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1

Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast  

ERIC Educational Resources Information Center

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

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

2006-01-01

2

Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin-proteasome pathway.  

PubMed Central

The inducible cAMP early repressor (ICER) is a powerful transcriptional inhibitor that plays an important role in the regulation of the cAMP-dependent transcriptional response in the neuroendocrine system. ICER activity is primarily determined by its intracellular concentration, rather than by post-translational modifications, such as phosphorylation. We investigated the mechanisms that regulate the levels of ICER transcript and polypeptides in cardiocytes, myogenic (C2C12) and pituitary-derived (GH3) cell lines. We show that in primary cardiocytes and GH3 cells ICER was inducible by cAMP but not by membrane depolarization. Moreover, lactacystin, a specific proteasome inhibitor, decreased the rate of ICER degradation. This effect was associated with the accumulation of ICER-ubiquitin conjugates. We conclude that the intracellular levels of ICER are controlled by the ubiquitin-proteasome pathway for protein breakdown.

Folco, E J; Koren, G

1997-01-01

3

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.  

PubMed Central

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.

Jeffers, M; Taylor, G A; Weidner, K M; Omura, S; Vande Woude, G F

1997-01-01

4

The significance of ubiquitin proteasome pathway in cancer development.  

PubMed

The ubiquitin proteasome pathway is the most significant intracellular proteolytic pathway. The target proteins are usually ubiquitinated prior to degradation by the proteasome; however, ubiquitin-independent targeting mechanisms have also been reported (e.g., the antizyme-mediated degradation of ornithine decarboxylase). Aberrations in the components of the ubiquitin proteasome pathway are commonly observed in many cancers, and uncontrolled growth of cancer cells can result either from stabilization of oncoproteins (e.g., c-jun) or increased degradation of tumor suppressor proteins (e.g., p53). In addition, due to the pleiotropic functions of the ubiquitin proteasome pathway in cells, there is great interest in developing inhibitors to specifically block this pathway for cancer treatment. This review summarizes the recent literature and several patented inventions on the ubiquitin proteasome pathway with respect to its role in cancer development and treatment. PMID:23061719

Yerlikaya, Azmi; Yöntem, Mustafa

2013-09-01

5

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels.  

PubMed

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels. PMID:15987767

Yan, Fei-Fei; Lin, Chia-Wei; Cartier, Etienne A; Shyng, Show-Ling

2005-11-01

6

Glutathiolation Enhances the Degradation of C-crystallin in Lens and Reticulocyte Lysates, Partially via the Ubiquitin-Proteasome Pathway  

Microsoft Academic Search

PURPOSE. S-glutathiolated proteins are formed in the lens during aging and cataractogenesis. The objective of this work was to explore the role of the ubiquitin-proteasome pathway in elim- inating S-glutathiolated C-crystallin. METHODS. Recombinant human C-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25°C for 1 hour. The extent of glutathiolation of the C- crystallin was determined

Madeleine Zetterberg; Xinyu Zhang; Allen Taylor; Bingfen Liu; Jack J. Liang; Fu Shang

7

The Ubiquitin-Proteasome Pathway and Proteasome Inhibitors  

PubMed Central

The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases.

Myung, Jayhyuk; Kim, Kyung Bo

2008-01-01

8

Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway?  

PubMed Central

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Gould, Faye; Harrison, Sally M.; Hewitt, Eric W.; Whitehouse, Adrian

2009-01-01

9

Genome-wide approaches to systematically identify substrates of the ubiquitin/proteasome pathway  

PubMed Central

The ubiquitin/proteasome system handles the majority of controlled proteolysis in eukaryotes. Defects in the ubiquitin/proteasome system have been implicated in diseases ranging from cancers to neurodegenerative disorders. However, the precise role of ubiquitin/proteasome-mediated degradation in health and disease is far from clear. A major challenge is to link specific substrates directly to a particular degradation pathway. Here, we review genome-wide approaches that have been developed in recent years to comprehensively identify ubiquitylated substrates of a particular pathway. The components of the ubiquitin/proteasome system are attractive drug targets, as illustrated by the efficacy of some proteasome inhibitors in the treatment of multiple myeloma. Information that has emerged from these studies could reveal novel drug targets and strategies for treating human diseases.

Liu, Chang; Choe, Vitnary; Rao, Hai

2010-01-01

10

The adaptor molecule CIN85 regulates Syk tyrosine kinase level by activating the ubiquitin-proteasome degradation pathway.  

PubMed

Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcepsilonRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcepsilonRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcepsilonRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcepsilonRI-induced tyrosine phosphorylation of phospholipase Cgamma, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity. PMID:17675467

Peruzzi, Giovanna; Molfetta, Rosa; Gasparrini, Francesca; Vian, Laura; Morrone, Stefania; Piccoli, Mario; Frati, Luigi; Santoni, Angela; Paolini, Rossella

2007-08-15

11

The Ubiquitin-Proteasome Pathway and Synaptic Plasticity  

ERIC Educational Resources Information Center

Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

Hegde, Ashok N.

2010-01-01

12

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells  

PubMed Central

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C.; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M.; Jones, John E.; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaetan; Sibley, David R.; Eisner, Gilbert; Felder, Robin A.; Jose, Pedro A.

2008-01-01

13

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.  

PubMed

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R. PMID:18464932

Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M; Jones, John E; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R; Eisner, Gilbert; Gildea, John J; Felder, Robin A; Jose, Pedro A

2008-06-01

14

The ubiquitin-proteasome pathway in cancer.  

PubMed Central

Degradation by the 26S proteasome of specific proteins that have been targeted by the ubiquitin pathway is the major intracellular non-lysosomal proteolytic mechanism and is involved in a broad range of processes, such as cell cycle progression, antigen presentation and control of gene expression. Recent work, reviewed here, has shown that this pathway is often the target of cancer-related deregulation and can underlie processes, such as oncogenic transformation, tumour progression, escape from immune surveillance and drug resistance.

Spataro, V.; Norbury, C.; Harris, A. L.

1998-01-01

15

Degradation signal diversity in the ubiquitin-proteasome system  

PubMed Central

Preface The ubiquitin-proteasome system degrades an enormous variety of proteins, which are targeted by specific degradation signals (degrons). Besides the degradation of regulatory proteins, virtually every protein suffers from sporadic biosynthetic errors or misfolding, and cells can recognize such aberrant proteins and rapidly degrade them. Structural and functional data on a handful of degrons allows some generalizations about their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons subject to signalling-dependent modification and those controlled by protein folding or assembly, as frequently occurs during protein quality control.

Ravid, Tommer; Hochstrasser, Mark

2008-01-01

16

The ubiquitin ligase FBG1 promotes the degradation of the disease-linked protein torsinA through the ubiquitin proteasome pathway and macroautophagy  

PubMed Central

DYT1 dystonia is a dominantly inherited, disabling neurological disorder with low penetrance that is caused by the deletion of a glutamic acid (?E) in the protein torsinA. We previously showed that torsinA(wt) is degraded through macroautophagy while torsinA(?E) is targeted to the ubiquitin proteasome pathway (UPP). The different catabolism of torsinA(wt) and (?E) potentially modulates torsinA(wt):torsinA(?E) stoichiometry. Therefore, gaining a mechanistic understanding on how the protein quality control machinery clears torsinA(?E) in neurons may uncover important regulatory steps in disease pathogenesis. Here, we asked whether FBG1, a ubiquitin ligase known to degrade neuronal glycoproteins, is implicated in the degradation of torsinA(?E) by the UPP. In a first set of studies completed in cultured cells, we show that FBG1 interacts with and influences the steady-state levels of torsinA(wt) and (?E). Interestingly, FBG1 achieves this effect promoting the degradation of torsinA not only through the UPP, but also by macroautophagy. To determine the potential clinical significance of these findings, we asked if eliminating expression of Fbg1 triggers a motor phenotype in torsinA(?E) knock in mice, a model of non-manifesting DYT1 mutation carriers. We detected differences in spontaneous locomotion between aged torsinA(?E) knock in-Fbg1 knock out and control mice. Furthermore, neuronal levels of torsinA were unaltered in Fbg1 null mice, indicating that redundant systems likely compensate in vivo for the absence of this ubiquitin ligase. In summary, our studies support a non-essential role for FBG1 on the degradation of torsinA and uncover a novel link of FBG1 to the autophagy pathway.

Gordon, Kara L; Glenn, Kevin A; Bode, Nicole; Wen, Hsiang M; Paulson, Henry L; Gonzalez-Alegre, Pedro

2012-01-01

17

Ubiquitin-proteasome pathway and cellular responses to oxidative stress  

PubMed Central

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

Taylor, Allen

2011-01-01

18

Pristimerin Induces Apoptosis in Prostate Cancer Cells by Down-regulating Bcl-2 through ROS-dependent Ubiquitin-proteasomal Degradation Pathway  

PubMed Central

Pristimerin is a quinonemethide triterpenoid with the potential of a promising anticancer agent. Pristimerin (PM) has shown anticancer activity against a range of cancer cell lines, but its activity for prostate cancer has not been adequately investigated. In the present study we have examined the underlying mechanisms of the apoptotic response of the hormone-sensitive (LNCaP) and hormone-refractory (PC-3) prostate cancer cell lines to PM. Treatment with PM induced apoptosis in both cell lines as characterized by increased annexin V-binding and cleavage of PARP-1 and procaspases-3 and -9. It also induced mitochondrial depolarization, cytochrome c release from mitochondria and generation of reactive oxygen species (ROS). Response to PM is regulated by Bcl-2 since it down-regulated Bcl-2 expression and overexpression of Bcl-2 rendered prostate cancer cells resistant to PM. ROS plays a role in down-regulation of Bcl-2, since treatment with PM in the presence of various ROS modulators, e.g., n-acetylcysteine (NAC), a general purpose antioxidant; diphenylene iodonium (DPI), a NADPH inhibitor; rotenone (ROT), a mitochondrial electron transport chain interrupter rotenone or MnTBAP, a O2 scavenger, attenuated the down-regulation of Bcl-2. Furthermore, ROS is also involved in the ubiquitination and proteasomal degradation of Bcl-2 as both of these events were blocked by O 2? scavenger MnTBAP. Thus, pristimerin induces apoptosis in prostate cancer cells predominately through the mitochondrial apoptotic pathway by inhibiting antiapoptic Bcl-2 through a ROS-dependent ubiquitin-proteasomal degradation pathway.

Liu, Yong Bo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S; Gautam, Subhash C

2014-01-01

19

Angiotensin-(1-7) through AT receptors mediates tyrosine hydroxylase degradation via the ubiquitin-proteasome pathway.  

PubMed

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1-7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1-7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1-7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from (3)H-l-tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1-7) showed a significant decrease in TH specific activity. Ang-(1-7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT(2) receptor antagonist and not by an AT(1) or Mas receptor antagonist, suggesting the involvement of AT(2) receptors. The proteasome inhibitor MG132 blocked the Ang-(1-7)-mediated TH reduction. In addition, Ang-(1-7) increased the amount of TH-ubiquitin complexes, indicating that the Ang-(1-7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1-7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity. PMID:19183250

Lopez Verrilli, María A; Pirola, Carlos J; Pascual, Mariano M; Dominici, Fernando P; Turyn, Daniel; Gironacci, Mariela M

2009-04-01

20

Plant ubiquitin-proteasome pathway and its role in gibberellin signaling  

PubMed Central

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

Wang, Feng; Deng, Xing Wang

2011-01-01

21

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.  

PubMed

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids. PMID:8700868

Isozaki, U; Mitch, W E; England, B K; Price, S R

1996-03-01

22

Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program  

PubMed Central

The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin–proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

Matsushita, Akane; Inoue, Haruhiko; Goto, Shingo; Nakayama, Akira; Sugano, Shoji; Hayashi, Nagao; Takatsuji, Hiroshi

2013-01-01

23

CSA-dependent degradation of CSB by the ubiquitin-proteasome pathway establishes a link between complementation factors of the Cockayne syndrome  

PubMed Central

Mutations in the CSA or CSB complementation genes cause the Cockayne syndrome, a severe genetic disorder that results in patients’ death in early adulthood. CSA and CSB act in a transcription-coupled repair (TCR) pathway, but their functional relationship is not understood. We have previously shown that CSA is a subunit of an E3 ubiquitin ligase complex. Here we demonstrate that CSB is a substrate of this ligase: Following UV irradiation, CSB is degraded at a late stage of the repair process in a proteasome- and CSA-dependent manner. Moreover, we demonstrate the importance of CSB degradation for post-TCR recovery of transcription and for the Cockayne syndrome. Our results unravel for the first time the functional relationship between CSA and CSB.

Groisman, Regina; Kuraoka, Isao; Chevallier, Odile; Gaye, Nogaye; Magnaldo, Thierry; Tanaka, Kiyoji; Kisselev, Alexei F.; Harel-Bellan, Annick; Nakatani, Yoshihiro

2006-01-01

24

The Ubiquitin Proteasome Pathway (UPP) in the regulation of cell cycle control and DNA damage repair and its implication in tumorigenesis  

PubMed Central

Accumulated evidence supports that the ubiquitin proteasome pathway (UPP) plays a crucial role in protein metabolism implicated in the regulation of many biological processes such as cell cycle control, DNA damage response, apoptosis, and so on. Therefore, alterations for the ubiquitin proteasome signaling or functional impairments for the ubiquitin proteasome components are involved in the etiology of many diseases, particularly in cancer development. In this minireview, we first give a brief outline for the ubiquitin proteasome pathway, we then discuss with focus for the ubiquitin proteasome pathway in the regulation of cell cycle control and DNA damage response, the relevance for the altered regulation of these signaling pathways in tumorigenesis is also reviewed. We finally assess and summarize the advancement for targeting the ubiquitin proteasome pathway in cancer therapy. A better understanding of the biological functions underlying ubiquitin regulatory mechanisms would provide us a wider prospective on cancer treatment.

Tu, Yaqin; Chen, Cai; Pan, Junru; Xu, Junfa; Zhou, Zhi-Guang; Wang, Cong-Yi

2012-01-01

25

Space shuttle flight (STS90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway  

Microsoft Academic Search

To elucidate the mechanisms of microgravity-induced muscle atrophy, we focused on fast-type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16-d spaceflight (STS-90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the

Madoka Ikemoto; Takeshi Nikawa; Shin' ichi Takeda; Chiho Watanabe; Takako Kitano; Kenneth M. Baldwin; Ryutaro Izumi; Ikuya Nonaka; Takae Towatari; Shigetada Teshima; Kazuhito Rokutan; Kyoichi Kishi

2001-01-01

26

?-catenin is a target for the ubiquitin–proteasome pathway  

Microsoft Academic Search

?-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless\\/Wnt signaling pathway. In the current model of this pathway, the amount of ?-catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the cadherin–catenin complex is low in the absence of Wingless\\/Wnt signal. Here we show that

Hermann Aberle; Andreas Bauer; Jörg Stappert; Andreas Kispert; Rolf Kemler

1997-01-01

27

Targeting Tumor Ubiquitin-Proteasome Pathway with Polyphenols for Chemosensitization  

PubMed Central

The development of tumor drug resistance is one of the biggest obstacles on the way to achieve a favorable outcome of chemotherapy. Among various strategies that have been explored to overcome drug resistance, the combination of current chemotherapy with plant polyphenols as a chemosensitizer has emerged as a promising one. Plant polyphenols are a group of phytochemicals characterized by the presence of more than one phenolic group. Mechanistic studies suggest that polyphenols have multiple intracellular targets, one of which is the proteasome complex. The proteasome is a proteolytic enzyme complex responsible for intracellular protein degradation and has been shown to play an important role in tumor growth and the development of drug resistance. Therefore, proteasome inhibition by plant polyphenols could be one of the mechanisms contributing to their chemosensitizing effect. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include (?)-epigallocatechins-3-gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin and tannic acid. These polyphenols have exhibited an appreciable effect on overcoming resistance to various chemotherapeutic drugs as well as multidrug resistance in a broad spectrum of tumors ranging from carcinoma and sarcoma to hematological malignances. The in vitro and in vivo studies on polyphenols with proteasome-inhibitory activity have built a solid foundation to support the idea that they could serve as a chemosensitizer for the treatment of cancer. In-depth mechanistic studies and identification of optimal regimen are needed in order to eventually translate this laboratory concept into clinical trials to actually benefit current chemotherapy.

Shen, Min; Chan, Tak Hang; Dou, Q. Ping

2012-01-01

28

Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice  

PubMed Central

Background The ubiquitin-proteasome pathway (UPP) is a major protein degradation pathway that is activated during sepsis and has been proposed as a therapeutic target for preventing skeletal muscle loss due to cachexia. Although several studies have investigated the modulation of proteasome activity in response to LPS administration, none have characterized the overall UPP response to LPS administration in the fate of proteasome inhibition. Methods Here, we determined the modulation pattern of the main key components of the UPP in the gastrocnemius (GAS) of mice during the acute phase of lipopolysaccharide (LPS)-mediated endotoxemia (7.5 mg/kg – 8 h) by measuring all three ?1, ?2 and ?5 activites of the 20S and 26S proteasomes, the levels of steady state polyubiquitinated proteins, mRNA levels of muscle ligases, as well as signaling pathways regulating the UPP. Another goal was to assess the effects of administration of a specific proteasome inhibitor (epoxomicin, 0.5 mg/kg) on UPP response to sepsis. Results The acute phase of LPS-induced endotoxemia lowered GAS/body weight ratio and increased MuRF1 and MAFbx mRNA concomitantly to an activation of the pathways known to regulate their expression. Unexpectedly, we observed a decrease in all 20S and 26S proteasome activities measured in GAS, which might be related to oxidative stress, as oxidized proteins (carbonyl levels) increase with LPS. While significantly inhibiting 20S and 26S proteasome ?5 activities in heart and liver, epoxomicin did not lower proteasome activity in GAS. However, the increase in mRNA expression of the muscle ligases MuRF1 and MAFbx were partially rescued without affecting the other investigated signaling pathways. LPS also strongly activated autophagy, which could explain the observed GAS atrophy with LPS-induced reduction of proteasome activity. Conclusions Our results highlight an opposite regulation of UPP in the early hours of LPS-induced muscle atrophy by showing reduced proteasome activities and increased mRNA expression of muscle specific ligases. Furthermore, our data do not support any preventive effect of epoxomicin in muscle atrophy due to acute cachexia since proteasome activities are not further repressed.

2014-01-01

29

Impairment of the ubiquitin-proteasome pathway in RPE alters the expression of inflammation related genes.  

PubMed

The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related factors. The UPP could be impaired by oxidative stress or chemical inhibition. Impairment of the UPP in RPE increased the expression of several inflammatory cytokines, such as IL-6 and IL-8. However, the expression of monocyte chemoattractant protein-1 (MCP-1) and complement factor H (CFH) and was reduced upon impairment of the UPP. These data suggest that impairment of the UPP in RPE may be one of the causes of retinal inflammation and abnormal functions of monocyte and the complement system during the pathogenesis of age-related macular degeneration. PMID:24664704

Liu, Zhenzhen; Qin, Tingyu; Zhou, Jilin; Taylor, Allen; Sparrow, Janet R; Shang, Fu

2014-01-01

30

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

SciTech Connect

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

Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O. [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701 (Korea, Republic of)] [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701 (Korea, Republic of); Kang, Seok-Seong, E-mail: sskang@ivi.int [Laboratory Sciences Division, International Vaccine Institute, Seoul 151-919 (Korea, Republic of)] [Laboratory Sciences Division, International Vaccine Institute, Seoul 151-919 (Korea, Republic of)

2011-01-14

31

Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.  

PubMed

Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP. PMID:19347873

Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

2009-07-01

32

Dysregulation of ubiquitin-proteasome pathway and apolipoprotein A metabolism in sickle cell disease-related pulmonary arterial hypertension  

PubMed Central

Abstract Pulmonary arterial hypertension (PAH) is a major complication of sickle cell disease (SCD). Low levels of apolipoprotein A1 (Apo-A1) have been implicated in the development of PAH in SCD. We speculate that lower levels of Apo-A1 are related to dysregulation of the ubiquitin-proteasome pathway (UPP). Of 36 recruited patients with SCD, 14 were found to have PAH on the basis of right heart catheterization. Levels of Apo-A1 and Apo-B, polyubiquitin, total protease, and specific and normalized activity of chymotrypsin-like, trypsin-like, and caspase-like proteases in plasma were measured. Levels of Apo-A1 were found to be lower and polyubiquitin levels were found to be significantly higher in the PAH group () in SCD. Apo-A levels were inversely correlated with polyubiquitin levels (, ). These results indicate that lower levels of Apo-A1 in SCD patients with PAH are likely related to enhance degradation by UPP, potentially contributing to pulmonary vascular pathology. These findings may provide significant insight in identifying suitable therapeutic targets in these patients.

