regulates its activity, this suggests that SPX4 senses the internal Pi concentration under diverse Pi conditions and regulates appropriate responses to maintain Pi homeostasis in plants. PMID:24692424

  6. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

    PubMed

    Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. PMID:26564029

  7. Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Fu, Lin; Qi, Xiaolong; Zhang, Zhenyi; Xia, Yuanxin; Jia, Jianhang; Jiang, Jin; Zhao, Yun; Wu, Geng

    2013-11-01

    Hedgehog (Hh) signalling regulates embryonic development and adult tissue homoeostasis. Mutations of its pathway components including Suppressor of Fused (Sufu) and Gli/Ci predispose to cancers and congenital anomalies. The Sufu-Gli protein complex occupies a central position in the vertebrate Hh signalling pathway, especially in mammals. Here structures of full-length human and Drosophila Sufu, the human Sufu-Gli complex, along with normal mode analysis and FRET measurement results, reveal that Sufu alternates between ‘open’ and ‘closed’ conformations. The ‘closed’ form of Sufu is stabilized by Gli binding and inhibited by Hh treatment, whereas the ‘open’ state of Sufu is promoted by Gli-dissociation and Hh signalling. Mutations of critical interface residues disrupt the Sufu-Gli complex and prevent Sufu from repressing Gli-mediated transcription, tethering Gli in the cytoplasm and protecting Gli from the 26S proteasome-mediated degradation. Our study thus provides mechanistic insight into the mutual recognition and regulation between Sufu and Gli/Ci.

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

    PubMed Central

    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

  9. On regulating perceived risk.

    PubMed

    van Andel, F G

    1985-01-01

    Modern society increasingly depends on government regulation to manage risks. Until recently, evaluation of risks of technology was primarily considered a technical problem. However, public controversy has politicized the issue of risk, raising questions about the role of experts. This paper briefly explores the nature of technical risks of aircraft, nuclear energy and medicines. It is contended that in the case of aircraft intensive regulation has led to a measurable improvement of its safety record. The constant call for more regulation in the areas of medicines and nuclear energy on the other hand seems more the result of public controversy, since the actual effect of regulatory measures on safety is too difficult to show. This stresses the important role of the media, a theme, which is elaborated by reviewing a number of cases. The general conclusion is concerned with the notion that public pressure is the only rationale which makes regulators step in. Regulatory decision-making about risk, then, is more anecdotal than systematic, because public controversy is unpredictable. As a consequence regulators can no longer seek to minimize harm, but must now move towards the aim of minimizing perceived harm. Finally, in the light of this assumption, some thought is given to costs and benefits of medicines and nuclear energy. It is appropriate to make a strong case for medicines in this context, for, as opposed to nuclear energy, alternatives are usually not available. PMID:10271778

  10. Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites*

    PubMed Central

    Li, Songhua; Izumi, Tadahide; Hu, Jane; Jin, Heather H.; Siddiqui, Ahmed-Abdul A.; Jacobson, Samuel G.; Bok, Dean; Jin, Minghao

    2014-01-01

    Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s. PMID:24849605

  11. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation. PMID:26172624

  12. Androgen receptor genomic regulation

    PubMed Central

    Jin, Hong-Jian; Kim, Jung

    2013-01-01

    The transcriptional activity of the androgen receptor (AR) is not only critical for the normal development and function of the prostate but also pivotal to the onset and progression of prostate cancer (PCa). The studies of AR transcriptional regulation were previously limited to a handful of AR-target genes. Owing to the development of various high-throughput genomic technologies, significant advances have been made in recent years. Here we discuss the discoveries of genome-wide androgen-regulated genes in PCa cell lines, animal models and tissues using expression microarray and sequencing, the mapping of genomic landscapes of AR using Combining Chromatin Immunoprecipitation (ChIP)-on-chip and ChIP-seq assays, the interplay of transcriptional cofactors in defining AR binding profiles, and the genomic regulation and AR reprogramming in advanced PCa. PMID:25237629

  13. Environmentally regulated aerospace coatings

    NASA Technical Reports Server (NTRS)

    Morris, Virginia L.

    1995-01-01

    Aerospace coatings represent a complex technology which must meet stringent performance requirements in the protection of aerospace vehicles. Topcoats and primers are used, primarily, to protect the structural elements of the air vehicle from exposure to and subsequent degradation by environmental elements. There are also many coatings which perform special functions, i.e., chafing resistance, rain erosion resistance, radiation and electric effects, fuel tank coatings, maskants, wire and fastener coatings. The scheduled promulgation of federal environmental regulations for aerospace manufacture and rework materials and processes will regulate the emissions of photochemically reactive precursors to smog and air toxics. Aerospace organizations will be required to identify, qualify and implement less polluting materials. The elimination of ozone depleting chemicals (ODC's) and implementation of pollution prevention requirements are added constraints which must be addressed concurrently. The broad categories of operations affected are the manufacture, operation, maintenance, and repair of military, commercial, general aviation, and space vehicles. The federal aerospace regulations were developed around the precept that technology had to be available to support the reduction of organic and air toxic emissions, i.e., the regulations cannot be technology forcing. In many cases, the regulations which are currently in effect in the South Coast Air Quality Management District (SCAQMD), located in Southern California, were used as the baseline for the federal regulations. This paper addresses strategies used by Southern California aerospace organizations to cope with these regulatory impacts on aerospace productions programs. All of these regulatory changes are scheduled for implementation in 1993 and 1994, with varying compliance dates established.

  14. Nuclear regulation and safety

    SciTech Connect

    Hendrie, J.M.

    1982-01-01

    Nuclear regulation and safety are discussed from the standpoint of a hypothetical country that is in the process of introducing a nuclear power industry and setting up a regulatory system. The national policy is assumed to be in favor of nuclear power. The regulators will have responsibility for economic, reliable electric production as well as for safety. Reactor safety is divided into three parts: shut it down, keep it covered, take out the afterheat. Emergency plans also have to be provided. Ways of keeping the core covered with water are discussed. (DLC)

  15. Search for reasonable regulations

    SciTech Connect

    Latz, J.