Hussain, M. Mahmood

2013-01-01

33

Dysregulation of ubiquitin-proteasome pathway and apolipoprotein A metabolism in sickle cell disease-related pulmonary arterial hypertension.  

PubMed

Pulmonary arterial hypertension (PAH) is a major complication of sickle cell disease (SCD). Low levels of apolipoprotein A1 (Apo-A1) have been implicated in the development of PAH in SCD. We speculate that lower levels of Apo-A1 are related to dysregulation of the ubiquitin-proteasome pathway (UPP). Of 36 recruited patients with SCD, 14 were found to have PAH on the basis of right heart catheterization. Levels of Apo-A1 and Apo-B, polyubiquitin, total protease, and specific and normalized activity of chymotrypsin-like, trypsin-like, and caspase-like proteases in plasma were measured. Levels of Apo-A1 were found to be lower and polyubiquitin levels were found to be significantly higher in the PAH group ([Formula: see text]) in SCD. Apo-A levels were inversely correlated with polyubiquitin levels ([Formula: see text], [Formula: see text]). These results indicate that lower levels of Apo-A1 in SCD patients with PAH are likely related to enhance degradation by UPP, potentially contributing to pulmonary vascular pathology. These findings may provide significant insight in identifying suitable therapeutic targets in these patients. PMID:25006400

Anjum, Fatima; Lazar, Jason; Soh, James; Albitar, Maher; Gowda, Satish; Hussain, M Mahmood; Wadgaonkar, Raj

2013-12-01

34

Activation of the Ubiquitin Proteasome Pathway in a Mouse Model of Inflammatory Myopathy  

PubMed Central

Objective Myositis is characterized by severe muscle weakness. We and others have previously shown that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of myositis. The present study was undertaken to identify perturbed pathways and assess their contribution to muscle disease in a mouse myositis model. Methods Stable isotope labeling with amino acids in cell culture (SILAC) was used to identify alterations in the skeletal muscle proteome of myositic mice in vivo. Differentially altered protein levels identified in the initial comparisons were validated using a liquid chromatography tandem mass spectrometry spike-in strategy and further confirmed by immunoblotting. In addition, we evaluated the effect of a proteasome inhibitor, bortezomib, on the disease phenotype, using well-standardized functional, histologic, and biochemical assessments. Results With the SILAC technique we identified significant alterations in levels of proteins belonging to the ER stress response, ubiquitin proteasome pathway (UPP), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. We validated the myositis-related changes in the UPP and demonstrated a significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) in myositis, but not in muscle affected by other dystrophies or normal muscle. Inhibition of the UPP with bortezomib significantly improved muscle function and also significantly reduced tumor necrosis factor ? expression in the skeletal muscle of mice with myositis. Conclusion Our findings indicate that ER stress activates downstream UPPs and contributes to muscle degeneration and that UCHL-1 is a potential biomarker for disease progression. UPP inhibition offers a potential therapeutic strategy for myositis.

Rayavarapu, Sree; Coley, William; der Meulen, Jack H. Van; Cakir, Erdinc; Tappeta, Kathyayini; Kinder, Travis B.; Dillingham, Blythe C.; Brown, Kristy J.; Hathout, Yetrib; Nagaraju, Kanneboyina

2014-01-01

35

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia.  

PubMed

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 microM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 microM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 microM) and MG132 (10 microM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome alpha-subunits, the 19S subunits MSS1 and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitortrade mark) (30 mg kg(-1)) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. PMID:16052213

Sanders, P M; Russell, S T; Tisdale, M J

2005-08-22

36

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.  

PubMed Central

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Tiao, G; Hobler, S; Wang, J J; Meyer, T A; Luchette, F A; Fischer, J E; Hasselgren, P O

1997-01-01

37

Modulation of p53 degradation via MDM2-mediated ubiquitylation and the ubiquitin–proteasome system during reperfusion after stroke: role of oxidative stress  

Microsoft Academic Search

The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin–proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown.

Atsushi Saito; Takeshi Hayashi; Shuzo Okuno; Tatsuro Nishi; Pak H Chan

2005-01-01

38

Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex  

PubMed Central

The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS), has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC) in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquibated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin ?-lactone) or a translation inhibitor (anisomycin) in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning and highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

Reis, David S.; Jarome, Timothy J.; Helmstetter, Fred J.

2013-01-01

39

Regulation of p63 Protein Stability via Ubiquitin-Proteasome Pathway  

PubMed Central

The p53-related p63 gene encodes multiple protein isoforms, which are involved in a variety of biological activities. p63 protein stability is mainly regulated by the ubiquitin-dependent proteasomal degradation pathway. Several ubiquitin E3 ligases have been identified and some protein kinases as well as other kinds of proteins are involved in regulation of p63 protein stability. These regulators are responsive to diverse extracellular signaling, resulting in changes of the p63 protein levels and impacting different biological processes.

Li, Chenghua; Xiao, Zhi-Xiong

2014-01-01

40

The Ubiquitin-Proteasome Pathway: Targets for Disease Treatment and New Tools for Discovery  

NSDL National Science Digital Library

Viewers of this webinar will learn more about the role of ubiquitin in cellular processes and about efforts to find new drugs that target the ubiquitin pathway. In addition, they will hear about the successful application of protein microarray technology for discovery of new substrates for ubiquitin ligases.

Alfred Goldberg (Harvard Medical School;Department of Cell Biology); Gregory Korbel (Invitrogen;); Paul Andrews (Amgen;); Sean Sanders (AAAS;)

2008-05-01

41

The vital link between the ubiquitin–proteasome pathway and DNA repair: Impact on cancer therapy  

Microsoft Academic Search

Proteasome-dependent protein degradation is involved in a variety of biological processes, including cell-cycle regulation, apoptosis, and stress-responses. Growing evidence from translational research and clinical trials proved the effectiveness of proteasome inhibitors (PIs) in treating several types of hematological malignancies. Although various key molecules in ubiquitin-dependent cellular processes have been proposed as relevant targets of therapeutic proteasome inhibition, our current understanding

Akira Motegi; Yasuhiro Murakawa; Shunichi Takeda

2009-01-01

42

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

PubMed Central

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

Ciechanover, Aaron

2012-01-01

43

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue.  

PubMed

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism. PMID:21149597

Gaus, Kristin; Hentschke, Moritz; Czymmeck, Nicole; Novikova, Lena; Trülzsch, Konrad; Valentin-Weigand, Peter; Aepfelbacher, Martin; Ruckdeschel, Klaus

2011-03-01

44

The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.  

PubMed

Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial intermembrane space proteins is coupled with their oxidative folding and governed by the mitochondrial intermembrane space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of intermembrane space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of intermembrane space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the intermembrane space of mitochondria. PMID:23508107

Bragoszewski, Piotr; Gornicka, Agnieszka; Sztolsztener, Malgorzata E; Chacinska, Agnieszka

2013-06-01

45

The Ubiquitin-Proteasome System Regulates Mitochondrial Intermembrane Space Proteins  

PubMed Central

Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial intermembrane space proteins is coupled with their oxidative folding and governed by the mitochondrial intermembrane space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of intermembrane space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of intermembrane space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the intermembrane space of mitochondria.

Bragoszewski, Piotr; Gornicka, Agnieszka; Sztolsztener, Malgorzata E.

2013-01-01

46

Functional Annotation of Small Noncoding RNAs Target Genes Provides Evidence for a Deregulated Ubiquitin-Proteasome Pathway in Spinocerebellar Ataxia Type 1  

PubMed Central

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by the expansion of CAG repeats in the ataxin 1 (ATXN1) gene. In affected cerebellar neurons of patients, mutant ATXN1 accumulates in ubiquitin-positive nuclear inclusions, indicating that protein misfolding is involved in SCA1 pathogenesis. In this study, we functionally annotated the target genes of the small noncoding RNAs (ncRNAs) that were selectively activated in the affected brain compartments. The primary targets of these RNAs, which exhibited a significant enrichment in the cerebellum and cortex of SCA1 patients, were members of the ubiquitin-proteasome system. Thus, we identified and functionally annotated a plausible regulatory pathway that may serve as a potential target to modulate the outcome of neurodegenerative diseases.

Persengiev, Stephan; Kondova, Ivanela; Bontrop, Ronald E.

2012-01-01

47

Seek and destroy: The ubiquitin-proteasome system in cardiac disease  

Microsoft Academic Search

The ubiquitin-proteasome system (UPS) is a major proteolytic system that regulates the degradation of intracellular proteins\\u000a in the heart. The UPS regulates the turnover of misfolded and damaged proteins, in addition to numerous cellular processes,\\u000a by affecting the stability of short-lived proteins such as transcription factors and cell signaling pathways. The UPS is tightly\\u000a regulated by the specificity of ubiquitin

Jessica E. Rodríoguez; Jonathan C. Schisler; Cam Patterson; Monte S. Willis

2009-01-01

48

Ubiquitin-proteasomal degradation of COX-2 in TGF-? stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.  

PubMed

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-? in the regulation of COX-2 in human uterine stromal cells. Each TGF-? isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-?. In addition, each TGF-? isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-?-induced COX-2 degradation. Taken together, these studies suggest that TGF-? promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways. PMID:22109885

Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

2012-01-01

49

Fbp1-mediated ubiquitin-proteasome pathway controls Cryptococcus neoformans virulence by regulating fungal intracellular growth in macrophages.  

PubMed

Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1? mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1? mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1? mutant is due to its inability to leave the lung. The fbp1? mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis. PMID:24478071

Liu, Tong-Bao; Xue, Chaoyang

2014-02-01

50

The Cytoplasmic Hsp70 Chaperone Machinery Subjects Misfolded and Endoplasmic Reticulum Import-incompetent Proteins to Degradation via the Ubiquitin-Proteasome System  

PubMed Central

The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import–defective mutated derivatives of carboxypeptidase yscY (?ssCPY* and ?ssCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (?ssCPY). All these protein species are rapidly degraded via the ubiquitin–proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssa1p function. Fusion of ?ssCPY* to GFP-cODC to form ?ssCPY*-GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation.

Park, Sae-Hun; Bolender, Natalia; Eisele, Frederik; Kostova, Zlatka; Takeuchi, Junko; Coffino, Philip

2007-01-01

51

The ubiquitin-proteasome system in glioma cell cycle control  

PubMed Central

A major determinant of cell fate is regulation of cell cycle. Tight regulation of this process is lost during the course of development and progression of various tumors. The ubiquitin-proteasome system (UPS) constitutes a universal protein degradation pathway, essential for the consistent recycling of a plethora of proteins with distinct structural and functional roles within the cell, including cell cycle regulation. High grade tumors, such as glioblastomas have an inherent potential of escaping cell cycle control mechanisms and are often refractory to conventional treatment. Here, we review the association of UPS with several UPS-targeted proteins and pathways involved in regulation of the cell cycle in malignant gliomas, and discuss the potential role of UPS inhibitors in reinstitution of cell cycle control.

2012-01-01

52

The degradation of kinesin-like calmodulin binding protein of D. salina (DsKCBP) is mediated by the ubiquitin-proteasome system.  

PubMed

Kinesin-like calmodulin binding protein (KCBP) is a member of kinesin-14 subfamily with unconventional domains distinct from other kinesins. This unique kinesin has the myosin tail homology 4 domain (MyTH4) and band4.1, ezrin, radixin and moesin domain (FERM) at the N-terminal which interact with several cytoskeleton proteins. Although KCBP is implicated in several microtubule-related cellular processes, studies on the KCBP of Dunaliella salina (DsKCBP) have not been reported. In this study, the roles of DsKCBP in flagella and cytoskeleton were investigated and the results showed that DsKCBP was present in flagella and upregulated during flagellar assembly indicting that it may be a flagellar kinesin and plays a role in flagellar assembly. A MyTH4-FERM domain of the DsKCBP was identified as a microtubule and actin interacting site. The interaction of DsKCBP with both microtubules and actin microfilaments suggests that this kinesin may be employed to coordinate these two cytoskeleton elements in algal cells. To gain more insights into the cellular function of the kinesin, DsKCBP-interacting proteins were examined using yeast two-hybrid screen. A 26S proteasome subunit Rpn8 was identified as a novel interacting partner of DsKCBP and the MyTH4-FERM domain was necessary for the interaction of DsKCBP with Rpn8. Furthermore, the DsKCBP was polyubiquitinated and up-regulated by proteasome inhibitor and degraded by ubiquitin-proteasome system indicating that proteasome is related to kinesin degradation. PMID:23271117

Shi, Ke; Li, Jie; Han, Kang; Jiang, Haili; Xue, Lexun

2013-04-01

53

Phosphorelay-Regulated Degradation of the Yeast Ssk1p Response Regulator by the Ubiquitin-Proteasome System  

Microsoft Academic Search

In Saccharomyces cerevisiae, a phosphorelay signal transduction pathway composed of Sln1p, Ypd1p, and Ssk1p, which are homologous to bacterial two-component signal transducers, is involved in the osmosensing mechanism. In response to high osmolarity, the phosphorelay system is inactivated and Ssk1p remains unphosphorylated. Unphosphorylated Ssk1p binds to and activates the Ssk2p mitogen-activated protein (MAP) kinase kinase kinase, which in turn activates

Naoto Sato; Hiroyuki Kawahara; Akio Toh-e; Tatsuya Maeda

2003-01-01

54

From ubiquitin-proteasomal degradation to CDK1 inactivation: requirements for the first polar body extrusion in mouse oocytes.  

PubMed

Completion of the first meiotic division, manifested by extrusion of the first polar body (PBI), depends on proteasomal degradation of cyclin B1 and securin and the subsequent respective CDK1 inactivation and chromosome segregation. We aimed at identifying the polyubiquitin signal that mediates proteasomal action and at a better characterization of the role of CDK1 inactivation at this stage of meiosis. Microinjections of mutated ubiquitin proteins into mouse oocytes revealed that interference with lysine-11 polyubiquitin chains abrogated chromosome segregation and reduced PBI extrusion by 63% as compared to WT ubiquitin-injected controls. Inactivation of CDK1 in oocytes arrested at first metaphase by a proteasome inhibitor fully rescued PBI extrusion. However, removal of CDK1 inhibition failed to allow progression to the second metaphase, rather, inducing PBI reengulfment in 62% of the oocytes. Inhibition of either PLK1 or MEK1/2 during the first anaphase changed spindle dimensions. The PLK1 inhibitor also blocked PBI emission and prevented RhoA translocation. Our results identified lysine-11 rather than the canonic lysine-48 ubiquitin chains as the degradation signal in oocytes resuming meiosis, further disclosing that CDK1 inactivation is necessary and sufficient for PBI emission. This information significantly contributes to our understanding of faulty chromosome segregation that may lead to aneuploidy. PMID:22859367

Pomerantz, Yael; Elbaz, Judith; Ben-Eliezer, Inbal; Reizel, Yitzhak; David, Yael; Galiani, Dalia; Nevo, Nava; Navon, Ami; Dekel, Nava

2012-11-01

55

No Splicing, No Dicing: Non-proteolytic Roles of the Ubiquitin-Proteasome System in Transcription*  

PubMed Central

The ubiquitin-proteasome pathway (UPP) is responsible for most programmed turnover of proteins in eukaryotic cells, and this activity has been known for some time to be involved in transcriptional regulation. More recently, intersections of the UPP and transcription have been discovered that are not proteolytic in nature and appear to revolve around the chaperonin-like activities of the ATPases in the 19 S regulatory subunit of the proteasome. Moreover, monoubiquitylation, which does not signal degradation, has been found to be a key modification of many transcription factors and histones. These various non-proteolytic roles of the UPP in transcription are reviewed here, and plausible mechanistic models are discussed.

Kodadek, Thomas

2010-01-01

56

The ubiquitin–proteasome pathway in thymocyte apoptosis: caspase-dependent processing of the deubiquitinating enzyme USP7 (HAUSP)  

Microsoft Academic Search

Programmed cell death (apoptosis) is crucial for thymocyte development. We analyzed the role of the ubiquitin (Ub)–proteasome pathway in dexamethasone-triggered and TCR-mediated apoptosis in fetal thymic organ culture (FTOC). Proteasome activity was increased in apoptotic thymocytes, as visualized by active-site labeling of proteasomal ? subunits. The activity of deubiquitinating enzymes in murine apoptotic thymocytes was likewise examined by active-site labeling.

Yulia Vugmeyster; Anna Borodovsky; Madelon M Maurice; René Maehr; Margo H Furman; Hidde L Ploegh

2002-01-01

57

Impairment of the Ubiquitin-Proteasome Pathway by Methyl N-(6-Phenylsulfanyl-1H-benzimidazol-2-yl)carbamate Leads to a Potent Cytotoxic Effect in Tumor Cells  

PubMed Central

In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and I?B? were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1?, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.

Dogra, Nilambra; Mukhopadhyay, Tapas

2012-01-01

58

Role of the ubiquitin-proteasome pathway and some peptidases during seed germination and copper stress in bean cotyledons.  

PubMed

The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 3.4.11.1 and EC 3.4.11.5, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 3.4.21.4 and EC 3.4.21.1, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress. PMID:24486582

Karmous, Inès; Chaoui, Abdelilah; Jaouani, Khadija; Sheehan, David; El Ferjani, Ezzedine; Scoccianti, Valeria; Crinelli, Rita

2014-03-01

59

The ubiquitin-proteasome system: Focus on the heart  

Microsoft Academic Search

Proteasomes are the main non-lysosomal multicatalytic protease complexes that are involved in the degradation of most intracellular proteins. The substrate proteins are marked by ubiquitin, which serves as a tag for their regulated proteasomal destruction. One major function of the ubiquitin-proteasome system (UPS) is to prevent accumulation of non-functional, potentially toxic proteins. Moreover, it has become clear that the UPS

Oliver Zolk; Carolus Schenke; Antonio Sarikas

2006-01-01

60

Targeting the ubiquitin-proteasome system for cancer therapy  

PubMed Central

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

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

2013-01-01

61

Livin promotes Smac\\/DIABLO degradation by ubiquitin–proteasome pathway  

Microsoft Academic Search

Livin, a member of the inhibitor of apoptosis protein (IAP) family, encodes a protein containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. It has been reported that Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain and is negatively regulated by Smac\\/DIABLO. Nonetheless, the detailed mechanism

L Ma; Y Huang; Z Song; S Feng; X Tian; W Du; X Qiu; K Heese; M Wu

2006-01-01

62

Degradation of the Met Tyrosine Kinase Receptor by the Ubiquitin-Proteasome Pathway  

Microsoft Academic Search

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular re- sponses following activation by its ligand, hepatocyte growth factor\\/scatter factor (HGF\\/SF). In this commu- nicationwedemonstratethatsignificantMetdegradationisinducedbyHGF\\/SFandthatthisdegradationcan be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquiti- nated in response to ligand and that polyubiquitinated Met molecules, which are normally

MICHAEL JEFFERS; GREGORY A. TAYLOR; K. MICHAEL WEIDNER; SATOSHI OMURA; ANDGEORGE F. VANDE WOUDE; Max Delbruck

1997-01-01

63

The role of allostery in the ubiquitin-proteasome system  

PubMed Central

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

Liu, Jin; Nussinov, Ruth

2012-01-01

64

Relationship between amyloid-beta and the ubiquitin-proteasome system in Alzheimer's disease.  

PubMed

Amyloid-beta (Abeta) peptide is the original causative factor of Alzheimer's disease (AD) according to the amyloid cascade hypothesis. The ubiquitin-proteasome system (UPS), the major intracellular protein quality control system in eukaryotic cells, is related to AD pathogenesis. There is growing evidence showing that there is a tight relationship between Abeta and UPS and this relationship plays an important role in AD pathogenesis. This article reviews the relationship between Abeta and the UPS in terms of the following three aspects: the interaction of the two factors, the ubiquitinating process of Abeta, and impact of dysfunctional UPS on Abeta production. The impairment in the UPS in AD could affect the degradation of Abeta and lead to an abnormal accumulation of Abeta. At the same time, Abeta inhibits the proteasomal activity and subsequently leads to impairment of multivesicular bodies (MVB) sorting pathway, forming an interacting relationship between Abeta and UPS. Mutant ubiquitin (Ub) and ubiquitin-like (UBL) ubiquilin-1 are related to Abeta accumulation. Meanwhile E2 conjugating enzymes, E3 ligases, and de-ubiquitinating enzymes, all of which function in the ubiquitination process, play a pivotal role in the proteasomal degradation of Abeta. Ubiquitin-proteasome system has an immense impact on the amyloidogenic pathway of amyloid precursor protein (APP) processing that generates Abeta. Upregulation in proteasomal degradation of BACE1 and components of gamma-secretase leads to decreased Abeta accumulation. A deep look into the mechanism underlying the interplay between Abeta and UPS may provide alternative therapeutic targets and lead to new drugs and therapies. PMID:24512022

Hong, Liang; Huang, Han-Chang; Jiang, Zhao-Feng

2014-03-01

65

Ubiquilin 2: A component of the ubiquitin-proteasome system with an emerging role in neurodegeneration.  

PubMed

Ubiquilin 2, which is encoded by the UBQLN2 gene, plays a critical role in protein clearance pathways including the ubiquitin-proteasome system and autophagy. Ubiquilin 2 physically associates with ubiquitin ligases and proteasomes to mediate protein degradation. It also plays a role in the regulation of cell signalling and cell cycle progression, and association with cytoskeletal elements. Recent studies have revealed that ubiquilin 2 also plays a pathogenic role in neurodegenerative disease, including amyotrophic lateral sclerosis (ALS), and ALS-frontotemporal dementia (ALS-FTD). Rare UBQLN2 mutations cause a small subset of ALS and ALS-FTD cases. More widespread is the presence of ubiquilin 2 positive inclusions in the affected neurons of some familial and sporadic ALS and ALS-FTD patients. These discoveries have led to the hypothesis that perturbation in protein clearance, mediated by ubiquilin 2, is an important pathogenic mechanism in neurodegeneration. PMID:24589709

Zhang, Katharine Y; Yang, Shu; Warraich, Sadaf T; Blair, Ian P

2014-05-01

66

MURF2B, a Novel LC3-Binding Protein, Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells  

PubMed Central

The ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways.

Pizon, Veronique; Rybina, Sofia; Gerbal, Fabien; Delort, Florence; Vicart, Patrick; Baldacci, Giuseppe; Karsenti, Eric

2013-01-01

67

Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin–proteasome system and onto human diseases and drug targeting  

Microsoft Academic Search

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental

Aaron Ciechanover

2005-01-01

68

Supplementation of ketoacids contributes to the up-regulation of the Wnt7a/Akt/p70S6K pathway and the down-regulation of apoptotic and ubiquitin-proteasome systems in the muscle of 5/6 nephrectomised rats.  

PubMed

Ketoacids (KA) are known to improve muscle mass among patients with chronic kidney disease (CKD) on a low-protein diet (CKD-LPD), but the mechanism of its preventive effects on muscle atrophy still remains unclear. Since muscle atrophy in CKD may be attributable to the down-regulation of the Wnt7a/Akt/p70S6K pathway and the activation of the ubiquitin-proteasome system (UPS) and the apoptotic signalling pathway, a hypothesis can readily be drawn that KA supplementation improves muscle mass by up-regulating the Wnt7a/Akt/p70S6K pathway and counteracting the activation of the UPS and caspase-3-dependent apoptosis in the muscle of CKD-LPD rats. Rats with 5/6 nephrectomy were randomly divided into three groups, and fed with either 22 % protein (normal-protein diet; NPD), 6 % protein (LPD) or 5 % protein plus 1 % KA for 24 weeks. Sham-operated rats with NPD intake were used as the control. The results demonstrated that KA supplementation improved protein synthesis and increased related mediators such as Wnt7a, phosphorylated Akt and p70S6K in the muscle of CKD-LPD rats. It also inhibited protein degradation, withheld the increase in ubiquitin and its ligases MAFbx (muscle atrophy F-box) and MuRF1 (muscle ring finger-1) as well as attenuated proteasome activity in the muscle of CKD-LPD rats. Moreover, KA supplementation gave rise to a reduction in DNA fragment, cleaved caspase-3 and 14 kDa actin fragment via the down-regulation of the Bax:Bcl-2 ratio in the muscle of CKD-LPD rats. The beneficial effects unveiled herein further consolidate that KA may be a better therapeutic strategy for muscle atrophy in CKD-LPD. PMID:24502851

Wang, Dong-Tao; Lu, Lu; Shi, Ying; Geng, Zhen-Bo; Yin, Yi; Wang, Ming; Wei, Lian-Bo

2014-05-01

69

Ligand-switchable Substrates for a Ubiquitin-Proteasome System*  

PubMed Central

Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.

Egeler, Emily L.; Urner, Lorenz M.; Rakhit, Rishi; Liu, Corey W.; Wandless, Thomas J.

2011-01-01

70

Hypoxia-induced inhibition of epithelial Na(+) channels in the lung. Role of Nedd4-2 and the ubiquitin-proteasome pathway.  

PubMed

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the ?-Liddle mouse strain mice carrying a truncation of ?-ENaC C-terminus abolishing the interaction between ?-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated ?-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in ?-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport. PMID:24093724

Gille, Thomas; Randrianarison-Pellan, Nadia; Goolaerts, Arnaud; Dard, Nicolas; Uzunhan, Yurdagül; Ferrary, Evelyne; Hummler, Edith; Clerici, Christine; Planès, Carole

2014-03-01

71

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

PubMed Central

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

Meller, Robert

2010-01-01

72

Role of ubiquitin-proteasome-mediated proteolysis in nervous system disease  

PubMed Central

Proteolysis by the ubiquitin-proteasome pathway (UPP) is now widely recognized as a molecular mechanism controlling myriad normal functions in the nervous system. Also, this pathway is intimately linked to many diseases and disorders of the brain. Among the diseases connected to the UPP are neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases. Perturbation in the UPP is also believed to play a causative role in mental disorders such as Angelman syndrome. The pathology of neurodegenerative diseases is characterized by abnormal deposition of insoluble protein aggregates or inclusion bodies within neurons. The ubiquitinated protein aggregates are believed to result from dysfunction of the UPP or from structural changes in the protein substrates which prevent their recognition and degradation by the UPP. An early effect of abnormal UPP in diseases of the nervous system is likely to be impairment of synaptic function. Here we discuss the UPP and its physiological roles in the nervous system and how alterations in the UPP relate to development of nervous system diseases.

Hegde, Ashok N.; Upadhya, Sudarshan C.

2010-01-01

73

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

PubMed

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

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

2013-09-01

74

Breaking It Down: The Ubiquitin Proteasome System in Neuronal Morphogenesis  

PubMed Central

The ubiquitin-proteasome system (UPS) is most widely known for its role in intracellular protein degradation; however, in the decades since its discovery, ubiquitination has been associated with the regulation of a wide variety of cellular processes. The addition of ubiquitin tags, either as single moieties or as polyubiquitin chains, has been shown not only to mediate degradation by the proteasome and the lysosome, but also to modulate protein function, localization, and endocytosis. The UPS plays a particularly important role in neurons, where local synthesis and degradation work to balance synaptic protein levels at synapses distant from the cell body. In recent years, the UPS has come under increasing scrutiny in neurons, as elements of the UPS have been found to regulate such diverse neuronal functions as synaptic strength, homeostatic plasticity, axon guidance, and neurite outgrowth. Here we focus on recent advances detailing the roles of the UPS in regulating the morphogenesis of axons, dendrites, and dendritic spines, with an emphasis on E3 ubiquitin ligases and their identified regulatory targets.

Hamilton, Andrew M.; Zito, Karen

2013-01-01

75

The ubiquitin proteasome system and myocardial ischemia  

PubMed Central

The ubiquitin proteasome system (UPS) has been the subject of intensive research over the past 20 years to define its role in normal physiology and in pathophysiology. Many of these studies have focused in on the cardiovascular system and have determined that the UPS becomes dysfunctional in several pathologies such as familial and idiopathic cardiomyopathies, atherosclerosis, and myocardial ischemia. This review presents a synopsis of the literature as it relates to the role of the UPS in myocardial ischemia. Studies have shown that the UPS is dysfunctional during myocardial ischemia, and recent studies have shed some light on possible mechanisms. Other studies have defined a role for the UPS in ischemic preconditioning which is best associated with myocardial ischemia and is thus presented here. Very recent studies have started to define roles for specific proteasome subunits and components of the ubiquitination machinery in various aspects of myocardial ischemia. Lastly, despite the evidence linking myocardial ischemia and proteasome dysfunction, there are continuing suggestions that proteasome inhibitors may be useful to mitigate ischemic injury. This review presents the rationale behind this and discusses both supportive and nonsupportive studies and presents possible future directions that may help in clarifying this controversy.

Calise, Justine

2013-01-01

76

The ubiquitin-proteasome machinery is essential for nuclear translocation of incoming minute virus of mice.  

PubMed

Minute virus of mice (MVM) infection is disrupted by proteasome inhibitors. Here, we show that inhibition of the ubiquitin-proteasome pathway did not affect viral entry and had influence neither on the natural proteolytic cleavage of VP2 to VP3 nor on the externalization of the N terminal of VP1. In both MG132-treated and untreated cells, MVM particles accumulated progressively in the perinuclear region. However, in MG132-treated cells, MVM was not able to penetrate into the nuclei, remaining blocked in the perinuclear region without capsid disassembly. MVM was similarly sensitive to MG132 in the two cell lines tested, A9 and NB324K. After releasing from the reversible MG132 block, MVM recovered the ability to translocate to the nuclei and replicate. Analysis of viral capsid proteins during internalization showed no evidence of capsid ubiquitination or degradation. We examined the effect of MG132 on two other parvoviruses, canine (CPV) and bovine parvovirus (BPV). Similarly to MVM, CPV infection was sensitive to MG132; however, BPV infection, as previously shown for adeno-associated viruses (AAVs), was not disturbed. These findings suggest that parvoviruses follow divergent strategies for nuclear transport, some of them requiring active proteasomes. PMID:15207621

Ros, Carlos; Kempf, Christoph

2004-07-01

77

Role of ubiquitin-proteasome system (UPS) in left ventricular hypertrophy (LVH)  

PubMed Central

Cardiac hypertrophy is a key compensatory mechanism acting in response to pressure or volume overload, involving some alterations in signaling transduction pathways and transcription factors-regulation. These changes result in enhanced proteins’ synthesis leading to Left Ventricular Hypertrophy (LVH). It is known that the main function of Ubiquitin-Proteasome System (UPS) is to prevent accumulation of damaged, misfolded and mutant proteins by proteolysis. But emerging evidences suggest that UPS also attends to the cells’ growth, favoring proteins’ synthesis, subsequently evolving in LVH. The role of the proteasome in to favor cellular hypertrophy consists in upregulation of the catalytic proteasome subunit, with prevalence of proteins-synthesis on proteins degradation. It is also evident that UPS inhibition may prevent cells’ growth opposing to the hypertrophy. In fact in several experimental models, UPS inhibition demonstrated to be able to prevent or reverse cardiac hypertrophy induced by abdominal aortic banding (AAB). That can happen with several proteasome inhibitors acting by multifactorial mechanisms. These evidences induce to hypothesize that, in the future, in patients with the increased volume overload by systemic hypertension, some proteasome-inhibitors could be used to antagonize or prevent LVH without reducing peripheral high blood pressure levels too.

Cacciapuoti, Federico

2014-01-01

78

Role of the ubiquitin proteasome system in renal cell carcinoma.  

PubMed

Renal cell carcinoma (RCC) accounts for approximately 2.6% of all cancers in the United States. While early stage disease is curable by surgery, the median survival of metastatic disease is only 13 months. In the last decade, there has been considerable progress in understanding the genetics of RCC. The VHL tumor suppressor gene is inactivated in the majority of RCC cases. The VHL protein (pVHL) acts as an E3 ligase that targets HIF-1, the hypoxia inducible transcription factor, for degradation by the ubiquitin proteasome system (UPS). In RCC cases with mutant pVHL, HIF-1 is stabilized and aberrantly expressed in normoxia, leading to the activation of pro-survival genes such as vascular endothelial growth factor (VEGF). This review will focus on the defect in the UPS that underlies RCC and describe the development of novel therapies that target the UPS. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com). PMID:18047741

Corn, Paul G

2007-01-01

79

Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species  

PubMed Central

ABSTRACT TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.

Scotter, Emma L.; Vance, Caroline; Nishimura, Agnes L.; Lee, Youn-Bok; Chen, Han-Jou; Urwin, Hazel; Sardone, Valentina; Mitchell, Jacqueline C.; Rogelj, Boris; Rubinsztein, David C.; Shaw, Christopher E.

2014-01-01

80

Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?  

PubMed

The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. PMID:24157661

Caldeira, Margarida V; Salazar, Ivan L; Curcio, Michele; Canzoniero, Lorella M T; Duarte, Carlos B

2014-01-01

81

Patented small molecule inhibitors in the ubiquitin proteasome system  

Microsoft Academic Search

Deregulation of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the proteasome inhibitor Velcade® (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented

Philippe Guédat; Frédéric Colland

2007-01-01

82

Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.  

PubMed

Certain types of human papillomaviruses have been etiologically associated with malignant lesions, most notably with cervical cancer. The major oncoproteins of these cancer-associated viruses are encoded by the viral E6 and E7 genes. Thorough characterization of these oncoproteins and their interaction with cellular proteins has shown that both E6 and E7 exploit the ubiquitin-proteasome system to degrade and, thus, to functionally inactivate negative cell-regulatory proteins including members of the p110(RB) family and p53. This act of piracy is assumed to contribute to both the efficient propagation of HPVs and HPV-induced carcinogenesis. PMID:12507557

Scheffner, Martin; Whitaker, Noel J

2003-02-01

83

Dysregulation of the ubiquitin-proteasome system by curcumin suppresses coxsackievirus B3 replication.  

PubMed

Curcumin (diferuloylmethane), a natural polyphenolic compound extracted from the spice turmeric, has been reported to have anti-inflammatory, antioxidant, and antiproliferative properties by modulating multiple cellular machineries. It inhibits several intracellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), casein kinase II (CKII), and the COP9 signalosome (CSN), in various cell types. It has also been recently demonstrated that exposure to curcumin leads to the dysregulation of the ubiquitin-proteasome system (UPS). Coxsackievirus infection is associated with various diseases, including myocarditis and dilated cardiomyopathy. In searching for new antiviral agents against coxsackievirus, we found that treatment with curcumin significantly reduced viral RNA expression, protein synthesis, and virus titer and protected cells from virus-induced cytopathic effect and apoptosis. We further demonstrated that reduction of viral infection by curcumin was unlikely due to inhibition of CVB3 binding to its receptors or CVB3-induced activation of MAPKs. Moreover, gene silencing of CKII and Jab1, a component of CSN, by small interfering RNAs did not inhibit the replication of coxsackievirus, suggesting that the antiviral action of curcumin is independent of these pathways. Finally, we showed that curcumin treatment reduced both the 20S proteasome proteolytic activities and the cellular deubiquitinating activities, leading to increased accumulation of ubiquitinated proteins and decreased protein levels of free ubiquitin. We have recently demonstrated that the UPS-mediated protein degradation and/or modification plays a critical role in the regulation of coxsackievirus replication. Thus, our results suggest an important antiviral effect of curcumin wherein it potently inhibits coxsackievirus replication through dysregulation of the UPS. PMID:17229707

Si, Xiaoning; Wang, Yahong; Wong, Jerry; Zhang, Jingchun; McManus, Bruce M; Luo, Honglin

2007-04-01

84

The ubiquitin–proteasome system contributes to the inflammatory injury in ischemic diabetic myocardium: the role of glycemic control  

Microsoft Academic Search

BackgroundBecause the ubiquitin–proteasome pathway (UPS) is required for activation of nuclear factor kappa beta (NFkB), a transcription factor that regulates inflammatory genes, we evaluated the UPS activity, NFkB activation, and tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, in ischemic specimens of diabetic myocardium and relate them to the glycemic control (HbA1c), oxidative stress (nitrotyrosine, a modified amino acid produced by

Raffaele Marfella; Clara Di Filippo; Michele Portoghese; Mario Siniscalchi; Simone Martis; Franca Ferraraccio; Salvatore Guastafierro; Gianfranco Nicoletti; Michelangela Barbieri; Antonino Coppola; Francesco Rossi; Giuseppe Paolisso; Michele D'Amico

2009-01-01

85

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

PubMed Central

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

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

2010-01-01

86

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

PubMed Central

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

Ballard, John William Oman; Yi, Young-Joo

2014-01-01

87

Gene expression profiling of sporadic Parkinson's disease substantia nigra pars compacta reveals impairment of ubiquitin-proteasome subunits, SKP1A, aldehyde dehydrogenase, and chaperone HSC-70.  

PubMed

Sporadic Parkinson's disease (PD) constitutes 99% of the disorder, while the remaining 1% of the cases is of familial (genetic) origin. The mutations reported to be associated with familial PD indicate impairment in protein processing and misfolding, as is handled by the ubiquitin-proteasome system (UPS), and in mitochondrial function. For these reasons, we have recently applied, for the first time, Affymetrix oligonucleotide microarray technique in the substantia nigra pars compacta of sporadic parkinsonian patients for studying global gene expression analysis and comparison to the alterations identified in inherited PD. This study identified decreased expression of 68 genes and elevation of 69 genes. Classification into functional groups revealed that the downregulated genes are related to signal transduction, protein degradation (e.g., ubiquitin-proteasome subunits), dopaminergic transmission/metabolism, iron transport, protein modification/phosphorylation, and energy pathways/glycolysis functional classes. A major finding is the decreased expressions of 5 subunits of the UPS, SKP1A, a member of the SCF (E3) ubiquitin ligase complex, and chaperone HSC-70, which can lead to a wide impairment in the function of an entire repertoire of proteins. The upregulated genes are clustered in cell adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/phosphorylation, protein metabolism and transcription, and inflammation/hypoxia (e.g., key iron and oxygen sensor EGLN1) classes. The study shows, for the first time, a convergence in the pathogenic processes that are observed in hereditary (familial) and sporadic PD, where abnormal iron metabolism, oxidative stress, and aggregation of proteins occur. An additional breakthrough in this research is the identification of a number of previously unsuspected crucial gene players that are also involved in the process of neurodegeneration, which can serve as specific biomarkers for PD and novel drug development. PMID:16179542

Mandel, Silvia; Grunblatt, Edna; Riederer, Peter; Amariglio, Ninette; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Youdim, Moussa B H

2005-08-01

88

Gambogic acid is cytotoxic to cancer cells through inhibition of the ubiquitin-proteasome system.  

PubMed

Gambogic acid (GA), displays cytotoxicity towards a wide variety of tumor cells and has been shown to affect many important cell-signaling pathways. In the present work, we investigated the mechanism of action of GA by analysis of drug-induced changes in gene expression profiles and identified GA and the derivative dihydro GA as possible inhibitors of the ubiquitin-proteasome system (UPS). Both GA and dihydro GA inhibited proteasome function in cells resulting in the accumulation of polyubiquitin complexes. In vitro experiments showed that both GA and dihydro GA inhibited 20S chymotrypsin activity and the inhibitory effects of GA and dihydro GA on proteasome function corresponded with apoptosis induction and cell death. In conclusion, our results show that GA and dihydro GA exert their cytotoxic activity through inhibition of the UPS, specifically by acting as inhibitors of the chymotrypsin activity of the 20S proteasome. PMID:23179339

Felth, Jenny; Lesiak-Mieczkowska, Karolina; D'Arcy, Padraig; Haglund, Caroline; Gullbo, Joachim; Larsson, Rolf; Linder, Stig; Bohlin, Lars; Fryknäs, Mårten; Rickardson, Linda

2013-06-01

89

The Ubiquitin-Proteasome System and the Autophagic-Lysosomal System in Alzheimer Disease  

PubMed Central

As neurons age, their survival depends on eliminating a growing burden of damaged, potentially toxic proteins and organelles—a capability that declines owing to aging and disease factors. Here, we review the two proteolytic systems principally responsible for protein quality control in neurons and their important contributions to Alzheimer disease pathogenesis. In the first section, the discovery of paired helical filament ubiquitination is described as a backdrop for discussing the importance of the ubiquitin–proteasome system in Alzheimer disease. In the second section, we review the prominent involvement of the lysosomal system beginning with pathological endosomal–lysosomal activation and signaling at the very earliest stages of Alzheimer disease followed by the progressive failure of autophagy. These abnormalities, which result in part from Alzheimer-related genes acting directly on these lysosomal pathways, contribute to the development of each of the Alzheimer neuropathological hallmarks and represent a promising therapeutic target.

Ihara, Yasuo; Morishima-Kawashima, Maho; Nixon, Ralph

2012-01-01

90

Ubiquitin Proteasome System Stress Underlies Synergistic Killing of Ovarian Cancer Cells by Bortezomib and a Novel HDAC6 Inhibitor  

PubMed Central

PURPOSE Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus ovarian cancer cell death can be triggered most effectively by co-inhibition of both the proteasome- and HDAC6-dependent protein degradation pathways. EXPERIMENTAL DESIGN To address this hypothesis we examined HDAC6 expression patterns in normal and cancerous ovarian tissues, and utilized a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer. RESULTS Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of ?-tubulin. CONCLUSION Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.