    1985-06-01

    The Linowes Commission recommendation that penalties be imposed to improve record keeping and security at production sites on federal and Indian lands led to the Federal Oil and Gas Royalty Management Act. The author outlines the background and provisions of the act. Public hearings to receive comments raised concerns that the regulations exceeded congressional intent and were too inflexible. The Bureau of Land Management responded with modifications which help to clarify and simplify the regulations and to eliminate automatic penalties. The next step is for the states to respond to the proposed rules and procedures.

  16. Regulation of plasminogen receptors.

    PubMed

    Herren, Thomas; Swaisgood, Carmen; Plow, Edward F

    2003-01-01

    Many eukaryotic and prokaryotic cells bind plasminogen in a specific and saturable manner. When plasminogen is bound to cell-surface proteins with C-terminal lysines via its lysine binding sites, its activation to plasmin is accelerated, and cell-bound plasmin is protected from inactivation by natural inhibitors. Plasmin mediates direct or indirect degradation of the extracellular matrix, and bound plasmin is used by cells to facilitate migration through extracellular matrices. Since cell migration and tissue remodelling are the underpinnings of many physiological and pathological responses, the modulation of plasminogen receptors may serve as a primary regulatory mechanism for control of many cellular responses. Specific examples of cell types on which plasminogen receptors undergo modulation include: fibroblasts, where modulation may contribute to cartilage and bone destruction in rheumatoid arthritis; leukemic cells, where enhanced plasminogen binding may contribute to the heightened fibrinolytic state in the patients; other tumor cells, where up-regulation may support invasion and metastasis; bacteria, where enhanced plasminogen binding may facilitate tissue destruction and invasion; platelets, where up-regulation of plasminogen binding may play a role in regulating clot lysis; and adipocytes, where the modulation of plasminogen receptor expression may regulate cell differentiation and fat accumulation. Two pathways for modulation of plasminogen receptors have been characterized: A protease-dependent pathway can either up-regulate or down-regulate plasminogen binding to cells by changing the availability of plasminogen-binding proteins with C-terminal lysines. New receptors may be generated by trypsin-like proteases, including plasmin, which create new C-terminal lysines; other enzymes may expose existing membrane proteins by altering the cell surface; or receptor function may be lost by removal of C-terminal lysines. The basic carboxypeptidases of blood

  17. The Impact of Regulating Social Science Research with Biomedical Regulations

    ERIC Educational Resources Information Center

    Durosinmi, Brenda Braxton

    2011-01-01

    The Impact of Regulating Social Science Research with Biomedical Regulations Since 1974 Federal regulations have governed the use of human subjects in biomedical and social science research. The regulations are known as the Federal Policy for the Protection of Human Subjects, and often referred to as the "Common Rule" because 18 Federal…

  18. Other-Regulation in Collaborative Groups: Implications for Regulation Quality

    ERIC Educational Resources Information Center

    Rogat, Toni Kempler; Adams-Wiggins, Karlyn R.

    2014-01-01

    The current study examines variation in other-regulation, conceptualized as efforts by one student to regulate their group's work. This study extends research which has conceptualized other-regulation as temporarily guiding others' conceptual understanding and skill development by broadening the spectrum of other-regulation to include…

  19. Regulation and Markets

    ERIC Educational Resources Information Center

    Gardner, Margaret; Wells, Julie

    2007-01-01

    There has been much critical comment in recent years about the tensions between the regulation imposed on public universities and the flexibility needed to compete effectively in international and national markets for students and funding. In the partisan world of politics each side points the finger at the other as the author of "too much"…

  20. METABOLIC PATHWAY REGULATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Research efforts in the past two decades have revealed the complex mechanisms employed by fungi to control gene activity. The tremendous expansion in our knowledge of the regulation of nitrogen metabolism and carbon metabolism, due largely to the powerful combination of genetics, biochemistry, and ...

  1. HIGH VOLTAGE REGULATOR

    DOEpatents

    Wright, B.T.

    1959-06-01

    A high voltage regulator for use with calutrons is described which rapidly restores accelerating voltage after a sudden drop such as is caused by sparking. The rapid restoration characteristic prevents excessive contamination of lighter mass receiver pockets by the heavier mass portion of the beam. (T.R.H.)

  2. Regulation of Energy Balance.

    ERIC Educational Resources Information Center

    Bray, George A.

    1985-01-01

    Explains relationships between energy intake and expenditure focusing on the cellular, chemical and neural mechanisms involved in regulation of energy balance. Information is referenced specifically to conditions of obesity. (Physicians may earn continuing education credit by completing an appended test). (ML)

  3. Focus on PTEN Regulation

    PubMed Central

    Bermúdez Brito, Miriam; Goulielmaki, Evangelia; Papakonstanti, Evangelia A.

    2015-01-01

    The role of phosphatase and tensin homolog on chromosome 10 (PTEN) as a tumor suppressor has been for a long time attributed to its lipid phosphatase activity against PI(3,4,5)P3, the phospholipid product of the class I PI3Ks. Besides its traditional role as a lipid phosphatase at the plasma membrane, a wealth of data has shown that PTEN can function independently of its phosphatase activity and that PTEN also exists and plays a role in the nucleus, in cytoplasmic organelles, and extracellularly. Accumulating evidence has shed light on diverse physiological functions of PTEN, which are accompanied by a complex regulation of its expression and activity. PTEN levels and function are regulated transcriptionally, post-transcriptionally, and post-translationally. PTEN is also sensitive to regulation by its interacting proteins and its localization. Herein, we summarize the current knowledge on mechanisms that regulate the expression and enzymatic activity of PTEN and its role in human diseases. PMID:26284192

  4. Lightweight Regulated Power Supply

    NASA Technical Reports Server (NTRS)

    Mclyman, C. W.

    1985-01-01

    Power-supply circuit regulates output voltage by adjusting frequency of chopper circuit according to variations. Currently installed in battery charger for electric wheelchair, circuit is well suited to other uses in which light weight is important - for example, in portable computers, radios, and test instruments.

  5. Metabolic regulation of yeast

    NASA Astrophysics Data System (ADS)

    Fiechter, A.