Bazzaro, Martina; Lin, Zhenhua; Santillan, Antonio; Lee, Michael K; Wang, Mei-Cheng; Chan, Kwun C; Bristow, Robert E; Mazitschek, Ralph; Bradner, James; Roden, Richard BS

2009-01-01

91

Autophagic-Lysosomal Inhibition Compromises Ubiquitin-Proteasome System Performance in a p62 Dependent Manner in Cardiomyocytes  

PubMed Central

Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test in vivo and in vitro the impact of inhibition of the ALP on UPS proteolytic performance in cardiomyocytes and to explore the underlying mechanism. Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo.

Tian, Zongwen; Wang, Changhua; Hu, Chengjun; Tian, Yihao; Liu, Jinbao; Wang, Xuejun

2014-01-01

92

Autophagic-Lysosomal Inhibition Compromises Ubiquitin-Proteasome System Performance in a p62 Dependent Manner in Cardiomyocytes.  

PubMed

Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test in vivo and in vitro the impact of inhibition of the ALP on UPS proteolytic performance in cardiomyocytes and to explore the underlying mechanism. Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo. PMID:24959866

Tian, Zongwen; Wang, Changhua; Hu, Chengjun; Tian, Yihao; Liu, Jinbao; Wang, Xuejun

2014-01-01

93

Equine Infectious Anemia Virus and the Ubiquitin-Proteasome System  

PubMed Central

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9Gag proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

Ott, David E.; Coren, Lori V.; Sowder, Raymond C.; Adams, Julian; Nagashima, Kunio; Schubert, Ulrich

2002-01-01

94

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.  

PubMed

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting. PMID:12019153

Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

2002-05-15

95

Plant Development: Regulation by Protein Degradation  

NSDL National Science Digital Library

Many aspects of eukaryotic development depend on regulated protein degradation by the ubiquitin-proteasome pathway. This highly conserved pathway promotes covalent attachment of ubiquitin to protein substrates through the sequential action of three enzymes called a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). Most ubiquitinated proteins are then targeted for degradation by the 26S proteasome. Recent studies have also shown that the ubiquitin-related protein RUB/Nedd8 and the proteasome-related COP9 signalosome complex cooperate with the ubiquitin-proteasome pathway to promote protein degradation. Most of these components are conserved in all three eukaryotic kingdoms. However, the known targets of the pathway in plants, and the developmental processes they regulate, are specific to the plant kingdom.

Hanjo Hellmann (University of Texas;Molecular Cell and Developmental Biology); Mark Estelle (University of Texas;Molecular Cell and Developmental Biology)

2002-08-02

96

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

PubMed Central

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

Timsit, Yoav E.; Negishi, Masahiko

2014-01-01

97

Molecular pathways: turning proteasomal protein degradation into a unique treatment approach.  

PubMed

Cancer treatment regimens have evolved from single cytotoxic substances affecting all proliferative tissues toward antibodies and kinase inhibitors targeting tumor-specific pathways. Treatment efficacy and cancer survival have improved overall, and side effects have become less frequent. The ubiquitin-proteasome system-mediated proteasomal protein degradation is the most critical pathway to regulate the quantity of signal proteins involved in carcinogenesis and tumor progression. These processes are, as well as protein recycling, highly regulated and offer targets for biomarker and drug development. Unspecific proteasome inhibitors such as bortezomib and carfilzomib have shown clinical efficacy and are approved for clinical use. Inhibitors of more substrate-specific enzymes of degradation processes are being developed and are now in early clinical trials. The novel compounds focus on the degradation of key regulatory proteins such as p53, p27(Kip1), and ß-catenin, and inhibitors specific for growth factor receptor kinase turnover are in preclinical testing. PMID:24756373

Stintzing, Sebastian; Lenz, Heinz-Josef

2014-06-15

98

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

Microsoft Academic Search

BACKGROUND: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. METHODS: We examined the effect

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

2008-01-01

99

The Ubiquitin-Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors  

PubMed Central

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin–proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the ?3, ?2, and ?4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of ?3 and the two ? subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the ?7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.

Rezvani, Khosrow; Teng, Yanfen

2010-01-01

100

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

PubMed Central

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

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

2013-01-01

101

Appraising the Roles of CBLL1 and the Ubiquitin/Proteasome System for Flavivirus Entry and Replication?  

PubMed Central

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.

Fernandez-Garcia, Maria-Dolores; Meertens, Laurent; Bonazzi, Matteo; Cossart, Pascale; Arenzana-Seisdedos, Fernando; Amara, Ali

2011-01-01

102

Rapid Turnover of Extracellular Signal-Regulated Kinase 3 by the Ubiquitin-Proteasome Pathway Defines a Novel Paradigm of Mitogen-Activated Protein Kinase Regulation during Cellular Differentiation  

Microsoft Academic Search

Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the

Philippe Coulombe; Genevieve Rodier; Stephane Pelletier; Johanne Pellerin; Sylvain Meloche

2003-01-01

103

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines  

Microsoft Academic Search

Summary.  Protein degradation mediated by the ubiquitin\\/proteasome system is the major route for the degradation of cellular proteins.\\u000a In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated\\u000a proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome\\u000a without ubiquitination,

C. Kahana

2007-01-01

104

Antiepileptic Drugs Reduce the Efficacy of Methotrexate Chemotherapy through Accelerated Degradation of the Reduced Folate Carrier by the Ubiquitin-Proteasome Pathway  

Microsoft Academic Search

Background\\/Aims: Concurrent treatment with methotrexate (MTX) and enzyme-inducing antiepileptic drugs including phenobarbital (PB) reduces the efficacy of MTX chemotherapy in cancer patients. We have shown that Reduced folate carrier (Rfc1)-mediated uptake of MTX, an essential determinant of MTX chemotherapy, is significantly reduced by PB via protein kinase C (PKC). However, whether PB treatment affects Rfc1 activity through regulation of carrier

Sandra Halwachs; Ingo Schaefer; Peter Seibel; Walther Honscha

2011-01-01

105

Regulation of the endothelial cell cycle by the ubiquitin-proteasome system.  

PubMed

Degradation of poly-ubiquitinated proteins by the 26S-proteasome complex represents a crucial quantitative control mechanism. The ubiquitin-proteasome system (UPS) plays a pivotal role in the complex molecular network regulating the progression both between and within each cell-cycle phase. Two major complexes are involved: the SKP1-CUL1-F-box-protein complex (SCF) and the anaphase-promoting complex/cyclosome (APC/C). Notwithstanding structural similarities, SCF and APC/C display different cellular functions and mechanisms of action. SCF modulates all cell-cycle stages and plays a prominent role at G1/S transition mainly through three regulatory subunits: Skp2, Fbw7, and beta-TRCP. APC/C, regulated by Cdc20 or Cdh1 subunits, has a crucial role in mitosis. In this review, we will describe how the endothelial cell cycle is regulated by the UPS. We will illustrate the principal SCF- and APC/C-dependent molecular mechanisms that modulate cell growth, allowing a unidirectional cell-cycle progression. Then, we will focus our attention on UPS modulation by oxidative stress, a pathogenic stimulus that causes endothelial dysfunction and is involved in numerous cardiovascular diseases. PMID:19617222

Fasanaro, Pasquale; Capogrossi, Maurizio C; Martelli, Fabio

2010-01-15

106

Piperlongumine induces inhibition of the ubiquitin-proteasome system in cancer cells.  

PubMed

Piperlongumine, a natural product from the plant Piperlongum, has demonstrated selective cytotoxicity to tumor cells and to show anti-tumor activity in animal models [1]. Cytotoxicity of piperlongumine has been attributed to increase in reactive oxygen species (ROS) in cancer cells. We here report that piperlongumine is an inhibitor of the ubiquitin-proteasome system (UPS). Exposure of tumor cells to piperlongumine resulted in accumulation of a reporter substrate known to be rapidly degraded by the proteasome, and of accumulation of ubiquitin conjugated proteins. However, no inhibition of 20S proteolytic activity or 19S deubiquitinating activity was observed at concentrations inducing cytotoxicity. Consistent with previous reports, piperlongumine induced strong ROS activation which correlated closely with UPS inhibition and cytotoxicity. Proteasomal blocking could not be mimicked by agents that induce oxidative stress. Our results suggest that the anti-cancer activity of piperlongumine involves inhibition of the UPS at a pre-proteasomal step, prior to deubiquitination of malfolded protein substrates at the proteasome, and that the previously reported induction of ROS is a consequence of this inhibition. PMID:23318177

Jarvius, Malin; Fryknäs, Mårten; D'Arcy, Pädraig; Sun, Chao; Rickardson, Linda; Gullbo, Joachim; Haglund, Caroline; Nygren, Peter; Linder, Stig; Larsson, Rolf

2013-02-01

107

Involvement of the ubiquitin-proteasome system in the formation of experimental postsurgical peritoneal adhesions.  

PubMed

We investigated the Ubiquitin-Proteasome System (UPS), major nonlysosomal intracellular protein degradation system, in the genesis of experimental postsurgical peritoneal adhesions. We assayed the levels of UPS within the adhered tissue along with the development of peritoneal adhesions and used the specific UPS inhibitor bortezomib in order to assess the effect of the UPS blockade on the peritoneal adhesions. We found a number of severe postsurgical peritoneal adhesions at day 5 after surgery increasing until day 10. In the adhered tissue an increased values of ubiquitin and the 20S proteasome subunit, NFkB, IL-6, TNF-? and decreased values of IkB-beta were found. In contrast, bortezomib-treated rats showed a decreased number of peritoneal adhesions, decreased values of ubiquitin and the 20S proteasome, NFkB, IL-6, TNF-?, and increased levels of IkB-beta in the adhered peritoneal tissue. The UPS system, therefore, is primarily involved in the formation of post-surgical peritoneal adhesions in rats. PMID:22496598

Di Filippo, Clara; Petronella, Pasquale; Freda, Fulvio; Scorzelli, Marco; Ferretti, Marco; Canonico, Sivestro; Rossi, Francesco; D'Amico, Michele

2012-01-01

108

Evidence of TRK-Fused Gene (TFG1) function in the ubiquitin-proteasome system.  

PubMed

A heterozygous mutation in the TRK-Fused Gene (TFG1) has recently been identified in hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P). TFG1 protein is reportedly localized at endoplasmic reticulum (ER) exit sites and modulates ER export, but the mechanism of its action in neurodegeneration remains unclear. To clarify the molecular pathogenesis of HMSN-P, we examined the biochemical and cellular characteristics of wild-type and mutant (P285L) TFG1 in vitro. A coexpression study of human TFG1 and ER substrates, which are degraded by the ubiquitin-proteasome system (UPS), showed that TFG1 is an inhibitory regulator of the UPS. Deletion mutant constructs revealed that the proline/glutamine-rich domain in TFG1 was critical for regulation of the UPS and proper localization at ER exit sites. Furthermore, overexpression of wild-type TFG1 increased ubiquitination of ER-resident proteins and led to ER stress. Mutant (P285L) TFG1, which is in the proline/glutamine-rich domain, enhanced the inhibitory effect on the UPS and the level of ER stress. These data provide new pathological insights into HMSN-P, and we suspect that the pathogenesis is tightly associated with disruption of intracellular protein homeostasis and ER stress. PMID:24613659

Yagi, Takuya; Ito, Daisuke; Suzuki, Norihiro

2014-06-01

109

Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo?  

PubMed

The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanisms responsible for the selective destruction of proteins within cells. PMID:10363646

Salvat, C; Aquaviva, C; Jariel-Encontre, I; Ferrara, P; Pariat, M; Steff, A M; Carillo, S; Piechaczyk, M

1999-04-01

110

Regulation of the calpain and ubiquitin proteasome systems in a canine model of muscular dystrophy  

PubMed Central

Introduction Previous studies have tested the hypothesis that calpain and/or proteasome inhibition is beneficial in Duchenne Muscular Dystrophy, based largely on evidence that calpain and proteasome activities are enhanced in the mdx mouse. Methods mRNA expression of ubiquitin proteasome and calpain system components were determined using RT-PCR in skeletal muscle and heart in the golden retriever muscular dystrophy model. Similarly, calpain 1/2 and proteasome activities were determined using fluorometric activity assays. Results We found that less than half of the muscles tested had increases in proteasome activity, and only one-half had increased calpain activity. Additionally, transcriptional regulation of the ubiquitin proteasome system was most pronounced in the heart, where numerous components were significantly decreased. Discussion This study illustrates the diversity of expression and activities of the ubiquitin proteasome and calpain systems, which may lead to unexpected consequences in response to pharmacologic inhibition.

Wadosky, Kristine M.; Li, Luge; Rodriguez, Jessica E.; Min, JinNa; Bogan, Dan; Gonzalez, Jason; Patterson, Cam; Kornegay, Joe N.; Willis, Monte

2011-01-01

111

Peroxisome Proliferator-Activated Receptor Gamma and Regulations by the Ubiquitin-Proteasome System in Pancreatic Cancer  

PubMed Central

Pancreatic cancer is one of the most lethal forms of human cancer. Although progress in oncology has improved outcomes in many forms of cancer, little progress has been made in pancreatic carcinoma and the prognosis of this malignancy remains grim. Several molecular abnormalities often present in pancreatic cancer have been defined and include mutations in K-ras, p53, p16, and DPC4 genes. Nuclear receptor Peroxisome Proliferator-Activated Receptor gamma (PPAR?) has a role in many carcinomas and has been found to be overexpressed in pancreatic cancer. It plays generally a tumor suppressor role antagonizing proteins promoting carcinogenesis such as NF-?B and TGF?. Regulation of pathways involved in pancreatic carcinogenesis is effectuated by the Ubiquitin Proteasome System (UPS). This paper will examine PPAR? in pancreatic cancer, the regulation of this nuclear receptor by the UPS, and their relationship to other pathways important in pancreatic carcinogenesis.

Stravodimou, Athina; Mazzoccoli, Gianluigi; Voutsadakis, Ioannis A.

2012-01-01

112

N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome  

Microsoft Academic Search

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its

Philippe Coulombe; Genevieve Rodier; Eric Bonneil; Pierre Thibault; Sylvain Meloche

2004-01-01

113

The possible role of the ubiquitin proteasome system in the development of atherosclerosis in diabetes  

Microsoft Academic Search

We have reviewed the impact of the ubiquitin proteasome system (UPS) on atherosclerosis progression of diabetic patients. A puzzle of many pieces of evidence suggests that UPS, in addition to its role in the removal of damaged proteins, is involved in a number of biological processes including inflammation, proliferation and apoptosis, all of which constitute important characteristics of atherosclerosis. From

Raffaele Marfella; Michele D' Amico; Clara Di Filippo; Mario Siniscalchi; Ferdinando Carlo sasso; Franca Ferraraccio; Francesco Rossi; Giuseppe Paolisso

2007-01-01

114

UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in ?-cells: Evidence of interplay between the ubiquitin/proteasome system and autophagy.  

PubMed

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in ?-cells and increased ?-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). ?-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of ?-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in ?-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in ?-cell mass caused by increased ?-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic ?-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in ?-cells. PMID:24879150

Costes, Safia; Gurlo, Tatyana; Rivera, Jacqueline F; Butler, Peter C

2014-06-01

115

Cap-dependent mRNA translation and the ubiquitin-proteasome system cooperate to promote ERBB2-dependent esophageal cancer phenotype  

PubMed Central

Pathological post-transcriptional control of the proteome composition is a central feature of malignancy. Two steps in this pathway, eIF4F-driven cap-dependent mRNA translation and the ubiquitin-proteasome system (UPS), are deregulated in most if not all cancers. We tested a hypothesis that eIF4F is aberrantly activated in human esophageal adenocarcinoma (EAC) and requires elevated rates of protein turnover and proteolysis and thereby activated UPS for its pro-neoplastic function. Here, we show that 80% of tumors and cell lines featuring amplified ERBB2 display an aberrantly activated eIF4F. Direct genetic targeting of the eIF4F in ERBB2-amplified EAC cells with a constitutively active form of the eIF4F repressor 4E-BP1 decreased colony formation and proliferation and triggered apoptosis. In contrast, suppression of m-TOR-kinase activity towards 4E-BP1with rapamycin only modestly inhibited eIF4F-driven cap-dependent translation and EAC malignant phenotype; and promoted feedback activation of other cancer pathways. Our data show that co-treatment with 2 FDA-approved agents, the m-TOR inhibitor rapamycin and the proteasome inhibitor bortezomib, leads to strong synergistic growth-inhibitory effects. Moreover, direct targeting of eIF4F with constitutively active 4E-BP1 is significantly more potent in collaboration with bortezomib than rapamycin. These data support the hypothesis that a finely tuned balance between eIF4F-driven protein synthesis and proteasome-mediated protein degradation is required for the maintenance of ERBB2-mediated EAC malignant phenotype. Altogether, our study supports the development of pharmaceuticals to directly target eIF4F as most efficient strategy; and provides a clear rationale for the clinical evaluation of combination therapy with m-TOR inhibitors and bortezomib for EAC treatment.

Issaenko, OA; Bitterman, PB; Polunovsky, VA; Dahlberg, PS

2013-01-01

116

Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion\\/cellular matrix and vesicle trafficking genes  

Microsoft Academic Search

Summary. Gene expression profiling of human substantia nigra pars compacta (SNpc) from Parkinson’s disease (PD) patients, was examined employing high density microarrays. We identified alterations in the expression of 137 genes, with 68 down regulated and 69 up regulated. The down regulated genes belong to signal transduction, protein degradation (e.g. ubiquitin-proteasome subunits), dopaminergic transmission\\/metabolism, ion transport, protein modification\\/phosphorylation and energy

E. Grünblatt; S. Mandel; J. Jacob-Hirsch; S. Zeligson; N. Amariglo; G. Rechavi; J. Li; R. Ravid; W. Roggendorf; P. Riederer; M. B. H. Youdim

2004-01-01

117

ATF4 Degradation Relies on a Phosphorylation-Dependent Interaction with the SCF TrCP Ubiquitin Ligase  

Microsoft Academic Search

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein bTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkBa and b-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on

IRINA LASSOT; EMMANUEL SEGERAL; CLARISSE BERLIOZ-TORRENT; HERVE DURAND; LIONEL GROUSSIN; TSONWIN HAI; RICHARD BENAROUS; FLORENCE MARGOTTIN-GOGUET

2001-01-01

118

Kaposi's Sarcoma-Associated Herpesvirus K3 Utilizes the Ubiquitin-Proteasome System in Routing Class I Major Histocompatibility Complexes to Late Endocytic Compartments  

PubMed Central

Human herpesvirus 8 (HHV8) downregulates major histocompatibility complex (MHC) class I complexes from the plasma membrane via two of its genes, K3 and K5. The N termini of K3 and K5 contain a plant homeodomain (PHD) predicted to be structurally similar to RING domains found in E3 ubiquitin ligases. In view of the importance of the ubiquitin-proteasome system in sorting within the endocytic pathway, we analyzed its role in downregulation of MHC class I complexes in cells expressing K3. Proteasome inhibitors as well as cysteine and aspartyl protease inhibitors stabilize MHC class I complexes in cells expressing K3. However, proteasome inhibitors differentially affect sorting of MHC class I complexes within the endocytic pathway and prevent their delivery to a dense endosomal compartment. In this compartment, the cytoplasmic tail of MHC class I complexes is cleaved by cysteine proteases. The complex is then cleaved within the plane of the membrane by an aspartyl protease, resulting in a soluble MHC class I fragment composed of the lumenal domain of the heavy chain, ?2-microglobulin (?2m), and peptide. We conclude that K3 not only directs internalization, but also targets MHC class I complexes to a dense endocytic compartment on the way to lysosomes in a ubiquitin-proteasome-dependent manner.

Lorenzo, Mayra E.; Jung, Jae U.; Ploegh, Hidde L.

2002-01-01

119

Defective regulation of the ubiquitin/proteasome system in the hypothalamus of obese male mice.  

PubMed

In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity. PMID:24892821

Ignacio-Souza, Leticia M; Bombassaro, Bruna; Pascoal, Livia B; Portovedo, Mariana A; Razolli, Daniela S; Coope, Andressa; Victorio, Sheila C; de Moura, Rodrigo F; Nascimento, Lucas F; Arruda, Ana P; Anhe, Gabriel F; Milanski, Marciane; Velloso, Licio A

2014-08-01

120

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

PubMed Central

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

Thomas, Sandhya S.; Mitch, William E.

2013-01-01

121

Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes.  