    1982-12-01

    Metabolic regulation which is based on endogeneous and exogeneous process variables which may act constantly or time dependently on the living cell is discussed. The observed phenomena of the regulation are the result of physical, chemical, and biological parameters. These parameters are identified. Ethanol is accumulated as an intermediate product and the synthesis of biomass is reduced. This regulatory effect of glucose is used for the aerobic production of ethanol. Very high production rates are thereby obtained. Understanding of the regulation mechanism of the glucose effect has improved. In addition to catabolite repression, several other mechanisms of enzyme regulation have been described, that are mostly governed by exogeneous factors. Glucose also affects the control of respiration in a third class of yeasts which are unable to make use of ethanol as a substrate for growth. This is due to the lack of any anaplerotic activity. As a consequence, diauxic growth behavior is reduced to a one-stage growth with a drastically reduced cell yield. The pulse chemostat technique, a systematic approach for medium design is developed and medium supplements that are essential for metabolic control are identified.

  6. Growth regulation of cancer

    SciTech Connect

    Lippman, M.E. )

    1988-01-01

    This book contains proceedings of an Ortho-UCLA Symposium on growth regulation of cancer. Included are the following chapters: Swiss 3T3 mouse embryo fibroblasts transfected with a human Prepro-GRP gene synthesize and secrete Pro-GRP rather than GRP, proto-oncogenes as mediators of growth and development: discussion summary, animal studies and clinical trials.

  7. Regulating the Internet

    ERIC Educational Resources Information Center

    Anderson, Byron

    2007-01-01

    The Internet's breakthrough to primetime usage beginning in the mid-1990s evolved in an era of openness. Unfettered access seemed key to Internet development. An important foundation for the 1996 Telecommunications Act was the theory that the telecom industry would work best if it were free of government regulation, a guiding principle that has…

  8. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jürgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  9. Regulated protein turnover: snapshots of the proteasome in action

    PubMed Central

    Bhattacharyya, Sucharita; Yu, Houqing; Mim, Carsten; Matouschek, Andreas

    2015-01-01

    The ubiquitin-proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and mis-folded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures bring some surprising insights into the functional mechanism of the proteasome and will provide invaluable guidance for genetic and biochemical studies of this key regulatory system. PMID:24452470

  10. ELECTRON EMISSION REGULATING MEANS

    DOEpatents

    Brenholdt, I.R.

    1957-11-19

    >An electronic regulating system is described for controlling the electron emission of a cathode, for example, the cathode in a mass spectrometer. The system incorporates a transformer having a first secondary winding for the above-mentioned cathode and a second secondary winding for the above-mentioned cathode and a second secondary winding load by grid controlled vacuum tubes. A portion of the electron current emitted by the cathode is passed through a network which develops a feedback signal. The system arrangement is completed by using the feedback signal to control the vacuum tubes in the second secondary winding through a regulator tube. When a change in cathode emission occurs, the feedback signal acts to correct this change by adjusting the load on the transformer.

  11. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  12. Siglecs and Immune Regulation

    PubMed Central

    Pillai, Shiv; Netravali, Ilka Arun; Cariappa, Annaiah; Mattoo, Hamid

    2013-01-01

    Sialic acid binding Ig-like lectins or Siglecs vary in their specificity for sialic acid containing ligands and are mainly expressed by cells of the immune system. Many siglecs are inhibitory receptors expressed in innate immune cells that regulate inflammation mediated by DAMPs and PAMPs. This family also includes molecules involved in adhesion and phagocytosis and receptors that can associate with the ITAM containing DAP12 adaptor. Siglecs contribute to the inhibition of immune cells both by binding to cis-ligands (expressed in the same cells) as well as by responding to pathogen derived sialoglycoconjugates. They can help maintain tolerance in B lymphocytes, modulate the activation of conventional and plasmacytoid dendritic cells, and contribute to the regulation of T cell function both directly and indirectly. Siglecs modulate immune responses influencing almost every cell in the immune system, and are of relevance both in health and disease. PMID:22224769

  13. Regulation of inflammasome signaling

    PubMed Central

    Rathinam, Vijay A K; Vanaja, Sivapriya Kailasan; Fitzgerald, Katherine A

    2012-01-01

    Innate immune responses have the ability to both combat infectious microbes and drive pathological inflammation. Inflammasome complexes are a central component of these processes through their regulation of interleukin 1β (IL-1β), IL-18 and pyroptosis. Inflammasomes recognize microbial products or endogenous molecules released from damaged or dying cells both through direct binding of ligands and indirect mechanisms. The potential of the IL-1 family of cytokines to cause tissue damage and chronic inflammation emphasizes the importance of regulating inflammasomes. Many regulatory mechanisms have been identified that act as checkpoints for attenuating inflammasome signaling at multiple steps. Here we discuss the various regulatory mechanisms that have evolved to keep inflammasome signaling in check to maintain immunological balance. PMID:22430786

  14. Redox regulated peroxisome homeostasis

    PubMed Central

    Wang, Xiaofeng; Li, Shuo; Liu, Yu; Ma, Changle

    2014-01-01

    Peroxisomes are ubiquitous organelles present in nearly all eukaryotic cells. Conserved functions of peroxisomes encompass beta-oxidation of fatty acids and scavenging of reactive oxygen species generated from diverse peroxisomal metabolic pathways. Peroxisome content, number, and size can change quickly in response to environmental and/or developmental cues. To achieve efficient peroxisome homeostasis, peroxisome biogenesis and degradation must be orchestrated. We review the current knowledge on redox regulated peroxisome biogenesis and degradation with an emphasis on yeasts and plants. PMID:25545794

  15. Fibrinogen gene regulation.

    PubMed

    Fish, Richard J; Neerman-Arbez, Marguerite

    2012-09-01

    The Aα, Bβ and γ polypeptide chains of fibrinogen are encoded by a three gene cluster on human chromosome four. The fibrinogen genes (FGB-FGA-FGG) are expressed almost exclusively in hepatocytes where their output is coordinated to ensure a sufficient mRNA pool for each chain and maintain an abundant plasma fibrinogen protein level. Fibrinogen gene expression is controlled by the activity of proximal promoters which contain binding sites for hepatocyte transcription factors, including proteins which influence fibrinogen transcription in response to acute-phase inflammatory stimuli. The fibrinogen gene cluster also contains cis regulatory elements; enhancer sequences with liver activities identified by sequence conservation and functional genomics. While the transcriptional control of this gene cluster is fascinating biology, the medical impetus to understand fibrinogen gene regulation stems from the association of cardiovascular disease risk with high level circulating fibrinogen. In the general population this level varies from about 1.5 to 3.5 g/l. This variation between individuals is influenced by genotype, suggesting there are genetic variants contributing to fibrinogen levels which reside in fibrinogen regulatory loci. A complete picture of how fibrinogen genes are regulated will therefore point towards novel sources of regulatory variants. In this review we discuss regulation of the fibrinogen genes from proximal promoters and enhancers, the influence of acute-phase stimulation, post-transcriptional regulation by miRNAs and functional regulatory variants identified in genetic studies. Finally, we discuss the fibrinogen locus in light of recent advances in understanding chromosomal architecture and suggest future directions for researching the mechanisms that control fibrinogen expression. PMID:22836683

  16. Regulating Telecommunications in Europe

    NASA Astrophysics Data System (ADS)

    Cave, Martin

    This chapter describes and assesses the new regime for regulating electronic communications services, which came into force in Europe in July 2003. The first two sections describe, respectively, the previous regime (the 1998 package) and the new regime. The third section discusses experience of the new system up to the end of 2007,1 whereas the fourth evaluates its operation and the plans, already in place, to reform it.

  17. Redox regulated peroxisome homeostasis.

    PubMed

    Wang, Xiaofeng; Li, Shuo; Liu, Yu; Ma, Changle

    2015-01-01

    Peroxisomes are ubiquitous organelles present in nearly all eukaryotic cells. Conserved functions of peroxisomes encompass beta-oxidation of fatty acids and scavenging of reactive oxygen species generated from diverse peroxisomal metabolic pathways. Peroxisome content, number, and size can change quickly in response to environmental and/or developmental cues. To achieve efficient peroxisome homeostasis, peroxisome biogenesis and degradation must be orchestrated. We review the current knowledge on redox regulated peroxisome biogenesis and degradation with an emphasis on yeasts and plants. PMID:25545794

  18. Ensembl regulation resources.

    PubMed

    Zerbino, Daniel R; Johnson, Nathan; Juetteman, Thomas; Sheppard, Dan; Wilder, Steven P; Lavidas, Ilias; Nuhn, Michael; Perry, Emily; Raffaillac-Desfosses, Quentin; Sobral, Daniel; Keefe, Damian; Gräf, Stefan; Ahmed, Ikhlak; Kinsella, Rhoda; Pritchard, Bethan; Brent, Simon; Amode, Ridwan; Parker, Anne; Trevanion, Steven; Birney, Ewan; Dunham, Ian; Flicek, Paul

    2016-01-01

    New experimental techniques in epigenomics allow researchers to assay a diversity of highly dynamic features such as histone marks, DNA modifications or chromatin structure. The study of their fluctuations should provide insights into gene expression regulation, cell differentiation and disease. The Ensembl project collects and maintains the Ensembl regulation data resources on epigenetic marks, transcription factor binding and DNA methylation for human and mouse, as well as microarray probe mappings and annotations for a variety of chordate genomes. From this data, we produce a functional annotation of the regulatory elements along the human and mouse genomes with plans to expand to other species as data becomes available. Starting from well-studied cell lines, we will progressively expand our library of measurements to a greater variety of samples. Ensembl's regulation resources provide a central and easy-to-query repository for reference epigenomes. As with all Ensembl data, it is freely available at http://www.ensembl.org, from the Perl and REST APIs and from the public Ensembl MySQL database server at ensembldb.ensembl.org. Database URL: http://www.ensembl.org. PMID:26888907

  19. Restructuring nuclear regulations.

    PubMed Central

    Mossman, Kenneth L

    2003-01-01

    Nuclear regulations are a subset of social regulations (laws to control activities that may negatively impact the environment, health, and safety) that concern control of ionizing radiation from radiation-producing equipment and from radioactive materials. The impressive safety record among nuclear technologies is due, in no small part, to the work of radiation safety professionals and to a protection system that has kept pace with the rapid technologic advancements in electric power generation, engineering, and medicine. The price of success, however, has led to a regulatory organization and philosophy characterized by complexity, confusion, public fear, and increasing economic costs. Over the past 20 years, regulatory costs in the nuclear sector have increased more than 250% in constant 1995 U.S. dollars. Costs of regulatory compliance can be reduced sharply, particularly when health and environmental benefits of risk reduction are questionable. Three key regulatory areas should be closely examined and modified to improve regulatory effectiveness and efficiency: a) radiation protection should be changed from a risk-based to dose-based system; b) the U.S. government should adopt the modern metric system (International System of Units), and radiation quantities and units should be simplified to facilitate international communication and public understanding; and c) a single, independent office is needed to coordinate nuclear regulations established by U.S. federal agencies and departments. PMID:12515683

  20. Aboveground storage tank regulations

    SciTech Connect

    Geyer, W. )

    1993-01-01

    There are critical differences between the potential for environmental impact of aboveground and underground oil storage. For example, while leaks from underground storage tanks (USTs) seep into soil or aquifers, the concern with aboveground storage tanks (ASTs) is that an overfill or tank rupture can cause product to escape into a navigable stream and immediately create an oil spill pollution incident. The US Environmental Protection Agency (EPA) has very distinct programs outlining regulation parameters for each type of storage, including source of authority, regulatory cutoffs and exclusions, definitions, prevention and response requirements, and penalties, etc. Engineers considering changes or recommending a change in type of storage, particularly from a UST to an AST, need to be aware of existing federal regulations. Since the federal UST program began, remediation costs have skyrocketed as a result of the need to clean up leaking tank and piping sites, backfill and surrounding soil or groundwater. Compliance with federal and state UST regulations has not been cheap, and is expected to top $23 billion, according to some estimates. Partly as a result, market demand has shifted toward use of aboveground storage tanks, a trend that is expected to continue. Industry figures show a 100% increase in factory fabricated aboveground tank activity during the last four years.