PubMed

Gene expression profiling of human substantia nigra pars compacta (SNpc) from Parkinson's disease (PD) patients, was examined employing high density microarrays. We identified alterations in the expression of 137 genes, with 68 down regulated and 69 up regulated. The down regulated genes belong to signal transduction, protein degradation (e.g. ubiquitin-proteasome subunits), dopaminergic transmission/metabolism, ion transport, protein modification/phosphorylation and energy pathways/glycolysis functional classes. Up-regulated genes, clustered mainly in biological processes involving cell adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/phosphorylation, protein metabolism, transcription and inflammation/stress (e.g. key iron and oxygen sensor EGLN1). One major finding in the present study is the particular decreased expression of SKP1A, a member of the SCF (E3) ligase complex specifically in the substantia nigra (SN) of sporadic parkinsonian patients, which may lead to a wide impairment in the function of an entire repertoire of proteins subjected to regulatory ubiquitination. These findings reveal novel players in the neurodegenerative scenario and provide potential targets for the development of novel drug compounds. PMID:15455214

Grünblatt, E; Mandel, S; Jacob-Hirsch, J; Zeligson, S; Amariglo, N; Rechavi, G; Li, J; Ravid, R; Roggendorf, W; Riederer, P; Youdim, M B H

2004-12-01

122

TNF? modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts  

PubMed Central

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNF? stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNF? stimulation in RA synovial fibroblasts compared with control fibroblasts. Methods Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNF? stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability. Results RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNF?. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNF?, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNF?, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells. Conclusions TNF? stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNF? on prolonging the survival of synovial fibroblasts in RA tissue.

2012-01-01

123

Baclofen, a GABAB receptor agonist, enhances ubiquitin-proteasome system functioning and neuronal survival in Huntington's disease model mice.  

PubMed

Huntington's disease (HD) is an autosomal neurodegenerative disease. Its manifestations is selective degeneration of medium-sized spiny neurons (MSN) in the striatum. The specificity of the vulnerability of these GABAergic MSNs can be explained by abnormal protein accumulation, excitotoxicity, mitochondrial dysfunction, and failure of trophic control, among other dysfunctions. In this study, we used in vitro and in vivo models of HD to study the effects of GABAergic neuron stimulation on the cellular protein degradation machinery. We administered the GABA(B) receptor agonist, baclofen, to wild-type or mutant huntingtin-expressing striatal cells (HD19 or HD43). Chymotrypsin-like proteasome activity and cell viability were significantly increased in the mutant huntingtin-expressing striatal cells (HD43) after GABA(B) receptor agonist treatment. In addition, we systemically administered baclofen to a HD model containing the entire human huntingtin gene with 128 CAG repeats (YAC128). Chymotrypsin-like proteasome activity was significantly increased in YAC128 transgenic mice after baclofen administration. Baclofen-injected mutant YAC128 mice also showed significantly reduced numbers of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum. Baclofen markedly improved behavioral abnormalities in mutant YAC128 mice as determined by the rotarod performance test. These data indicate that stimulation of GABAergic neurons with the GABAB receptor agonist, baclofen, enhances ubiquitin-proteasome system (UPS) function and cell survival in in vitro and in vivo models of HD. PMID:24333873

Kim, Woori; Seo, Hyemyung

2014-01-10

124

The F-Box Protein Skp2 Participates in c-Myc Proteosomal Degradation and Acts as a Cofactor for c-Myc-Regulated Transcription  

Microsoft Academic Search

The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin\\/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S

Natalie von der Lehr; Sara Johansson; Siqin Wu; Fuad Bahram; Alina Castell; Cihan Cetinkaya; Per Hydbring; Ingrid Weidung; Keiko Nakayama; Keiichi I Nakayama; Ola Söderberg; Tom K Kerppola; Lars-Gunnar Larsson

2003-01-01

125

Neuroendocrine differentiation and the ubiquitin-proteasome system in cancer: Partners or enemies?  

PubMed Central

Neuroendocrine (NE) differentiation of cancer and deregulation of the ubiquitin-proteasome system (UPS) are two processes that have been independently linked to the development of aggressive and treatment-resistant tumors. Striking data suggest a plausible interconnection between these two mechanisms, based on indirect evidence of neuropeptide-induced effects on UPS, reversed by proteasome inhibition and deubiquitinase-like properties of NE markers. Deciphering the model of their exact interactions is one of the keys to targeting the NE malignant phenotype more effectively.

Vlachostergios, Panagiotis J; Papandreou, Christos N

2011-01-01

126

Hepatitis E Virus Replication Requires an Active Ubiquitin-Proteasome System  

PubMed Central

The mechanism of hepatitis E virus (HEV) replication remains largely unknown. Here we demonstrate that HEV replication requires an active ubiquitin-proteasome system and that proteasome inhibitors affect HEV replication, possibly by inhibition of viral transcription or/and translation without a significant effect on cellular translation. Overexpression of ubiquitin in inhibitor-treated cells partially reverses the inhibitor effect on HEV replication. The results suggest that HEV replication requires interactions with proteasome machinery, which could be a potential therapeutic target against HEV.

Karpe, Yogesh A.

2012-01-01

127

The Ubiquitin-Proteasome System of Saccharomyces cerevisiae  

PubMed Central

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

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

2012-01-01

128

Narrative Review: Protein Degradation and Human Diseases: The Ubiquitin Connection  

NSDL National Science Digital Library

Physiology in Medicine review article. When researchers discovered the organelle lysosome, they assumed that cellular proteins were degraded within it. However, several independent lines of evidence strongly suggested that intracellular proteolysis was largely nonlysosomal. It is now recognized that degradation of intracellular proteins by the ubiquitin proteasome system (UPS) is involved in the regulation of a broad array of cellular processes,drugs that target the degradation pathways of a single or a few proteins without affecting other proteins. It seems that pharmacologic manipulation of the UPS might alter the outcome of many diseases, especially malignant conditions and possibly neurodegenerative and chronic inflammatory diseases.

MD/PhD Eyal Reinstein (Tel-Aviv University Deparrment of Internal Medicine); MD/PhD Aaron Ciechanover (Technion-Israel Institute of Technology)

2006-11-07

129

Activation of the ubiquitin-proteasome system against arsenic trioxide cardiotoxicity involves ubiquitin ligase Parkin for mitochondrial homeostasis.  

PubMed

Parkin is an E3 ubiquitin ligase involved in the elimination of damaged mitochondria. Ubiquitination of mitochondrial substrates by Parkin results in proteasomal as well as lysosomal degradation of mitochondria, the latter of which is executed by the autophagy machinery and is called as mitophagy (mitochondrial autophagy). The aim of this study is to examine the possible role of Parkin against cardiotoxicity elicited by arsenic trioxide (ATO) exposure in HL-1 mouse atrial cardiomyocytes. HL-1 cells were administered 1-10?M ATO for up to 24h, and the involvements of apoptosis, and the ubiquitin-proteasome and autophagy-lysosome systems (UPS and ALS) were examined. ATO dose-dependently reduced mitochondrial membrane potentials (??m) in HL-1 cells, indicating that ATO works as a mitochondrial toxin in these cells. Apoptosis was evident in cells exposed to more than 6?M ATO for 24h. Levels of Parkin in mitochondria-rich fractions were increased, suggesting the recruitment of Parkin to mitochondria. Ubiquitination of the voltage-dependent anion channel1 (VDAC1), a substrate of Parkin, was also proved by immunoprecipitation. Accumulation of ubiquitinated proteins including both K48- and K63-lineages was observed in HL-1 cells after ATO exposure, implying an increased demand for proteasomal as well as lysosomal degradation of cellular proteins. Although UPS was activated by ATO as proved by increased proteasomal activity, only slight activation of the ALS marker LC3 was observed, suggesting differential reactions of UPS and ALS to ATO toxicity. The abrogation of UPS by the proteasome inhibitor bortezomib significantly sensitized HL-1 cells to ATO toxicity, showing the contribution of UPS to the maintenance of cellular homeostasis during ATO exposure. Taken together, our results reveal the activation of Parkin as well as UPS during ATO exposure in HL-1 cardiomyocytes, which contributes to the maintenance of mitochondrial as well as cellular homeostasis. PMID:24801902

Watanabe, Mayumi; Funakoshi, Takeshi; Unuma, Kana; Aki, Toshihiko; Uemura, Koichi

2014-08-01

130

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

PubMed Central

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

Lau, Alan F.

2009-01-01

131

Non-canonical ?-catenin degradation mediates reactive oxygen species-induced epidermal cell death  

PubMed Central

?-catenin is constantly degraded through the ubiquitin-proteasomal pathway. We here report that a different type of ?-catenin degradation is causally involved in epidermal cell death. We observed that reactive oxygen species (ROS) caused ?-catenin degradation in the epidermal cells through a caspase-dependent mechanism, which results in disruption of cell adhesion. Disruption of cell adhesion increased ROS and activated caspases. Upregulation of the intact ?-catenin blocked ROS accumulation and caspase activation. These results indicate that a feed-forward loop consisting of ROS, caspases activation and ?-catenin degradation induces epidermal cell death.

Omori, Emily; Matsumoto, Kunihiro; Ninomiya-Tsuji, Jun

2011-01-01

132

Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis.  

PubMed

In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression. PMID:18826962

Cheroni, Cristina; Marino, Marianna; Tortarolo, Massimo; Veglianese, Pietro; De Biasi, Silvia; Fontana, Elena; Zuccarello, Laura Vitellaro; Maynard, Christa J; Dantuma, Nico P; Bendotti, Caterina

2009-01-01

133

Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment.  

PubMed

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt. PMID:19666572

Maynard, Christa J; Böttcher, Claudia; Ortega, Zaira; Smith, Ruben; Florea, Bogdan I; Díaz-Hernández, Miguel; Brundin, Patrik; Overkleeft, Hermen S; Li, Jia-Yi; Lucas, Jose J; Dantuma, Nico P

2009-08-18

134

Ubiquitin-Proteasome System Profiling in Acute Leukemias and its Clinical Relevance  

PubMed Central

The ubiquitin-proteasome system (UPS) plays a major role in the homeostasis of cellular protein. We demonstrate that each of the major hematologic diseases (AML, ALL, and MDS) has a specific and different plasma profile of UPS protein and enzymatic activities. While high levels of proteasome and ubiquitin proteins and enzymatic activities are detected in the plasma samples from patients, normalizing enzymatic activities, show that each proteasome has lower enzymatic activities in these diseases as compared with normal controls. Proteasome protein levels in AML are strong predictor of survival independently of cytogenetics, performance status and age. The Ch-L activity when normalized to the level of proteasome protein show significant negative correlation with survival in ALL.

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuiang; Estrov, Zeev; O'Brien, Susan; Albitar, Maher

2014-01-01

135

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice.  

PubMed

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models. PMID:18362179

Wang, Jianjun; Wang, Chuan-En; Orr, Adam; Tydlacka, Suzanne; Li, Shi-Hua; Li, Xiao-Jiang

2008-03-24

136

Cystic fibrosis transmembrane conductance regulator degradation: cross-talk between the ubiquitylation and SUMOylation pathways.  

PubMed

Defining the significant checkpoints in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis should identify targets for therapeutic intervention with CFTR folding mutants such as F508del. Although the role of ubiquitylation and the ubiquitin proteasome system is well established in the degradation of this common CFTR mutant, the part played by SUMOylation is a novel aspect of CFTR biogenesis/quality control. We identified this post-translational modification of CFTR as resulting from its interaction with small heat shock proteins (Hsps), which were found to selectively facilitate the degradation of F508del through a physical interaction with the SUMO (small ubiquitin-like modifier) E2 enzyme, Ubc9. Hsp27 promoted the SUMOylation of mutant CFTR by the SUMO-2 paralogue, which can form poly-chains. Poly-SUMO chains are then recognized by the SUMO-targeted ubiquitin ligase, RNF4, which elicited F508del degradation in a Hsp27-dependent manner. This work identifies a sequential connection between the SUMO and ubiquitin modifications of the CFTR mutant: Hsp27-mediated SUMO-2 modification, followed by ubiquitylation via RNF4 and degradation of the mutant via the proteasome. Other examples of the intricate cross-talk between the SUMO and ubiquitin pathways are discussed with reference to other substrates; many of these are competitive and lead to different outcomes. It is reasonable to anticipate that further research on SUMO-ubiquitin pathway interactions will identify additional layers of complexity in the process of CFTR biogenesis and quality control. PMID:23809253

Ahner, Annette; Gong, Xiaoyan; Frizzell, Raymond A

2013-09-01

137

The ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy  

PubMed Central

Cardiomyopathies represent an important cause of cardiovascular morbidity and mortality due to heart failure, arrhythmias, and sudden death. Most forms of hypertrophic cardiomyopathy (HCM) are familial with an autosomal-dominant mode of inheritance. Over the last 20 years, the genetic basis of the disease has been largely unravelled. HCM is considered as a sarcomeropathy involving mutations in sarcomeric proteins, most often ?-myosin heavy chain and cardiac myosin-binding protein C. ‘Missense’ mutations, more common in the former, are associated with dysfunctional proteins stably integrated into the sarcomere. ‘Nonsense’ and frameshift mutations, more common in the latter, are associated with low mRNA and protein levels derived from the diseased allele, leading to haploinsufficiency of the remaining healthy allele. The two quality control systems responsible for the removal of the affected mRNAs and proteins are the nonsense-mediated mRNA decay (NMD) and the ubiquitin-proteasome system (UPS), respectively. This review discusses clinical and genetic aspects of HCM and the role of NMD and UPS in the regulation of mutant proteins, evidence for impairment of UPS as a pathogenic factor, as well as potential therapies for HCM.

Carrier, Lucie; Schlossarek, Saskia; Willis, Monte S.; Eschenhagen, Thomas

2010-01-01

138

Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy  

PubMed Central

Although intracellular beta amyloid (A?) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which A?deposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (A?PP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of A?PP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that A?PP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

Li, Honghao; Chen, Qi; Liu, Fuchen; Zhang, Xuemei; Li, Wei; Liu, Shuping; Zhao, Yuying; Gong, Yaoqin; Yan, Chuanzhu

2013-01-01

139

Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes.  

PubMed Central

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway. Images

Mitch, W E; Medina, R; Grieber, S; May, R C; England, B K; Price, S R; Bailey, J L; Goldberg, A L

1994-01-01

140

Cytokine-induced oxidative stress in cardiac inflammation and heart failure--how the ubiquitin proteasome system targets this vicious cycle  

PubMed Central

The ubiquitin proteasome system (UPS) is critical for the regulation of many intracellular processes necessary for cell function and survival. The absolute requirement of the UPS for the maintenance of protein homeostasis and thereby for the regulation of protein quality control is reflected by the fact that deviation of proteasome function from the norm was reported in cardiovascular pathologies. Inflammation is a major factor contributing to cardiac pathology. Herein, cytokines induce protein translation and the production of free radicals, thereby challenging the cellular protein equilibrium. Here, we discuss current knowledge on the mechanisms of UPS-functional adaptation in response to oxidative stress in cardiac inflammation. The increasing pool of oxidant-damaged degradation-prone proteins in cardiac pathology accounts for the need for enhanced protein turnover by the UPS. This process is accomplished by an up-regulation of the ubiquitylation machinery and the induction of immunoproteasomes. Thereby, the inflamed heart muscle is cleared from accumulating misfolded proteins. Current advances on immunoproteasome-specific inhibitors in this field question the impact of the proteasome as a therapeutic target in heart failure.

Voigt, Antje; Rahnefeld, Anna; Kloetzel, Peter M.; Kruger, Elke

2013-01-01

141

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

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

142

Direct intranigral administration of an ubiquitin proteasome system inhibitor in rat: behavior, positron emission tomography, immunohistochemistry.  

PubMed

Several independent lines of research suggest that disruption of the ubiquitin proteasome system (UPS) may play a role in the pathophysiology of Parkinson's disease. Direct intracerebral injection of UPS inhibitors (e.g. lactacystin) in animals has consistently produced important features of the disease. In this study, a range of lactacystin doses (0.5, 1, 2, 10 and 20 ?g) were injected into the right substantia nigra in rats to determine the ideal dose required to produce a robust and specific lesion of the dopamine nigro-striatal system and motor deficits. Motor behavior, assessed with the tapered ledged beam task, was severely affected in animals that received high doses (10 and 20 ?g) but only mild, impairments were observed in animals that received low doses (0.5, 1, and 2 ?g). Positron emission tomography was performed with a dedicated small animal scanner on the rats following the injection of the radio-labeled tracer (±)[(11)C]dihydrotetrabenazine (DTBZ) which labels vesicular monoamine transporter type 2. Severe loss of [(11)C]DTBZ binding in the ipsilateral striatum was observed in the higher dose groups and mild loss was observed in the low dose groups. Stereological cell counting of tyrosine hydroxylase immunoreactive cells in the substantia nigra and the ventral tegmental area indicated a dose dependent loss of dopaminergic neurons. Significant correlations were found between the behavioral motor deficits, striatal [(11)C]DTBZ binding and cell counts of tyrosine hydroxylase immunoreactive cells. Taken together these results indicate that intranigral injection of lactacystin produces dose dependent effects on the dopamine nigro-striatal system and a dose of 10 ?g will produce a consistent severe lesion. PMID:23557600

Mackey, Scott; Jing, Yu; Flores, Joseph; Dinelle, Katie; Doudet, Doris J

2013-09-01

143

Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure.  

PubMed

Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28? subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S ?? chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF. PMID:23729213

Rajagopalan, Viswanathan; Zhao, Mingming; Reddy, Sushma; Fajardo, Giovanni; Wang, Xuejun; Dewey, Shannamar; Gomes, Aldrin V; Bernstein, Daniel

2013-08-15

144

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

PubMed Central

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

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

2014-01-01

145

Involvement of the ubiquitin-proteasome system in the stabilization of cell-cell contacts in human keratinocytes.  

PubMed

Desmosomes are highly organized intercellular junctions composed of a number of interacting proteins that provide mechanical integrity to epithelial tissues. Mutations in genes encoding desmosomal proteins, including desmoplakin (DP), are associated with human hereditary diseases affecting skin integrity. The detailed mechanism of desmosome assembly remains, despite many efforts, incompletely understood. Recently, the ubiquitin-proteasome system (UPS) has been suggested to be an important regulatory system for the proper intracellular trafficking of proteins. Here, we provide evidence for a calcium-independent, but UPS-dependent, stabilization of cell-cell contacts in human keratinocytes, which might be mediated by the maintenance of DP at desmosomes. PMID:22882483

Löffek, Stefanie; Bruckner-Tuderman, Leena; Magin, Thomas M

2012-10-01

146

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells  

PubMed Central

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their ?-? unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

Anchoori, Ravi K.; Khan, Saeed R.; Sueblinvong, Thanasak; Felthauser, Alicia; Iizuka, Yoshie; Gavioli, Riccardo; Destro, Federica; Isaksson Vogel, Rachel; Peng, Shiwen; Roden, Richard B. S.; Bazzaro, Martina

2011-01-01

147

The Mammalian Endoplasmic Reticulum-Associated Degradation System  

PubMed Central

The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.

Olzmann, James A.; Kopito, Ron R.; Christianson, John C.

2013-01-01

148

Sequestosome 1\\/p62 Is a Polyubiquitin Chain Binding Protein Involved in Ubiquitin Proteasome Degradation  

Microsoft Academic Search

Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1\\/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydro- phobic patch MGF as well

M. Lamar Seibenhener; Jeganathan Ramesh Babu; Thangiah Geetha; Hing C. Wong; N. Rama Krishna; Marie W. Wooten

2004-01-01

149

Estrogen and progesterone regulate p27kip1 levels via the ubiquitin-proteasome system: pathogenic and therapeutic implications for endometrial cancer.  

PubMed

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA. PMID:23029392

Huang, Kuang-Tzu; Pavlides, Savvas C; Lecanda, Jon; Blank, Stephanie V; Mittal, Khushbakhat R; Gold, Leslie I

2012-01-01

150

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCFAtSKP2 Pathway in Response to Light  

Microsoft Academic Search

Selective ubiquitin-mediated proteolysis through the cell cycle controls the availability, and therefore the activity, of several cell proliferation proteins. E2F transcription factors play distinct roles in both proliferating and differentiated cells by regulating gene expression. Here, we report that Arabidopsis AtE2Fc is regulated by a balance between gene expression and ubiquitin-proteasome proteolysis. AtE2Fc degradation implicates the function of the E3

Juan Carlos del Pozo; Maria Beatrice Boniotti; Crisanto Gutierrez

2002-01-01

151

Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the ubiquitin proteasome pathway  

Microsoft Academic Search

Objectives:To determine if peripheral blood monocytes from patients with ankylosing spondylitis (AS) differed in protein expression compared to rheumatoid arthritis (RA) and healthy controls (HC).Methods:Monocyte protein expression was characterised by 2D gel electrophoresis and by label-free quantitative expression profiling, using nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MSE, where E refers to low\\/high collision energy switching). Data

C Wright; M Edelmann; K diGleria; S Kollnberger; H Kramer; S McGowan; K McHugh; S Taylor; B Kessler; P Bowness

2009-01-01

152

Regulation of Interferon-gamma-Activated STAT1 by the Ubiquitin Proteasome Pathway  

Microsoft Academic Search

STAT proteins (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that are phosphorylated by Janus kinases in response to cytokines. Phosphorylated STAT proteins translocate to the nucleus, where they transiently turn on specific sets of cytokine-inducible genes. The mechanism that controls the amounts of activated STAT proteins is not understood. STAT1 proteins activated by interferon-gamma treatment in

Tae Kook Kim; Tom Maniatis

1996-01-01

153

CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation.  