  1. Ensembl regulation resources

    PubMed Central

    Zerbino, Daniel R.; Johnson, Nathan; Juetteman, Thomas; Sheppard, Dan; Wilder, Steven P.; Lavidas, Ilias; Nuhn, Michael; Perry, Emily; Raffaillac-Desfosses, Quentin; Sobral, Daniel; Keefe, Damian; Gräf, Stefan; Ahmed, Ikhlak; Kinsella, Rhoda; Pritchard, Bethan; Brent, Simon; Amode, Ridwan; Parker, Anne; Trevanion, Steven; Birney, Ewan; Dunham, Ian; Flicek, Paul

    2016-01-01

    New experimental techniques in epigenomics allow researchers to assay a diversity of highly dynamic features such as histone marks, DNA modifications or chromatin structure. The study of their fluctuations should provide insights into gene expression regulation, cell differentiation and disease. The Ensembl project collects and maintains the Ensembl regulation data resources on epigenetic marks, transcription factor binding and DNA methylation for human and mouse, as well as microarray probe mappings and annotations for a variety of chordate genomes. From this data, we produce a functional annotation of the regulatory elements along the human and mouse genomes with plans to expand to other species as data becomes available. Starting from well-studied cell lines, we will progressively expand our library of measurements to a greater variety of samples. Ensembl’s regulation resources provide a central and easy-to-query repository for reference epigenomes. As with all Ensembl data, it is freely available at http://www.ensembl.org, from the Perl and REST APIs and from the public Ensembl MySQL database server at ensembldb.ensembl.org. Database URL: http://www.ensembl.org PMID:26888907

  2. Regulation reform slows down

    SciTech Connect

    1995-03-29

    Regulatory reformers in Congress are easing off the accelerator as they recognize that some of their more far-reaching proposals lack sufficient support to win passage. Last week the proposed one-year moratorium on new regulations was set back in the Senate by it main sponsor, Sen. Non Nickles (R., OK), who now seeks to replace it with a more moderate bill. Nickel`s substitute bill would give Congress 45 days after a regulation is issued to decide whether to reject it. It also retroactively allows for review of 80 regulations issued since last November 9, 1994. Asked how his new proposal is superior to a moratorium, which is sharply opposed by the Clinton Administration, Nickles says he thinks it is better because its permanent. The Chemical Manufacturer`s Association (CMA) has not publicly made a regulatory moratorium a top priority, but has quietly supported it by joining with other industry groups lobbying on the issue. A moratorium would halt EPA expansion of the Toxics Release Inventory (TRI) and alloys the delisting of several TRI chemicals.

  3. Flow compensating pressure regulator

    NASA Technical Reports Server (NTRS)

    Baehr, E. F. (Inventor)

    1978-01-01

    An apparatus for regulating pressure of treatment fluid during ophthalmic procedures is described. Flow sensing and pressure regulating diaphragms are used to modulate a flow control valve. The pressure regulating diaphragm is connected to the flow control valve to urge the valve to an open position due to pressure being applied to the diaphragm by bias means such as a spring. The flow sensing diaphragm is mechanically connected to the flow control valve and urges it to an opened position because of the differential pressure on the diaphragm generated by a flow of incoming treatment fluid through an orifice in the diaphragm. A bypass connection with a variable restriction is connected in parallel relationship to the orifice to provide for adjusting the sensitivity of the flow sensing diaphragm. A multiple lever linkage system is utilized between the center of the second diaphragm and the flow control valve to multiply the force applied to the valve by the other diaphragm and reverse the direction of the force.

  4. Taiwan Regulation of Biobanks.

    PubMed

    Fan, Chien-Te; Hung, Tzu-Hsun; Yeh, Chan-Kun

    2015-01-01

    This paper introduces legal framework and governance structure in relation to the management and development of biobanks in Taiwan. At first, we briefly describe Taiwan's population, political system and health care system. Secondly, this research introduces biobanking framework of Taiwan including 25 biobanks established with the approval of the Ministry of Health and Welfare. In those biobanks, "Taiwan Biobank" is the first and the largest government-supported biobank which comprises population-based cohort study and disease- oriented study. Since the collection of information, data, and biological specimen of biobanks often involve highly sensitive personal information, in the legal framework of Taiwan, there is a specific regulation, "Human Biobank Management Act" (HBMA), which plays an important role in regulating biobanks in Taiwan. HBMA, the Personal Information Act and other regulations constitute a comprehensive legal and regulatory privacy framework of biobanks. Through the introduction and analysis of the current legal framework applicable to biobanks, we found that there are several challenges that need to be solved appropriately that involve duplicate review systems, the obstacles in the international collaboration, and data sharing between biobanks in Taiwan. PMID:26711420

  5. Improving CS regulations.

    SciTech Connect

    Nesse, R.J.; Scheer, R.M.; Marasco, A.L.; Furey, R.

    1980-10-01

    President Carter issued Executive Order 12044 (3/28/78) that required all Federal agencies to distinguish between significant and insignificant regulations, and to determine whether a regulation will result in major impacts. This study gathered information on the impact of the order and the guidelines on the Office of Conservation and Solar Energy (CS) regulatory practices, investigated problems encountered by the CS staff when implementing the order and guidelines, and recommended solutions to resolve these problems. Major tasks accomplished and discussed are: (1) legislation, Executive Orders, and DOE Memoranda concerning Federal administrative procedures relevant to the development and analysis of regulations within CS reviewed; (2) relevant DOE Orders and Memoranda analyzed and key DOE and CS staff interviewed in order to accurately describe the current CS regulatory process; (3) DOE staff from the Office of the General Counsel, the Office of Policy and Evaluation, the Office of the Environment, and the Office of the Secretary interviewed to explore issues and problems encountered with current CS regulatory practices; (4) the regulatory processes at five other Federal agencies reviewed in order to see how other agencies have approached the regulatory process, dealt with specific regulatory problems, and responded to the Executive Order; and (5) based on the results of the preceding four tasks, recommendations for potential solutions to the CS regulatory problems developed. (MCW)

  6. Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

    PubMed Central

    Hartmann, Thomas; Xu, Xinsong; Kronast, Mira; Muehlich, Susanne; Meyer, Kathleen; Zimmermann, Wolfgang; Hurwitz, Jerard; Pan, Zhen-Qiang; Engelhardt, Stefan; Sarikas, Antonio

    2014-01-01

    Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT–CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LTΔ69–83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways. PMID:24550499

  7. Regulation of adipocyte lipolysis.

    PubMed

    Frühbeck, Gema; Méndez-Giménez, Leire; Fernández-Formoso, José-Antonio; Fernández, Secundino; Rodríguez, Amaia

    2014-06-01

    In adipocytes the hydrolysis of TAG to produce fatty acids and glycerol under fasting conditions or times of elevated energy demands is tightly regulated by neuroendocrine signals, resulting in the activation of lipolytic enzymes. Among the classic regulators of lipolysis, adrenergic stimulation and the insulin-mediated control of lipid mobilisation are the best known. Initially, hormone-sensitive lipase (HSL) was thought to be the rate-limiting enzyme of the first lipolytic step, while we now know that adipocyte TAG lipase is the key enzyme for lipolysis initiation. Pivotal, previously unsuspected components have also been identified at the protective interface of the lipid droplet surface and in the signalling pathways that control lipolysis. Perilipin, comparative gene identification-58 (CGI-58) and other proteins of the lipid droplet surface are currently known to be key regulators of the lipolytic machinery, protecting or exposing the TAG core of the droplet to lipases. The neuroendocrine control of lipolysis is prototypically exerted by catecholaminergic stimulation and insulin-induced suppression, both of which affect cyclic AMP levels and hence the protein kinase A-mediated phosphorylation of HSL and perilipin. Interestingly, in recent decades adipose tissue has been shown to secrete a large number of adipokines, which exert direct effects on lipolysis, while adipocytes reportedly express a wide range of receptors for signals involved in lipid mobilisation. Recently recognised mediators of lipolysis include some adipokines, structural membrane proteins, atrial natriuretic peptides, AMP-activated protein kinase and mitogen-activated protein kinase. Lipolysis needs to be reanalysed from the broader perspective of its specific physiological or pathological context since basal or stimulated lipolytic rates occur under diverse conditions and by different mechanisms. PMID:24872083

  8. Gastrointestinal hormones regulating appetite.

    PubMed

    Chaudhri, Owais; Small, Caroline; Bloom, Steve

    2006-07-29

    The role of gastrointestinal hormones in the regulation of appetite is reviewed. The gastrointestinal tract is the largest endocrine organ in the body. Gut hormones function to optimize the process of digestion and absorption of nutrients by the gut. In this capacity, their local effects on gastrointestinal motility and secretion have been well characterized. By altering the rate at which nutrients are delivered to compartments of the alimentary canal, the control of food intake arguably constitutes another point at which intervention may promote efficient digestion and nutrient uptake. In recent decades, gut hormones have come to occupy a central place in the complex neuroendocrine interactions that underlie the regulation of energy balance. Many gut peptides have been shown to influence energy intake. The most well studied in this regard are cholecystokinin (CCK), pancreatic polypeptide, peptide YY, glucagon-like peptide-1 (GLP-1), oxyntomodulin and ghrelin. With the exception of ghrelin, these hormones act to increase satiety and decrease food intake. The mechanisms by which gut hormones modify feeding are the subject of ongoing investigation. Local effects such as the inhibition of gastric emptying might contribute to the decrease in energy intake. Activation of mechanoreceptors as a result of gastric distension may inhibit further food intake via neural reflex arcs. Circulating gut hormones have also been shown to act directly on neurons in hypothalamic and brainstem centres of appetite control. The median eminence and area postrema are characterized by a deficiency of the blood-brain barrier. Some investigators argue that this renders neighbouring structures, such as the arcuate nucleus of the hypothalamus and the nucleus of the tractus solitarius in the brainstem, susceptible to influence by circulating factors. Extensive reciprocal connections exist between these areas and the hypothalamic paraventricular nucleus and other energy-regulating centres of the

  9. [Regulation of terpene metabolism

    SciTech Connect

    Croteau, R.

    1992-01-01

    This report describes accomplishments over the past year on understanding of terpene synthesis in mint plants and sage. Specifically reported are the fractionation of 4-S-limonene synthetase, the enzyme responsible for the first committed step to monoterpene synthesis, along with isolation of the corresponding RNA and DNA cloning of its gene; the localization of the enzyme within the oil glands, regulation of transcription and translation of the synthetase, the pathway to camphor biosynthesis,a nd studies on the early stages and branch points of the isoprenoid pathway.

  10. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  11. Self-regulating valve

    DOEpatents

    Humphreys, D.A.

    1982-07-20

    A variable, self-regulating valve having a hydraulic loss coefficient proportional to a positive exponential power of the flow rate. The device includes two objects in a flow channel and structure which assures that the distance between the two objects is an increasing function of the flow rate. The range of spacing between the objects is such that the hydraulic resistance of the valve is an increasing function of the distance between the two objects so that the desired hydraulic loss coefficient as a function of flow rate is obtained without variation in the flow area.

  12. REGULATION OF VASCULOGENESIS AND ANGIOGENESIS

    EPA Science Inventory

    Regulation of vasculogenesis and angiogenesis.
    B.D. Abbott
    Reproductive Toxicology Division, Environmental Protection Agency, Research Triangle Park, North Carolina, USA
    Vasculogenesis and angiogenesis are regulated by a complex, interactive family of receptors and lig...

  13. Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation.

    PubMed

    Sen, Rwik; Ferdoush, Jannatul; Kaja, Amala; Bhaumik, Sukesh R

    2016-06-01

    FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression. PMID:27044865

  14. Temperature controlled high voltage regulator

    DOEpatents

    Chiaro, Jr., Peter J.; Schulze, Gerald K.

    2004-04-20

    A temperature controlled high voltage regulator for automatically adjusting the high voltage applied to a radiation detector is described. The regulator is a solid state device that is independent of the attached radiation detector, enabling the regulator to be used by various models of radiation detectors, such as gas flow proportional radiation detectors.

  15. [Regulation of terpene metabolism

    SciTech Connect

    Croteau, R.