PubMed

Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1(Cdt2)-dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1(Cdt2) were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co-immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1(Cdt2) complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G(2) phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1(Cdt2). Our findings provide a new aspect of proteolytic control of p21 during the cell cycle. PMID:18703516

Nishitani, Hideo; Shiomi, Yasushi; Iida, Hiroka; Michishita, Masato; Takami, Toshihiro; Tsurimoto, Toshiki

2008-10-24

154

Regulation of cyclin D2 expression and degradation by follicle-stimulating hormone during rat granulosa cell proliferation in vitro.  

PubMed

Cyclin D2 (CCND2, encoded by Ccnd2) plays an important role in the induction of early-to-mid G1 phase transition and is required for granulosa cell proliferation during ovarian folliculogenesis. In the present study, we investigated the role of follicle-stimulating hormone (FSH) in the regulation of cyclin D2 expression and degradation during rat granulosa cell proliferation in vitro. FSH acutely increased granulosa cell Ccnd2 mRNA abundance and CCND2 protein content as well as proliferation. FSH-induced granulosa cell CCND2 protein content and proliferation were mimicked by forskolin and attenuated by inhibitors of protein kinase A (PKA; H89) and phosphatidylinositol 3-kinase (PI3K; LY294002) as well as PKA catalytic subunit (PRKACA) small interfering RNA (siRNA) and dominant-negative Akt (dn-Akt) but were not affected by mitogen-activated protein kinase kinase 1/2 (MEK1/2; U0126). Interestingly, FSH also enhanced CCND2 protein degradation in granulosa cells, a process involving a PKA-mediated ubiquitin-proteasome degradation pathway. Taken together, these results demonstrate that FSH acutely regulated CCND2 expression through both PKA and PI3K signaling pathways during granulosa cell proliferation and also accelerated its ubiquitination-proteasomal degradation, which may prevent overstimulation of granulosa cell proliferation and follicular growth. PMID:23349233

Han, Yingying; Xia, Guoliang; Tsang, Benjamin K

2013-03-01

155

Insulin/insulin-like growth factor (IGF) stimulation abrogates an association between a deubiquitinating enzyme USP7 and insulin receptor substrates (IRSs) followed by proteasomal degradation of IRSs.  

PubMed

Insulin receptor substrates (IRSs) play central roles in insulin/insulin-like growth factor (IGF) signaling and mediate a variety of their bioactivities. IRSs are tyrosine-phosphorylated by activated insulin receptor/IGF-I receptor tyrosine kinase in response to insulin/IGF, and are recognized by signaling molecules possessing the SH2 domain such as phosphatidylinositol 3-kinase (PI3K), leading to the activation of downstream pathways. Recent studies have suggested that degradation of IRSs by the ubiquitin-proteasome pathway leads to impaired insulin/IGF signaling, but the precise mechanism underlying the process is still unclear. In this study, we identified deubiquitinating enzyme ubiquitin specific protease 7 (USP7) as an IRS-2-interacting protein and demonstrated that deubiquitinase activity of USP7 plays important roles in IRS-2 stabilization through the ubiquitin-proteasome pathway. In addition, insulin treatment dissociated USP7 from IRS-2, leading to degradation of IRS-2. This dissociation was prevented by treatment with LY294002, a PI3K inhibitor, indicating that insulin activation of the PI3K pathway leads to dissociation of IRS-2 from USP7 and IRS-2 degradation. We obtained similar results for IRS-1 in cells treated with insulin and for IRS-2 in cells treated with IGF-I. Taken together, this is the first report demonstrating that USP7 is an IRS-1/2 deubiquitinating enzyme forming a negative feedback loop in insulin/IGF signaling. PMID:22634009

Yoshihara, Hidehito; Fukushima, Toshiaki; Hakuno, Fumihiko; Saeki, Yasushi; Tanaka, Keiji; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-ichiro; Natsume, Tohru; Asano, Tomoichiro; Chida, Kazuhiro; Girnita, Leonard; Takahashi, Shin-Ichiro

2012-06-22

156

Ubiquitin-proteasome-dependent slingshot 1 downregulation in neuronal cells inactivates cofilin to facilitate HSV-1 replication.  

PubMed

Actin and its regulators are critical for neuronal function. Infection with herpes simplex virus 1 (HSV-1) remodels neuronal cell actin dynamics, which may relate virus-induced pathological processes in the nervous system. We previously demonstrated that cofilin is an actin regulator that participates in HSV-1-induced actin dynamics in neuronal cells, but how HSV-1 regulates cofilin has remained unclear. In the present study, we demonstrated the HSV-1-induced the inactivation of cofilin and the accumulation of phosphorylated cofilin in the nucleus, which together benefited viral replication. This consistent cofilin inactivation was achieved by the downregulation of slingshot 1 (SSH1). Notably, virus-induced SSH1 downregulation depended on the ubiquitin-proteasome system. Cofilin inactivation is therefore critical for HSV-1 replication during neuronal infection and is maintained by SSH1 downregulation. Moreover, these results provide new insight into the HSV-1-induced neurological pathogenesis and suggest potential new strategies to inhibit HSV-1 replication. PMID:24418541

Xiang, Yangfei; Zheng, Kai; Zhong, Meigong; Chen, Jia; Wang, Xiao; Wang, Qiaoli; Wang, Shaoxiang; Ren, Zhe; Fan, Jianglin; Wang, Yifei

2014-01-20

157

The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro  

PubMed Central

Transforming growth factor-? (TGF-?) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-? signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-?/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-? were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-? were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-? activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.

Huang, Wei; Yang, Chen; Nan, Qinling; Gao, Chenlin; Feng, Hong; Gou, Fang; Chen, Guo; Zhang, Zhihong; Yan, Pijun; Peng, Juan

2014-01-01

158

The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.  

PubMed

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens. PMID:23940030

Teixeira, Felipe R; Manfiolli, Adriana O; Soares, Cláudia S; Baqui, Munira M A; Koide, Tie; Gomes, Marcelo D

2013-09-27

159

The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis.  

PubMed

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. Here we link the CSN to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, we show that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes similar defects as mutations in CSN or SCF(Ago) subunits: extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation. PMID:12737805

Doronkin, Sergey; Djagaeva, Inna; Beckendorf, Steven K

2003-05-01

160

Influence of electrical stimulation on calpain and ubiquitin-proteasome systems in the denervated and unloaded rat tibialis anterior muscles.  

PubMed

The influence of electrical stimulation on calpain and ubiquitin-proteasome systems was examined in the denervated and unloaded tibialis anterior muscles of male Wistar rats. Animals were divided into 5 groups: control, denervation, denervation plus electrical stimulation, unloading, and hindlimb unloading plus electrical stimulation groups. Due to denervation and unloading for 14 days, muscle atrophy markedly occurred in the denervated and unloading animals, and the atrophy in the former was significantly more severe than that in the latter. In the denervated muscle, the atrophy was significantly attenuated by the electrical stimulation, but not in the unloaded muscle. Overexpression of calpain-2 and ubiquitinated proteins was observed only in denervated muscles. In the unloaded animals, though the expression level of calpain-2 appeared to be slightly higher than that in the control, the expression level of ubiquitinated proteins was almost the same as that in the control. The overexpression of calpain-1, calpain-2, and ubiquitinated proteins in the denervated muscle was inhibited by the electrical stimulation. However, there was no difference in these expressions between the unloaded and unloaded plus electrical stimulation groups. The mechanism of the preventive effect of the electrical stimulation on muscle atrophy might differ between the denervated and unloaded muscles. PMID:24745757

Matsumoto, Ayaka; Fujita, Naoto; Arakawa, Takamitsu; Fujino, Hidemi; Miki, Akinori

2014-06-01

161

Exercise Training Prevents Oxidative Stress and Ubiquitin-Proteasome System Overactivity and Reverse Skeletal Muscle Atrophy in Heart Failure  

PubMed Central

Background Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF. Methods/Principal Findings Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels. Conclusions Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.

Cunha, Telma F.; Bacurau, Aline V. N.; Moreira, Jose B. N.; Paixao, Nathalie A.; Campos, Juliane C.; Ferreira, Julio C. B.; Leal, Marcelo L.; Negrao, Carlos E.; Moriscot, Anselmo S.; Wisl?ff, Ulrik; Brum, Patricia C.

2012-01-01

162

Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin-proteasome system and ?-synuclein  

PubMed Central

In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and ?-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking ?-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an ?-synuclein-dependent manner.

Fornai, Francesco; Schluter, Oliver M.; Lenzi, Paola; Gesi, Marco; Ruffoli, Riccardo; Ferrucci, Michela; Lazzeri, Gloria; Busceti, Carla L.; Pontarelli, Fabrizio; Battaglia, Giuseppe; Pellegrini, Antonio; Nicoletti, Ferdinando; Ruggieri, Stefano; Paparelli, Antonio; Sudhof, Thomas C.

2005-01-01

163

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

PubMed

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

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

2010-12-29

164

Casein Kinase 1?-dependent Wee1 Protein Degradation.  

PubMed

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1?-dependent Wee1 degradation. Small-molecule CK1? inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1? also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1? in controlling the cell cycle. PMID:24817118

Penas, Clara; Ramachandran, Vimal; Simanski, Scott; Lee, Choogon; Madoux, Franck; Rahaim, Ronald J; Chauhan, Ruchi; Barnaby, Omar; Schurer, Stephan; Hodder, Peter; Steen, Judith; Roush, William R; Ayad, Nagi G

2014-07-01

165

Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair.  

PubMed

Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of beta-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Delta and sec61-2) did not inhibit the degradation of the beta-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p. PMID:10982838

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

2000-10-01

166

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

PubMed Central

Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of ?-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3? and sec61-2) did not inhibit the degradation of the ?-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p.

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

2000-01-01

167

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

PubMed Central

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

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

2014-01-01

168

The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process  

PubMed Central

In response to hormonal stimuli, a cascade of hierarchical post-translational modifications of nuclear receptors are required for the correct expression of target genes. Here, we show that the transcription factor TFIIH, via its cdk7 kinase, phosphorylates the androgen receptor (AR) at position AR/S515. Strikingly, this phosphorylation is a key step for an accurate transactivation that includes the cyclic recruitment of the transcription machinery, the MDM2 E3 ligase, the subsequent ubiquitination of AR at the promoter of target genes and its degradation by the proteasome machinery. Impaired phosphorylation disrupts the transactivation, as observed in cells either overexpressing the non-phosphorylated AR/S515A, isolated from xeroderma pigmentosum patient (bearing a mutation in XPD subunit of TFIIH), or in which cdk7 kinase was silenced. Indeed, besides affecting the cyclic recruitment of the transcription machinery, the AR phosphorylation defect favourizes to the recruitment of the E3 ligase CHIP instead of MDM2, at the PSA promoter, that will further attract the proteasome machinery. These observations illustrate how the TFIIH phosphorylation might participate to the transactivation by regulating the nuclear receptors turnover.

Chymkowitch, Pierre; Le May, Nicolas; Charneau, Pierre; Compe, Emmanuel; Egly, Jean-Marc

2011-01-01

169

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

SciTech Connect

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.

Mi, Lixin, E-mail: lm293@georgetown.edu [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)] [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Gan, Nanqin; Chung, Fung-Lung [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)] [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)

2009-10-16

170

Fates-shifted is an F-box protein that targets Bicoid for degradation and regulates developmental fate determination in Drosophila embryos.  

PubMed

Bicoid (Bcd) is a morphogenetic protein that instructs patterning along the anterior-posterior (A-P) axis in Drosophila melanogaster embryos. Despite extensive studies, what controls the formation of a normal concentration gradient of Bcd remains an unresolved and controversial question. Here, we show that Bcd protein degradation is mediated by the ubiquitin-proteasome pathway. We have identified an F-box protein, encoded by fates-shifted (fsd), that has an important role in Bcd protein degradation by targeting it for ubiquitylation. Embryos from females lacking fsd have an altered Bcd gradient profile, resulting in a shift of the fatemap along the A-P axis. Our study is an experimental demonstration that, contrary to an alternative hypothesis, Bcd protein degradation is required for normal gradient formation and developmental fate determination. PMID:21170036

Liu, Junbo; Ma, Jun

2011-01-01

171

Lys(203) and Lys(382) are essential for the proteasomal degradation of BACE1.  

PubMed

Amyloid ? protein (A?) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. A? is generated through proteolysis of ?-amyloid precursor protein (APP) by two proteases: ?-secretase and ?-secretase. BACE1, the ?-secretase in vivo and the key rate-limiting enzyme that initiates the formation of A?, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at ? site and A? production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential. PMID:22299711

Wang, Rong; Ying, Zhengxin; Zhao, Juan; Zhang, Yanyan; Wang, Ran; Lu, Hong; Deng, Yulin; Song, Weihong; Qing, Hong

2012-06-01

172

Smurf2-mediated ubiquitination and degradation of Id1 regulates p16 expression during senescence  

PubMed Central

Summary The Id (inhibitor of differentiation or DNA binding) family of transcription regulators plays an important role in cell proliferation, differentiation and senescence. However, regulation of Id expression during these processes is poorly understood. Id proteins are known to undergo rapid turnover mediated by the ubiquitin-proteasome pathway. Anaphase-promoting complex has been shown to ubiquitinate Id2, but E3 ubiquitin ligase(s) that ubiquitinate other Id family members are not known. Here we report for the first time the identification of Smurf2 as the E3 ligase that ubiquitinates Id1 and Id3. Smurf2-mediated ubiquitination and consequent degradation of Id1 or Id3 plays an important role in the regulation of Id expression in senescent cells. Furthermore, we found that Id1 is the mediator through which Smurf2 regulates p16 expression, providing a mechanistic link between Smurf2 and p16 expression during senescence.

Kong, Yahui; Cui, Hang; Zhang, Hong

2011-01-01

173

Molecular profiling of malignant peritoneal mesothelioma identifies the ubiquitin–proteasome pathway as a therapeutic target in poor prognosis tumors  

Microsoft Academic Search

Malignant mesothelioma is an aggressive neoplastic proliferation derived from cells lining serosal membranes. The biological and clinical characteristics of epithelial type malignant mesothelioma are distinct from those of biphasic and sarcomatous type tumors. The goal of our study was to examine the molecular basis for this distinction. Microarray analysis confirmed that the molecular signatures of epithelial and biphasic histologic subtypes

A C Borczuk; G C A Cappellini; H K Kim; M Hesdorffer; R N Taub; C A Powell

2007-01-01

174

Genetic myostatin decrease in the golden retriever muscular dystrophy model does not significantly affect the ubiquitin proteasome system despite enhancing the severity of disease  

PubMed Central

Recent studies suggest that inhibiting the protein myostatin, a negative regulator of skeletal muscle mass, may improve outcomes in patients with Duchenne muscular dystrophy by enhancing muscle mass. When the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog was bred with whippets having a heterozygous mutation for the myostatin gene, affected GRMD dogs with decreased myostatin (GRippets) demonstrated an accelerated physical decline compared to related affected GRMD dogs with full myostatin. To examine the role of the ubiquitin proteasome and calpain systems in this accelerated decline, we determined the expression of the muscle ubiquitin ligases MuRF1, Atrogin-1, RNF25, RNF11, and CHIP: the proteasome subunits PSMA6, PSMB4, and PSME1: and calpain 1/2 by real time PCR in the cranial sartorius and vastus lateralis muscles in control, affected GRMD, and GRippet dogs. While individual affected GRMD and GRippet dogs contributed to an increased variability seen in ubiquitin ligase expression, neither group was significantly different from the control group. The affected GRMD dogs demonstrated significant increases in caspase-like and trypsin-like activity in the cranial sartorius; however, all three proteasome activities in the GRippet muscles did not differ from controls. Increased variability in calpain 1 and calpain 2 expression and activity in the affected GRMD and GRippet groups were identified, but no statistical differences from the control group were seen. These studies suggest a role of myostatin in the disease progression of GRMD, which does not significantly involve key components of the ubiquitin proteasome and calpain systems involved in the protein quality control of sarcomere and other structural skeletal muscle proteins.

Cotten, Steven W; Kornegay, Joe N; Bogan, Daniel J; Wadosky, Kristine M; Patterson, Cam; Willis, Monte S

2014-01-01

175

Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein  

PubMed Central

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8+ cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2181?188) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2181?188. GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28?/?. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2181?188, by activating CD8+ CTL specific for TRP-2181?188. TRP-2181?188 fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8+ T cells specific for the CTL epitope. Furthermore, CD4+ T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the ‘ubiquitin-proteasome pathway’. It is noteworthy to document that genetic immunization with pGFP plus pTRP-2181?188 failed to exert the antitumour immunity.

Zhang, Manxin; Ishii, Kazunari; Hisaeda, Hajime; Murata, Shigeo; Chiba, Tomoki; Tanaka, Keiji; Li, Yang; Obata, Chikage; Furue, Masutaka; Himeno, Kunisuke

2004-01-01

176

Glucocorticoids differentially regulate degradation of MyoD and Id1 by N-terminal ubiquitination to promote muscle protein catabolism  

PubMed Central

Accelerated protein degradation via the ubiquitin–proteasome pathway is the principal cause of skeletal muscle wasting associated with common human disease states and pharmacological treatment with glucocorticoids. Although many protein regulatory factors essential for muscle development and regeneration are degraded via the ubiquitin system, little is known about the mechanisms and regulation of this pathway that promote wasting muscle. Here, we demonstrate that, in differentiated myotubes, glucocorticoid, via the glucocorticoid receptor, selectively induces a decrease in protein abundance of MyoD, a master switch for muscle development and regeneration, but not that of its negative regulator Id1. This decrease in MyoD protein results from accelerated degradation after glucocorticoid exposure. Using MyoD and Id1 mutants deficient in either N terminus-dependent or internal lysine-dependent ubiquitination, we further show that these ubiquitination pathways of MyoD degradation are regulated differently from those of Id1 degradation. Specifically, glucocorticoid activates the N-terminal ubiquitination pathway in MyoD degradation in myotubes, without concomitant effects on Id1 degradation. This effect of glucocorticoid on MyoD and Id1 protein degradation is associated with the distinct cellular compartments in which their degradation occurs. Taken together, these results support a key role for the N terminus-dependent ubiquitination pathway in the physiology of muscle protein degradation.

Sun, Liping; Trausch-Azar, Julie S.; Muglia, Louis J.; Schwartz, Alan L.

2008-01-01

177

ER-Golgi Traffic Is a Prerequisite for Efficient ER Degradation  

PubMed Central

Protein quality control is an essential function of the endoplasmic reticulum. Misfolded proteins unable to acquire their native conformation are retained in the endoplasmic reticulum, retro-translocated back into the cytosol, and degraded via the ubiquitin-proteasome system. We show that efficient degradation of soluble malfolded proteins in yeast requires a fully competent early secretory pathway. Mutations in proteins essential for ER-Golgi protein traffic severely inhibit ER degradation of the model substrate CPY*. We found ER localization of CPY* in WT cells, but no other specific organelle for ER degradation could be identified by electron microscopy studies. Because CPY* is degraded in COPI coat mutants, only a minor fraction of CPY* or of a proteinaceous factor required for degradation seems to enter the recycling pathway between ER and Golgi. Therefore, we propose that the disorganized structure of the ER and/or the mislocalization of Kar2p, observed in early secretory mutants, is responsible for the reduction in CPY* degradation. Further, we observed that mutations in proteins directly involved in degradation of malfolded proteins (Der1p, Der3/Hrd1p, and Hrd3p) lead to morphological changes of the endoplasmic reticulum and the Golgi, escape of CPY* into the secretory pathway and a slower maturation rate of wild-type CPY.

Taxis, Christof; Vogel, Frank; Wolf, Dieter H.

2002-01-01

178

Degradation of Activated Protein Kinases by Ubiquitination  

PubMed Central

Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

Lu, Zhimin; Hunter, Tony

2009-01-01

179

Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome, Translation, and RNA Processing System Homologs  

PubMed Central

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNALysUUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1? and a ?-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNALysUUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Chavarria, Nikita E.; Hwang, Sungmin; Cao, Shiyun; Fu, Xian; Holman, Mary; Elbanna, Dina; Rodriguez, Suzanne; Arrington, Deanna; Englert, Markus; Uthandi, Sivakumar; Soll, Dieter; Maupin-Furlow, Julie A.

2014-01-01

180

Triggering Fbw7-mediated proteasomal degradation of c-Myc by oridonin induces cell growth inhibition and apoptosis.  

PubMed

The transcription factor c-Myc is important in cell fate decisions and is frequently overexpressed in cancer cells, making it an attractive therapeutic target. Natural compounds are among the current strategies aimed at targeting c-Myc, but their modes of action still need to be characterized. To explore the mechanisms underlying the anticancer activity of a natural diterpenoid, oridonin, we conducted miRNA expression profiling and statistical analyses that strongly suggested that c-Myc was a potential molecular target of oridonin. Furthermore, experimental data showed that oridonin significantly reduced c-Myc protein levels in vitro and in vivo and that this reduction was mediated by the ubiquitin-proteasome system. Fbw7, a component of the ubiquitin-proteasome system and an E3 ubiquitin ligase of c-Myc, was upregulated rapidly in K562 cells and other leukemia and lymphoma cells, resulting in the rapid turnover of c-Myc. In cell lines harboring mutations in the WD domain of Fbw7, the degradation of c-Myc induced by oridonin was attenuated during short-term treatment. GSK-3, an Fbw7 priming kinase, was also activated by oridonin, along with an increase in T58-phosphorylated c-Myc. Furthermore, the knockdown of Fbw7 or the forced expression of stable c-Myc resulted in reduced sensitization to oridonin-induced apoptosis. Our observations help to clarify the anticancer mechanisms of oridonin and shed light on the application of this natural compound as an Fbw7-c-Myc pathway targeting agent in cancer treatment. PMID:22389469

Huang, Hui-Lin; Weng, Heng-You; Wang, Lu-Qin; Yu, Chun-Hong; Huang, Qiao-Juan; Zhao, Pan-Pan; Wen, Jun-Zhi; Zhou, Hui; Qu, Liang-Hu

2012-05-01

181

Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation.  

PubMed

The accumulation of protein deposits in neurons, in vitro proteasome assays and over-expression studies suggest that impairment of the ubiquitin-proteasome system (UPS) may be a common mechanism of pathogenesis in polyglutamine diseases such as Huntington disease and spinocerebellar ataxias (SCAs). Using a knock-in mouse model that recapitulates the clinical features of human SCA7, including selective neuronal dysfunction, we assessed the UPS at cellular resolution using transgenic mice that express a green fluorescent protein (GFP)-based reporter substrate (Ub(G76V)-GFP) of the UPS. The levels of the reporter remained low during the initial phase of disease, suggesting that neuronal dysfunction occurs in the presence of a functional UPS. Late in disease, we observed a significant increase in reporter levels specific to the most vulnerable neurons. Surprisingly, the basis for the increase in Ub(G76V)-GFP protein can be explained by a corresponding increase in Ub(G76V)-GFP mRNA in the vulnerable neurons. An in vitro assay also showed normal proteasome proteolytic activity in the vulnerable neurons. Thus, no evidence for general UPS impairment or reduction of proteasome activity was seen. The differential increase of Ub(G76V)-GFP among individual neurons directly correlated with the down-regulation of a marker of selective pathology and neuronal dysfunction in SCA7. Furthermore, we observed a striking inverse correlation between the neuropathology revealed by this reporter and ataxin-7 nuclear inclusions in the vulnerable neurons. Altogether, these data show a protective role against neuronal dysfunction for polyglutamine nuclear inclusions and exclude significant impairment of the UPS as a necessary step for polyglutamine neuropathology. PMID:15661755

Bowman, Aaron B; Yoo, Seung-Yun; Dantuma, Nico P; Zoghbi, Huda Y

2005-03-01

182

Effect of inhibition of the Ubiquitin-Proteasome System and Hsp90 on growth and survival of Rhabdomyosarcoma cells in vitro  

PubMed Central

Background The ubiquitin-proteasome system (UPS) and the heat shock response (HSR) are two critical regulators of cell homeostasis, as their inhibition affects growth and survival of normal cells, as well as stress response and invasiveness of cancer cells. We evaluated the effects of the proteasome inhibitor Bortezomib and of 17-DMAG, a competitive inhibitor of Hsp90, in rhabdomyosarcoma (RMS) cells, and analyzed the efficacy of single-agent exposures with combination treatments. Methods To assess cytotoxicity induced by Bortezomib and 17-DMAG in RMS cells, viability was measured by MTT assay after 24, 48 and 72 hours. Western blotting and immunofluorescence analyses were carried out to elucidate the mechanisms of action. Apoptosis was measured by FACS with Annexin-V-FITC and Propidium Iodide. Results Bortezomib and 17-DMAG, when combined at single low-toxic concentrations, enhanced growth inhibition of RMS cells, with signs of autophagy that included intensive cytoplasmic vacuolization and conversion of cytosolic LC3-I protein to its autophagosome-associated form. Treatment with lysosomal inhibitor chloroquine facilitates apoptosis, whereas stimulation of autophagy by rapamycin prevents LC3-I conversion and cell death, suggesting that autophagy is a resistance mechanism in RMS cells exposed to proteotoxic drugs. However, combination treatment also causes caspase-dependent apoptosis, PARP cleavage and Annexin V staining, as simultaneous inhibition of both UPS and HSR systems limits cytoprotective autophagy, exacerbating stress resulting from accumulation of misfolded proteins. Conclusion The combination of proteasome inhibitor Bortezomib with Hsp90 inhibitor 17-DMAG, appears to have important therapeutic advantages in the treatment of RMS cells compared with single-agent exposure, because compensatory survival mechanisms that occur as side effects of treatment may be prevented.

2012-01-01

183

p38 Regulates Pigmentation via Proteasomal Degradation of Tyrosinase*  

PubMed Central

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by ?-melanocyte-stimulating hormone (?-MSH) and ultraviolet irradiation. Previous studies have shown that ?-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and ?-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.

Bellei, Barbara; Maresca, Vittoria; Flori, Enrica; Pitisci, Angela; Larue, Lionel; Picardo, Mauro

2010-01-01

184

p38 regulates pigmentation via proteasomal degradation of tyrosinase.  

PubMed

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by alpha-melanocyte-stimulating hormone (alpha-MSH) and ultraviolet irradiation. Previous studies have shown that alpha-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and alpha-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA. PMID:20053998

Bellei, Barbara; Maresca, Vittoria; Flori, Enrica; Pitisci, Angela; Larue, Lionel; Picardo, Mauro

2010-03-01

185

BACE is degraded via the lysosomal pathway.  

PubMed

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes. PMID:16033761

Koh, Young Ho; von Arnim, Christine A F; Hyman, Bradley T; Tanzi, Rudolph E; Tesco, Giuseppina

2005-09-16

186

Emerging role of the ubiquitin proteasome system in the control of shoot apical meristem function(f).  

PubMed

The shoot apical meristem (SAM) is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant's life. A plethora of different families of transcription factors (TFs) play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system (UPS). However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors. PMID:23164365

Di Giacomo, Elisabetta; Serino, Giovanna; Frugis, Giovanna

2013-01-01

187

A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system  

PubMed Central

Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2–E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2–E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2–E3 interactions of the human ubiquitination system.

van Wijk, Sjoerd J L; de Vries, Sjoerd J; Kemmeren, Patrick; Huang, Anding; Boelens, Rolf; Bonvin, Alexandre M J J; Timmers, H Th Marc

2009-01-01

188

Assaying Proteasomal Degradation in a Cell-free System in Plants  

PubMed Central

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Garcia-Cano, Elena; Zaltsman, Adi; Citovsky, Vitaly

2014-01-01

189

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

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

190

Alpha-synuclein aggregation involves a bafilomycin A1-sensitive autophagy pathway  

PubMed Central

Synucleinopathies like Parkinson disease and dementia with Lewy bodies (DLB) are characterized by ?-synuclein aggregates within neurons (Lewy bodies) and their processes (Lewy neurites). Whereas ?-synuclein has been genetically linked to the disease process, the pathological relevance of ?-synuclein aggregates is still debated. Impaired degradation is considered to result in aggregation of ?-synuclein. In addition to the ubiquitin-proteasome degradation, the autophagy-lysosomal pathway (ALP) is involved in intracellular degradation processes for ?-synuclein. Here, we asked if modulation of ALP affects ?-synuclein aggregation and toxicity. We have identified an induction of the ALP markers LAMP-2A and LC3-II in human brain tissue from DLB patients, in a transgenic mouse model of synucleinopathy, and in a cell culture model for ?-synuclein aggregation. ALP inhibition using bafilomycin A1 (BafA1) significantly potentiates toxicity of aggregated ?-synuclein species in transgenic mice and in cell culture. Surprisingly, increased toxicity is paralleled by reduced aggregation in both in vivo and in vitro models. The dichotomy of effects on aggregating and nonaggregating species of ?-synuclein was specifically sensitive to BafA1 and could not be reproduced by other ALP inhibitors. The present study expands on the accumulating evidence regarding the function of ALP for ?-synuclein degradation by isolating an aggregation specific, BafA1-sensitive, ALP-related pathway. Our data also suggest that protein aggregation may represent a detoxifying event rather than being causal for cellular toxicity.

Klucken, Jochen; Poehler, Anne-Maria; Ebrahimi-Fakhari, Darius; Schneider, Jacqueline; Nuber, Silke; Rockenstein, Edward; Schlotzer-Schrehardt, Ursula; Hyman, Bradley T.; McLean, Pamela J.; Masliah, Eliezer; Winkler, Juergen

2012-01-01

191

The E3 ubiquitin ligase HOS1 regulates Arabidopsis flowering by mediating CONSTANS degradation under cold stress.  

PubMed

The timing of flowering is coordinated by a web of gene regulatory networks that integrates developmental and environmental cues in plants. Light and temperature are two major environmental determinants that regulate flowering time. Although prolonged treatment with low nonfreezing temperatures accelerates flowering by stable repression of FLOWERING LOCUS C (FLC), repeated brief cold treatments delay flowering. Here, we report that intermittent cold treatments trigger the degradation of CONSTANS (CO), a central activator of photoperiodic flowering; daily treatments caused suppression of the floral integrator FLOWERING LOCUS T (FT) and delayed flowering. Cold-induced CO degradation is mediated via a ubiquitin/proteasome pathway that involves the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). HOS1-mediated CO degradation occurs independently of the well established cold response pathways. It is also independent of the light signaling repressor CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase and light wavelengths. CO has been shown to play a key role in photoperiodic flowering. Here, we demonstrated that CO served as a molecular hub, integrating photoperiodic and cold stress signals into the flowering genetic pathways. We propose that the HOS1-CO module contributes to the fine-tuning of photoperiodic flowering under short term temperature fluctuations, which often occur during local weather disturbances. PMID:23135282

Jung, Jae-Hoon; Seo, Pil Joon; Park, Chung-Mo

2012-12-21

192

ABA modulates the degradation of phosphoenolpyruvate carboxylase kinase in sorghum leaves.  

PubMed

Salt stresses strongly enhance the phosphoenolpyruvate carboxylase kinase (PEPC-k) activity of sorghum leaves. This work shows that (1) abscisic acid (ABA) increased the rise in kinase activity in illuminated leaf disks of the non-stressed plant, (2) ABA decreased the disappearance of PEPC-k activity in the dark, (3) two PEPC-k genes expressed in sorghum leaves, PPCK1 and PPCK2, were not up-regulated by the phytohormone and, (4) ABA effects were mimicked by MG132, a powerful inhibitor of the ubiquitin-proteasome pathway. Collectively these data support a role for the ubiquitin-proteasome pathway in the rapid turnover of PEPC-k. The negative control by ABA on this pathway might account for the increase of kinase activity observed in salt-treated plants. PMID:17618627

Monreal, José Antonio; Feria, Ana Belén; Vinardell, José María; Vidal, Jean; Echevarría, Cristina; García-Mauriño, Sofía

2007-07-24

193

Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1.  

PubMed

Cells have a variety of strategies for dealing with misfolded proteins. Heat shock response involves transcriptional induction of chaperones to promote and/or correct folding, and also activation of the ubiquitin/proteasome system to degrade defective proteins. In the secretory pathway, it is primarily luminal misfolded or unassembled proteins that trigger the unfolded protein response which, like heat shock, induces chaperones and components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. To understand cellular response to a misfolded polytopic membrane protein of the secretory pathway, we studied Pma1-D378S, a model ERAD substrate. Expression of misfolded Pma1 induces heat shock response in the absence of increased temperature. Overexpression of HSF1, the transcription factor that mediates heat shock response, increases degradation of Pma1-D378S without temperature upshift. Nevertheless, efficient Pma1-D378S degradation occurs in an hsf1 mutant that maintains basal transcription levels but cannot mediate transcriptional activation. Thus, heat shock protein induction enhances but is not necessary for ERAD. The Ssa group of cytoplasmic Hsp70 chaperones is required for ERAD of both Pma1-D378S and another transmembrane ERAD substrate, Ste6*. In the absence of Ssa chaperones, ubiquitination of both substrates is impaired, resulting in stabilization. We suggest a role for Hsp70 cytoplasmic chaperones in recognition by the endoplasmic reticulum-associated ubiquitination machinery. PMID:17631501

Han, Sumin; Liu, Yu; Chang, Amy

2007-09-01

194

Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis  

PubMed Central

Bioactive gibberellins (GAs) are diterpene phytohormones that modulate growth and development throughout the whole life cycle of the plant. Arabidopsis genes encoding most GA biosynthesis and catabolism enzymes, as well as GA receptors (GIBBERELLIN INSENSITIVE DWARF1, GID1) and early GA signaling components have been identified. Expression studies on the GA biosynthesis genes are beginning to reveal the potential sites of GA biosynthesis during plant development. Biochemical and genetic analyses demonstrate that GA de-represses its signaling pathway by binding to GID1s, which induce degradation of GA signaling repressors (DELLAs) via an ubiquitin-proteasome pathway. To modulate plant growth and development, the GA pathway is also regulated by endogenous signals (other hormones) and environmental cues (such as light, temperature and salt stress). In many cases, these internal and external cues directly affect GA metabolism and bioactive GA levels, and indirectly alter DELLA accumulation and GA responses. Importantly, direct negative interaction between DELLA and PIF3 and PIF4 (2 phytochrome interacting transcription factors) appears to integrate the effects of light and GA on hypocotyl elongation.

Sun, Tai-ping

2008-01-01

195

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

PubMed Central

Background The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation. Results Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development. Conclusions The rapid growth of neonatal muscle is in part due to the positive balance between the activation of protein synthesis and degradation signaling. Insulin, amino acids, and, particularly, leucine, act as signals to modulate muscle protein synthesis and degradation in neonates.

2014-01-01

196

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination  

PubMed Central

McKusick–Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet–Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin–proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

Hirayama, Shoshiro; Yamazaki, Yuji; Kitamura, Akira; Oda, Yukako; Morito, Daisuke; Okawa, Katsuya; Kimura, Hiroshi; Cyr, Douglas M.; Nagata, Kazuhiro

2008-01-01

197

A novel protein modification pathway related to the ubiquitin system.  

PubMed Central

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

Liakopoulos, D; Doenges, G; Matuschewski, K; Jentsch, S

1998-01-01

198

Negative regulation of DAB2IP by Akt and SCFFbw7 pathways.  

PubMed

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCFFbw7. DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1?. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development. PMID:24912918

Dai, Xiangping; North, Brian J; Inuzuka, Hiroyuki

2014-05-30

199

Negative regulation of DAB2IP by Akt and SCFFbw7 pathways  

PubMed Central

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCFFbw7. DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1?. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development.

Inuzuka, Hiroyuki

2014-01-01

200

S. pombe TORC1 activates the ubiquitin-proteasomal degradation of the meiotic regulator Mei2 in cooperation with Pat1 kinase.  

PubMed

Target of rapamycin (TOR) kinase regulates cell metabolism and growth, acting as a subunit of two multi-protein complexes, TORC1 and TORC2. Known TORC substrates are either kinases or general factors involved in growth control. Here, we show that fission yeast TORC1, which promotes vegetative growth and suppresses sexual development, can phosphorylate Mei2 (a specific factor involved in switching the cell fate) in vitro. Alanine substitutions at the nine Mei2 phosphorylation sites stabilize the protein and promote mating and meiosis in vivo. We found that Mei2 is polyubiquitylated in vivo in a TORC1-dependent manner. Based on these data, we propose that TORC1 contributes to the suppression of sexual development by phosphorylating Mei2, in addition to controlling the cellular metabolic status. PMID:24741065

Otsubo, Yoko; Yamashita, Akira; Ohno, Hayao; Yamamoto, Masayuki

2014-06-15

201

Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin  

PubMed Central

Biosynthesis and folding of multidomain transmembrane proteins is a complex process. Structural fidelity is monitored by endoplasmic reticulum (ER) quality control involving the molecular chaperone calnexin. Retained misfolded proteins undergo ER-associated degradation (ERAD) through the ubiquitin-proteasome pathway. Our data show that the major degradation pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) with F508del (the most frequent mutation found in patients with the genetic disease cystic fibrosis) from the ER is independent of calnexin. Moreover, our results demonstrate that inhibition of mannose-processing enzymes, unlike most substrate glycoproteins, does not stabilize F508del-CFTR, although wild-type (wt) CFTR is drastically stabilized under the same conditions. Together, our data support a novel model by which wt and F508del-CFTR undergo ERAD from two distinct checkpoints, the mutant being disposed of independently of N-glycosidic residues and calnexin, probably by the Hsc70/Hsp70 machinery, and wt CFTR undergoing glycan-mediated ERAD.

Farinha, Carlos M.; Amaral, Margarida D.

2005-01-01

202

Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G.  

PubMed

The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner. PMID:21228271

Sugiyama, Ryuichi; Nishitsuji, Hironori; Furukawa, Ayako; Katahira, Masato; Habu, Yuichiro; Takeuchi, Hiroaki; Ryo, Akihide; Takaku, Hiroshi

2011-03-25

203

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

PubMed

Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood. Here using a high-coverage genome-wide shRNA library, we identify the uncharacterized protein TMEM129 and the ubiquitin-conjugating E2 enzyme UBE2J2 to be essential for US11-mediated HLA class I downregulation. TMEM129 is an unconventional C4C4-type RING finger E3 ubiquitin ligase that resides within a complex containing various other ERAD components, including Derlin-1, Derlin-2, VIMP and p97, indicating that TMEM129 is an integral part of the ER-resident dislocation complex mediating US11-induced HLA class I degradation. PMID:24807418

van de Weijer, Michael L; Bassik, Michael C; Luteijn, Rutger D; Voorburg, Cornelia M; Lohuis, Mirjam A M; Kremmer, Elisabeth; Hoeben, Rob C; LeProust, Emily M; Chen, Siyuan; Hoelen, Hanneke; Ressing, Maaike E; Patena, Weronika; Weissman, Jonathan S; McManus, Michael T; Wiertz, Emmanuel J H J; Lebbink, Robert Jan

2014-01-01

204

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

PubMed Central

Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin–proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood. Here using a high-coverage genome-wide shRNA library, we identify the uncharacterized protein TMEM129 and the ubiquitin-conjugating E2 enzyme UBE2J2 to be essential for US11-mediated HLA class I downregulation. TMEM129 is an unconventional C4C4-type RING finger E3 ubiquitin ligase that resides within a complex containing various other ERAD components, including Derlin-1, Derlin-2, VIMP and p97, indicating that TMEM129 is an integral part of the ER-resident dislocation complex mediating US11-induced HLA class I degradation.

van de Weijer, Michael L.; Bassik, Michael C.; Luteijn, Rutger D.; Voorburg, Cornelia M.; Lohuis, Mirjam A.M.; Kremmer, Elisabeth; Hoeben, Rob C.; LeProust, Emily M.; Chen, Siyuan; Hoelen, Hanneke; Ressing, Maaike E.; Patena, Weronika; Weissman, Jonathan S.; McManus, Michael T.; Wiertz, Emmanuel J.H.J.; Lebbink, Robert Jan

2014-01-01

205

Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein  

PubMed Central

Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

Xu, Chongfeng; Sun, Lei; Chen, Jilong; Zhang, Lianfeng; Liu, Wenjun

2012-01-01

206

Proteolysis by calpains: a possible contribution to degradation of p53.  

PubMed Central

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Pariat, M; Carillo, S; Molinari, M; Salvat, C; Debussche, L; Bracco, L; Milner, J; Piechaczyk, M

1997-01-01

207

Geldanamycin-induced degradation of Chk1 is mediated by proteasome  

SciTech Connect

Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3 h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24 h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.