    1991-01-01

    During the last grant period, we have completed studies on the key pathways of monoterpene biosynthesis and catabolism in sage and peppermint, and have, by several lines of evidence, deciphered the rate-limiting step of each pathway. We have at least partially purified and characterized the relevant enzymes of each pathway. We have made a strong case, based on analytical, in vivo, and in vitro studies, that terpene accumulation depends upon the balance between biosynthesis and catabolism, and provided supporting evidence that these processes are developmentally-regulated and very closely associated with senescence of the oil glands. Oil gland ontogeny has been characterized at the ultrastructural level. We have exploited foliar-applied bioregulators to delay gland senescence, and have developed tissue explant and cell culture systems to study several elusive aspects of catabolism. We have isolated pure gland cell clusters and localized monoterpene biosynthesis and catabolism within these structures, and have used these preparations as starting materials for the purification to homogeneity of target regulatory'' enzymes. We have thus developed the necessary background knowledge, based on a firm understanding of enzymology, as well as the necessary experimental tools for studying the regulation of monoterpene metabolism at the molecular level. Furthermore, we are now in a position to extend our systematic approach to other terpenoid classes (C[sub 15]-C[sub 30]) produced by oil glands.

  16. Environmental regulations on chlorofluorocarbons

    SciTech Connect

    Hoffman, J.S.; Wells, J.B. )

    1989-05-01

    In August 1988, the US Environmental Protection Agency issued final regulations that implement the Montreal Protocol on Substances that Deplete the Ozone Layer. The regulations require a 50% reduction in consumption of fully halogenated chlorofluorocarbons (CFCs) within 10 years and a freeze on consumption of halons within 4 years. The Montreal Protocol provisions were designed in September 1987 based on the results of a 2-year international series of scientific, technical, and economic workshops. As would be expected, scientific investigations continued during this period. While these investigations suggested that significant global depletion had already occurred, these preliminary findings were not taken into account during negotiations or rulemaking. In March 1988, however, the international Ozone Trends Panel confirmed the findings. Depletion greater than that projected under the Montreal Protocol has already occurred. An early reassessment of the Protocol provisions appears to be inevitable. Restrictions on CFCs will affect the refrigeration and air-conditioning industries. Emerging alternatives to CFCs include newly developed refrigerants, innovative designs, and engineering controls. Key issues in evaluating these alternatives include energy efficiency, capital costs, service to consumers, and compatibility with existing designs.

  17. Buck/boost regulator

    NASA Technical Reports Server (NTRS)

    Paulkovich, J.; Rodriguez, G. E. (Inventor)

    1981-01-01

    A voltage regulated DC to DC converter uses an inductor and a capacitor as storage elements. The inductor is composed of two windings having a common junction. A transformer with a center tap connected to the common junction of the two windings is connected at either end of its winding to ground through controlled switches. One winding of the inductor and either end of the transformer winding are connected by power diodes to the capacitor which supplies the output voltage to a load. The other winding of the inductor is connected to a fourth power diode as a clamping diode. Input voltage is supplied to the inductor through a third controlled switch. A pulse width modulator connected to the output of the converter alternately closes and opens the switches connected to either end of the transformer winding and also closes the switch supplying input voltage to the inductor each time either of the switches connected to the ends of the transformer winding are closed. The duty cycle of the closing and opening of the several switches is adjusted by the pulse modulator to regulate the output voltage.

  18. The regulation of public utilities

    SciTech Connect

    Phillips, C.F. Jr.

    1992-01-01

    The current edition of [open quotes]The Regulation of Public Utilities[close quotes] is divided into 17 chapters which provide an historical analysis of the economic and legal concepts of public utility regulation, rate of return, rate base, operating expenses, rate structure, the electric power and natural gas industries, as well as the telecommunications and water industries. The value of the third edition is limited by the changes that have taken place in public utility regulation since 1992; current topic such as cogeneration, independent power production, and the sea-change in oil pipeline regulations are not discussed. The volume does, however, provide a comprehensive historical background of utility regulation.

  19. 75 FR 10997 - Cuban Assets Control Regulations; Sudanese Sanctions Regulations; Iranian Transactions Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-10

    ...The Department of the Treasury's Office of Foreign Assets Control (``OFAC'') is amending the Sudanese Sanctions Regulations and the Iranian Transactions Regulations to authorize the exportation of certain services and software incident to the exchange of personal communications over the Internet. Similarly, OFAC is amending the Cuban Assets Control Regulations to authorize the exportation of......

  20. The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Ravid, T; Doolman, R; Avner, R; Harats, D; Roitelman, J

    2000-11-17

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR. PMID:10964918

  1. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

    Microglia are resident macrophages of the central nervous system (CNS) that display high functional similarities to other tissue macrophages. However, it is especially important to create and maintain an intact tissue homeostasis to support the neuronal cells, which are very sensitive even to minor changes in their environment. The transition from the “resting” but surveying microglial phenotype to an activated stage is tightly regulated by several intrinsic (e.g., Runx-1, Irf8, and Pu.1) and extrinsic factors (e.g., CD200, CX3CR1, and TREM2). Under physiological conditions, minor changes of those factors are sufficient to cause fatal dysregulation of microglial cell homeostasis and result in severe CNS pathologies. In this review, we discuss recent achievements that gave new insights into mechanisms that ensure microglia quiescence. PMID:23630462

  2. Pubertal development and regulation.

    PubMed

    Abreu, Ana Paula; Kaiser, Ursula B

    2016-03-01

    Puberty marks the end of childhood and is a period when individuals undergo physiological and psychological changes to achieve sexual maturation and fertility. The hypothalamic-pituitary-gonadal axis controls puberty and reproduction and is tightly regulated by a complex network of excitatory and inhibitory factors. This axis is active in the embryonic and early postnatal stages of life and is subsequently restrained during childhood, and its reactivation culminates in puberty initiation. The mechanisms underlying this reactivation are not completely known. The age of puberty onset varies between individuals and the timing of puberty initiation is associated with several health outcomes in adult life. In this Series paper, we discuss pubertal markers, epidemiological trends of puberty initiation over time, and the mechanisms whereby genetic, metabolic, and other factors control secretion of gonadotropin-releasing hormone to determine initiation of puberty. PMID:26852256

  3. Magnetostrictive Pressure Regulating System

    NASA Technical Reports Server (NTRS)

    Richard, James A. (Inventor); Pickens, Herman L. (Inventor)

    2013-01-01

    A magnetostrictive pressure regulating system includes a magnetostrictive valve that incorporates a magnetostrictive actuator with at least one current-carrying coil disposed thereabout. A pressure force sensor, in fluid communication with the fluid exiting the valve, includes (i) a magnetostrictive material, (ii) a magnetic field generator in proximity to the magnetostrictive material for inducing a magnetic field in and surrounding the magnetostrictive material wherein lines of magnetic flux passing through the magnetostrictive material are defined, and (iii) a sensor positioned adjacent to the magnetostrictive material and in the magnetic field for measuring changes in at least one of flux angle and flux density when the magnetostrictive material experiences an applied force that is aligned with the lines of magnetic flux. The pressure of the fluid exiting the valve causes the applied force. A controller coupled to the sensor and to the current-carrying coil adjusts a current supplied to the current-carrying coil based on the changes so-measured.

  4. Regulation of Potassium Homeostasis

    PubMed Central

    2015-01-01

    Potassium is the most abundant cation in the intracellular fluid, and maintaining the proper distribution of potassium across the cell membrane is critical for normal cell function. Long-term maintenance of potassium homeostasis is achieved by alterations in renal excretion of potassium in response to variations in intake. Understanding the mechanism and regulatory influences governing the internal distribution and renal clearance of potassium under normal circumstances can provide a framework for approaching disorders of potassium commonly encountered in clinical practice. This paper reviews key aspects of the normal regulation of potassium metabolism and is designed to serve as a readily accessible review for the well informed clinician as well as a resource for teaching trainees and medical students. PMID:24721891

  5. Regulation of Terpene Metabolism

    SciTech Connect

    Rodney Croteau

    2004-03-14

    OAK-B135 Research over the last four years has progressed fairly closely along the lines initially proposed, with progress-driven expansion of Objectives 1, 2 and 3. Recent advances have developed from three research thrusts: 1. Random sequencing of an enriched peppermint oil gland cDNA library has given access to a large number of potential pathway and regulatory genes for test of function; 2. The availability of new DNA probes and antibodies has permitted investigation of developmental regulation and organization of terpenoid metabolism; and 3. The development of a transformation system for peppermint by colleagues at Purdue University has allowed direct transgenic testing of gene function and added a biotechnological component to the project. The current status of each of the original research objectives is outlined below.

  6. Transcription Regulation in Archaea.

    PubMed

    Gehring, Alexandra M; Walker, Julie E; Santangelo, Thomas J

    2016-07-15

    The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. PMID:27137495

  7. TFEB regulates lysosomal proteostasis.

    PubMed

    Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura

    2013-05-15

    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs. PMID:23393155

  8. [Regulation of terpene metabolism

    SciTech Connect

    Croteau, R.

    1989-11-09

    Terpenoid oils, resins, and waxes from plants are important renewable resources. The objective of this project is to understand the regulation of terpenoid metabolism using the monoterpenes (C[sub 10]) as a model. The pathways of monoterpene biosynthesis and catabolism have been established, and the relevant enzymes characterized. Developmental studies relating enzyme levels to terpene accumulation within the oil gland sites of synthesis, and work with bioregulators, indicate that monoterpene production is controlled by terpene cyclases, the enzymes catalyzing the first step of the monoterpene pathway. As the leaf oil glands mature, cyclase levels decline and monoterpene biosynthesis ceases. Yield then decreases as the monoterpenes undergo catabolism by a process involving conversion to a glycoside and transport from the leaf glands to the root. At this site, the terpenoid is oxidatively degraded to acetate that is recycled into other lipid metabolites. During the transition from terpene biosynthesis to catabolism, the oil glands undergo dramatic ultrastructural modification. Degradation of the producing cells results in mixing of previously compartmentized monoterpenes with the catabolic enzymes, ultimately leading to yield decline. This regulatory model is being applied to the formation of other terpenoid classes (C[sub 15] C[sub 20], C[sub 30], C[sub 40]) within the oil glands. Preliminary investigations on the formation of sesquiterpenes (C[sub 15]) suggest that the corresponding cyclases may play a lesser role in determining yield of these products, but that compartmentation effects are important. From these studies, a comprehensive scheme for the regulation of terpene metabolism is being constructed. Results from this project wail have important consequences for the yield and composition of terpenoid natural products that can be made available for industrial exploitation.

  9. Endocannabinoids in cerebrovascular regulation.

    PubMed

    Benyó, Zoltán; Ruisanchez, Éva; Leszl-Ishiguro, Miriam; Sándor, Péter; Pacher, Pál

    2016-04-01

    The cerebral blood flow is tightly regulated by myogenic, endothelial, metabolic, and neural mechanisms under physiological conditions, and a large body of recent evidence indicates that inflammatory pathways have a major influence on the cerebral blood perfusion in certain central nervous system disorders, like hemorrhagic and ischemic stroke, traumatic brain injury, and vascular dementia. All major cell types involved in cerebrovascular control pathways (i.e., smooth muscle, endothelium, neurons, astrocytes, pericytes, microglia, and leukocytes) are capable of synthesizing endocannabinoids and/or express some or several of their target proteins [i.e., the cannabinoid 1 and 2 (CB1 and CB2) receptors and the transient receptor potential vanilloid type 1 ion channel]. Therefore, the endocannabinoid system may importantly modulate the regulation of cerebral circulation under physiological and pathophysiological conditions in a very complex manner. Experimental data accumulated since the late 1990s indicate that the direct effect of cannabinoids on cerebral vessels is vasodilation mediated, at least in part, by CB1 receptors. Cannabinoid-induced cerebrovascular relaxation involves both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium. However, under stress conditions (e.g., in conscious restrained animals or during hypoxia and hypercapnia), cannabinoid receptor activation was shown to induce a reduction of the cerebral blood flow, probably via inhibition of the electrical and/or metabolic activity of neurons. Finally, in certain cerebrovascular pathologies (e.g., subarachnoid hemorrhage, as well as traumatic and ischemic brain injury), activation of CB2 (and probably yet unidentified non-CB1/non-CB2) receptors appear to improve the blood perfusion of the brain via attenuating vascular inflammation. PMID:26825517

  10. Regulating the regulators: serine/arginine-rich proteins under scrutiny.

    PubMed

    Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella

    2012-10-01

    Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908