Nomura, M. [Department of Neurosurgery, Yokohama Sakae Kyosai Hospital, Yokohama (Japan); Department of Neurosurgery, Kanazawa University School of Medicine, Kanazawa (Japan); E-mail: nomura413jp@yahoo.co.jp; Nomura, N. [Department of Ophthalmology, Kanazawa Social Insurance Hospital, Kanazawa (Japan); Yamashita, J. [Department of Neurosurgery, Kanazawa University School of Medicine, Kanazawa (Japan)

2005-09-30

208

Vitamin C Degradation Products and Pathways in the Human Lens*  

PubMed Central

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

Nemet, Ina; Monnier, Vincent M.

2011-01-01

209

Kaposi Sarcoma-associated Herpesvirus Degrades Cellular Toll-Interleukin-1 Receptor Domain-containing Adaptor-inducing ?-Interferon (TRIF)*  

PubMed Central

Kaposi sarcoma-associated herpesvirus (KSHV) is a human ?-herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing ?-interferon (TRIF, also called TIR-domain-containing adaptor molecule-1 (TICAM-1)) is a signaling adaptor molecule that is critically involved in the Toll-like receptor 3 (TLR-3) and TLR-4 signaling pathways for type I interferon (IFN) production, a key component of innate immunity against microbial infection. In this report, we find a new mechanism by which RTA blocks innate immunity by targeting cellular TRIF. RTA specifically degrades TRIF by shortening the half-life of TRIF protein. This RTA-mediated degradation is at least partially mediated through the ubiquitin-proteasome pathway because proteasome inhibitors as well as knockdown of cellular ubiquitin expression alleviate the degradation. RTA may not directly interact with TRIF and may activate TRIF degradation indirectly through an unknown mediator(s). RTA targets multiple regions of TRIF and may use its ubiquitin ligase domain for the degradation. In addition, physiological levels of TRIF protein are down-regulated during KSHV lytic replication when RTA is expressed. Finally, RTA down-regulates double-stranded RNA-initiated activation of TLR-3 pathway, in the absence of degradation of IFN regulatory factor 7 (IRF-7). Taken together, these data suggest that KSHV employs a novel mechanism to block the innate immunity by degrading TRIF protein. This work may contribute to our understandings on how KSHV evades host immunity for its survival in vivo.

Ahmad, Humera; Gubbels, Rachel; Ehlers, Erica; Meyer, Florencia; Waterbury, Thomas; Lin, Rongtuan; Zhang, Luwen

2011-01-01

210

The PHD Domain of MEKK1 Acts as an E3 Ubiquitin Ligase and Mediates Ubiquitination and Degradation of ERK1\\/2  

Microsoft Academic Search

ERK1\\/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1\\/2 activity is downregulated by ubiquitin\\/proteasome-mediated degradation of ERK1\\/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity

Zhimin Lu; Shuichan Xu; Claudio Joazeiro; Melanie H Cobb; Tony Hunter

2002-01-01

211

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

SciTech Connect

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

T Wang; K Heran Darwin; H Li

2011-12-31

212

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

SciTech Connect

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

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

2010-11-01

213

Selective protein degradation in the yeast exocytic pathway.  

PubMed Central

Protein degradation in the exocytic pathway was studied in Saccharomyces cerevisiae using human alpha-1-protease inhibitor (A1Pi) as a reporter molecule. Yeast cells transformed with A1Pi cDNA genes synthesized A1Pi that entered the secretion pathway and accumulated in the endoplasmic reticulum (ER). Cells expressing A1PiM (wild-type) accumulated about 10-fold more A1Pi than cells expressing A1PiZ (secretion defective variant). Analyses of A1Pi mRNA indicated that the low level of A1PiZ relative to A1PiM was not the result of differential gene transcription. Pulse-chase A1Pi radiolabeling showed that A1PiM and A1PiZ were degraded at different rates and suggested a rapid specific turnover of newly synthesized A1PiZ in the ER. Accumulated A1Pi was degraded at comparable rates in both wild-type cells and cells deficient in vacuolar protease activity, indicating that degradation of A1Pi did not occur in the vacuole. Studies to investigate the intracellular location of the degradative process, using temperature-sensitive secretion defective yeast strains, suggested the possibility that degradation occurs not only in the ER but at a second site accessed by vesicle transport. Together, these results demonstrate that a selective protein degradation process operates early in the yeast cell exocytic pathway. Images

McCracken, A A; Kruse, K B

1993-01-01

214

When RNA and protein degradation pathways meet  

PubMed Central

RNA silencing has become a major focus of molecular and biomedical research in the last decade. This mechanism, which is conserved in most eukaryotes, has been extensively studied and is associated to various pathways implicated in the regulation of development, in the control of transposition events, heterochromatin maintenance and also playing a role in defense against viruses. Despite of its importance, the regulation of the RNA silencing machinery itself remains still poorly explored. Recently several reports in both plants and metazoans revealed that key components of RNA silencing, such as RNA-induced silencing complex component ARGONAUTE proteins, but also the endonuclease Dicer are subjected to proteasomal and autophagic pathways. Here we will review these post-translational proteolytic regulations with a special emphasis on plant research and also discuss their functional relevance.

Derrien, Benoit; Genschik, Pascal

2014-01-01

215

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation.  

PubMed

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation. PMID:21145412

Wang, Jiannan; Salem, Mohamed; Qi, Nan; Kenney, P Brett; Rexroad, Caird E; Yao, Jianbo

2011-03-01

216

The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1  

PubMed Central

Plant responses to cold stress are mediated by a transcriptional cascade, in which the transcription factor ICE1 and possibly related proteins activate the expression of C-repeat (CRT)-binding factors (CBFs), leading to the transcription of downstream effector genes. The variant RING finger protein high expression of osmotically responsive gene (HOS)1 was identified genetically as a negative regulator of cold responses. We present evidence here that HOS1 is an E3 ligase required for the ubiquitination of ICE1. HOS1 physically interacts with ICE1 and mediates the ubiquitination of ICE1 both in vitro and in vivo. We found that cold induces the degradation of ICE1 in plants, and this degradation requires HOS1. Consistent with enhanced cold-responsive gene expression in loss-of-function hos1 mutant plants, overexpression of HOS1 represses the expression of CBFs and their downstream genes and confers increased sensitivity to freezing stress. Our results indicate that cold stress responses in Arabidopsis are attenuated by a ubiquitination/proteasome pathway in which HOS1 mediates the degradation of the ICE1 protein.

Dong, Chun-Hai; Agarwal, Manu; Zhang, Yiyue; Xie, Qi; Zhu, Jian-Kang

2006-01-01

217

Meta-pathway degradation of phenolics by thermophilic Bacilli  

Microsoft Academic Search

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain,

Saiqa Ali; Roberto Fernandez-Lafuente; Don A Cowan

1998-01-01

218

Validation of microarray data in human lymphoblasts shows a role of the ubiquitin-proteasome system and NF-kB in the pathogenesis of Down syndrome  

PubMed Central

Background Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. Methods To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. Results We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. Conclusions The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS.

2013-01-01

219

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways  

Microsoft Academic Search

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. The authors have investigate the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass

B. E. Haigler; J. C. Spain

1991-01-01

220

Stabilization of RNT-1 protein, runt-related transcription factor (RUNX) protein homolog of Caenorhabditis elegans, by oxidative stress through mitogen-activated protein kinase pathway.  

PubMed

RUNX proteins are evolutionarily conserved transcription factors known to be involved in various developmental processes. Here we report a new role for a RUNX protein: a role in stress response. We show that RNT-1, the Caenorhabditis elegans RUNX homolog, is constantly produced and degraded by the ubiquitination-proteasome pathway in the intestine of the nematode. RNT-1 was rapidly stabilized by oxidative stress, and the rnt-1-mutant animals were more sensitive to oxidative stress, indicating that rapid RNT-1 stabilization is a defense response against the oxidative stress. The MAP kinase pathway is required for RNT-1 stabilization, and RNT-1 was phosphorylated by SEK-1/PMK-1 in vitro. ChIP-sequencing analysis revealed a feedback loop mechanism of the MAP kinase pathway by the VHP-1 phosphatase in the RNT-1-mediated oxidative stress response. We propose that rnt-1 is regulated at the protein level for its role in the immediate response to environmental challenges in the intestine. PMID:22308034

Lee, Kiho; Shim, Jiwon; Bae, Jaebum; Kim, Young-Joon; Lee, Junho

2012-03-23

221

Stabilization of RNT-1 Protein, Runt-related Transcription Factor (RUNX) Protein Homolog of Caenorhabditis elegans, by Oxidative Stress through Mitogen-activated Protein Kinase Pathway*  

PubMed Central

RUNX proteins are evolutionarily conserved transcription factors known to be involved in various developmental processes. Here we report a new role for a RUNX protein: a role in stress response. We show that RNT-1, the Caenorhabditis elegans RUNX homolog, is constantly produced and degraded by the ubiquitination-proteasome pathway in the intestine of the nematode. RNT-1 was rapidly stabilized by oxidative stress, and the rnt-1-mutant animals were more sensitive to oxidative stress, indicating that rapid RNT-1 stabilization is a defense response against the oxidative stress. The MAP kinase pathway is required for RNT-1 stabilization, and RNT-1 was phosphorylated by SEK-1/PMK-1 in vitro. ChIP-sequencing analysis revealed a feedback loop mechanism of the MAP kinase pathway by the VHP-1 phosphatase in the RNT-1-mediated oxidative stress response. We propose that rnt-1 is regulated at the protein level for its role in the immediate response to environmental challenges in the intestine.

Lee, Kiho; Shim, Jiwon; Bae, Jaebum; Kim, Young-Joon; Lee, Junho

2012-01-01

222

Evolution of a new bacterial pathway for 4-nitrotoluene degradation.  

PubMed

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases. PMID:21895789

Ju, Kou-San; Parales, Rebecca E

2011-10-01

223

Degradation of aromatic compounds and degradative pathway of 4-nitrocatechol by Ochrobactrum sp. B2.  

PubMed

The potential capacity of a soil methyl parathion-degrading bacterium strain, Ochrobactrum sp. B2, for degrading various aromatic compounds were investigated. The results showed B2 was capable of degrading diverse aromatic compounds, but amino-substituted benzene compounds, at a concentration up to 100 mg L(-1) in 4 days. B2 could use 4-nitrocatechol (4-NC) as a sole carbon and energy source with release of nitrite ion. The pathway for 4-NC degradation via 1,2,4-benzenetriol (BT) and hydroquinone (HQ) formation in B2 was proposed based on the identification and quantification of intermediates by gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography (HPLC). Degradation studies carried out on a plasmid-cured derivative showed that the genes for 4-NC degradative pathway was plasmid-borne in B2, suggesting that B2 degrades both p-nitrophenol and 4-NC by enzymes encoded by genes on the same plasmid. PMID:18074282

Zhong, Qiuzan; Zhang, Haiyan; Bai, Wenqin; Li, Mei; Li, Baotong; Qiu, Xinghui

2007-12-01

224

The transcription factor STAT2 enhances proteasomal degradation of RCAN1 through the ubiquitin E3 ligase FBW7.  

PubMed

Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-? reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability. PMID:22426484

Lee, Jee Won; Kang, Hye Seon; Lee, Jae Youn; Lee, Eun Jung; Rhim, Hyewhon; Yoon, Joo Heon; Seo, Su Ryeon; Chung, Kwang Chul

2012-04-01

225

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

PubMed Central

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

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

2013-01-01

226

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene  

SciTech Connect

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. The authors report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.

Nishino, S.F.; Paoli, G.C.; Spain, J.C.

2000-05-01

227

BACE2 degradation mediated by the macroautophagy-lysosome pathway.  

PubMed

Neuritic plaque is the pathological hallmark in Alzheimer's disease (AD). Amyloid-? protein (A?), the central component of neuritic plaques, is generated from amyloid-? precursor protein (APP) by ?-site APP cleaving enzyme 1 (BACE1) and ?-secretase. ?-site APP cleaving enzyme 2 (BACE2), a homolog of BACE1, functions differently from BACE1 in APP processing. BACE1 is the ?-secretase essential for A? production, and BACE2, a ?-secretase, cleaves APP within the A? domain, preventing A? production. Elucidation of the mechanism underlying BACE2 degradation is important for defining its biological features and its potential role in Alzheimer's disease drug development. In this report we first showed that the half-life of BACE2 is approximately 20 h. Lysosomal inhibition increased BACE2 protein levels whereas proteasomal inhibition had no effect on BACE2 protein expression. Furthermore, we identified that macroautophagy mediated BACE2 degradation. Finally, we showed that lysosomal inhibition increased BACE2 cleavage of APP. Taken together, our in vitro study showed that BACE2 is degraded through the macrophagy-lysosome pathway and that lysosomal inhibition affects BACE2 processing of APP. Modulation of BACE2 degradation via the lysosomal pathway could be a new target for AD drug development. PMID:23773066

Liu, Xi; Wang, Zhe; Wu, Yili; Wang, Jianping; Song, Weihong

2013-06-01

228

Proteasomal and lysosomal protein degradation and heart disease.  

PubMed

In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". PMID:24239609

Wang, Xuejun; Robbins, Jeffrey

2014-06-01

229

Tyrosinase Degradation Is Prevented when EDEM1 Lacks the Intrinsically Disordered Region  

PubMed Central

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I ?1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121–598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40–119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

Marin, Marioara B.; Ghenea, Simona; Spiridon, Laurentiu N.; Chiritoiu, Gabriela N.; Petrescu, Andrei-Jose; Petrescu, Stefana-Maria

2012-01-01

230

Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I.  

PubMed Central

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

Kitzmuller, Claudia; Caprini, Andrea; Moore, Stuart E H; Frenoy, Jean-Pierre; Schwaiger, Eva; Kellermann, Odile; Ivessa, N Erwin; Ermonval, Myriam

2003-01-01

231

Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes.  

PubMed Central

The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.

Stancovski, I; Gonen, H; Orian, A; Schwartz, A L; Ciechanover, A

1995-01-01

232

Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I.  

PubMed

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation. PMID:12952521

Kitzmüller, Claudia; Caprini, Andrea; Moore, Stuart E H; Frénoy, Jean-Pierre; Schwaiger, Eva; Kellermann, Odile; Ivessa, N Erwin; Ermonval, Myriam

2003-12-15

233

Tetracycline degradation by ozonation in the aqueous phase: Proposed degradation intermediates and pathway  

Microsoft Academic Search

During the ozonation of tetracycline (TC) in aqueous media at pHs 2.2 and 7.0, the effects of pH variations, protonation and dissociation of functional groups and variation in free radical exposure were investigated to elucidate the transformation pathway. Liquid chromatography–triple quadrupole mass spectrometry detected around 15 ozonation products, and uncovered their production and subsequent degradation patterns. During ozonation at pH

M. Hammad Khan; Hyokwan Bae; Jin-Young Jung

2010-01-01

234

Degradation pathway(s) of chlorophyll: what has gene cloning revealed?  

PubMed

The mechanism responsible for the degreening of plants and the degradation of chlorophyll was unclear for many years. However, recent studies have identified the colorless intermediates and helped to construct a basic pathway for degradation. After the successive removal of phytol and Mg21 from the chlorophyll molecule by chlorophyllase and 'Mg dechelatase', pheophorbide a is cleaved and reduced to yield a colorless, open tetrapyrrole intermediate. After further modifications, this is finally transported to the vacuole. Cloning the genes for chlorophyllase isozymes and the reductase should help to elucidate the physiological roles of each enzyme at a molecular level. PMID:11044719

Takamiya, K I; Tsuchiya, T; Ohta, H

2000-10-01

235

RIG-G as a key mediator of the antiproliferative activity of interferon-related pathways through enhancing p21 and p27 proteins  

PubMed Central

The RIG-G gene, originally isolated from an acute promyelocytic leukemia cell line NB4, codes for a 60-kDa cytoplasmic protein that is induced by all-trans retinoic acid (ATRA) treatment along with the induction of morphological differentiation of NB4 cells. Here, we provide evidence that ectopic expression of Rig-G in U937 cells can lead to a significant accumulation of cells at G1/S transition. Growth arrest seems to occur by modulating several major cell cycle regulatory players. Interestingly, Rig-G alters JAB1 cellular distribution through interacting with this protein and increases the intracellular level of p27 by preventing it from the JAB-1-dependent and ubiquitin/proteasome-mediated degradation. Furthermore, we demonstrate a role of Rig-G for c-myc down-regulation that results in an up-regulation of p21, tightly associated with cell cycle arrest. In addition, our studies reveal that Rig-G is a direct target of STAT1, a key transcription factor in regulating IFN responses, and may be one of the first experimentally proven molecular mediators for the antiproliferative effect of IFN-?. Considering that IFN-? and ATRA synergistically inhibit growth along the intracellular pathways triggered by the two compounds in many cell types, we suggest that Rig-G may also represent one of the key molecular nodes of signaling cross-talk between ATRA and IFN-?.

Xiao, Shu; Li, Dong; Zhu, Hai-Qing; Song, Man-Gen; Pan, Xiao-Rong; Jia, Pei-Min; Peng, Lin-Ling; Dou, Ai-Xia; Chen, Guo-Qiang; Chen, Sai-Juan; Chen, Zhu; Tong, Jian-Hua

2006-01-01

236

Degradation of Ionic Pathway in PEM Fuel Cell Cathode  

SciTech Connect

The degradation of the ionic pathway throughout the catalyst layer in proton exchange membrane fuel cells was studied under an accelerated stress test of catalyst support (potential hold at 1.2 V). Electrochemical behaviors of the cathode based on graphitic mesoporous carbon supported Pt catalyst were examined using electrochemical impedance spectroscopy and cyclic voltammetry. Impedance data were plotted and expressed in the complex capacitance form to determine useful parameters in the transmission line model: the double-layer capacitance, peak frequency, and ionic resistance. Electrochemical surface area and hydrogen crossover current through the membrane were estimated from cyclic voltammogram, while cathode Faradaic resistance was compared with ionic resistance as a function of test time. It was observed that during an accelerated stress test of catalyst support, graphitic mesoporous carbon becomes hydrophilic which increases interfacial area between the ionomer and the catalyst up to 100 h. However, the ionic resistance in the catalyst layer drastically increases after 100 h with further carbon support oxidation. The underlying mechanism has been studied and it was found that significant degradation of ionic pathway throughout the catalyst layer due to catalyst support corrosion induces uneven hydration and mechanical stress in the ionomer.

Park, Seh Kyu; Shao, Yuyan; Wan, Haiying; Viswanathan, Vilayanur V.; Towne, Silas A.; Rieke, Peter C.; Liu, Jun; Wang, Yong

2011-11-12

237

Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6.  

PubMed

Stenotrophomonas maltophilia strain C6, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from creosote-contaminated sites at Hilo, Hawaii. Twenty-two metabolites of phenanthrene, covering from dihydrodiol to protocatechuic acid, were isolated and characterized. Phenanthrene was degraded via an initial dioxygenation on 1,2-, 3,4-, and 9,10-C, where the 3,4-dioxygenation and subsequent metabolisms were most dominant. The metabolic pathways were further branched by ortho- and meta-cleavage of phenanthrenediols to produce 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, and naphthalene-1,2-dicarboxylic acid. These intermediates were then transformed to naphthalene-1,2-diol. 1-Hydroxy-2-naphthoic acid was also degraded via a direct ring cleavage. Naphthalene-1,2-diol underwent primarily ortho-cleavage to produce trans-2-carboxycinnamic acid and then to form phthalic acid, 4,5-dihydroxyphthalic acid and protocatechuic acid. Accumulation of salicylic acid in prolonged incubation indicated that a limited extent of meta-cleavage of naphthalene-1, 2-diol also occurred. This is the first study of detailed phenanthrene metabolic pathways by Stenotrophomonas maltophilia. PMID:23539472

Gao, Shumei; Seo, Jong-Su; Wang, Jun; Keum, Young-Soo; Li, Jianqiang; Li, Qing X

2013-04-01

238

Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6  

PubMed Central

Stenotrophomonas maltophilia strain C6, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from creosote-contaminated sites at Hilo, Hawaii. Twenty-two metabolites of phenanthrene, covering from dihydrodiol to protocatechuic acid, were isolated and characterized. Phenanthrene was degraded via an initial dioxygenation on 1,2-, 3,4-, and 9,10-C, where the 3,4-dioxygenation and subsequent metabolisms were most dominant. The metabolic pathways were further branched by ortho- and meta-cleavage of phenanthrenediols to produce 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, and naphthalene-1,2-dicarboxylic acid. These intermediates were then transformed to naphthalene-1,2-diol. 1-Hydroxy-2-naphthoic acid was also degraded via a direct ring cleavage. Naphthalene-1,2-diol underwent primarily ortho-cleavage to produce trans-2-carboxycinnamic acid and then to form phthalic acid, 4,5-dihydroxyphthalic acid and protocatechuic acid. Accumulation of salicylic acid in prolonged incubation indicated that a limited extent of meta-cleavage of naphthalene-1, 2-diol also occurred. This is the first study of detailed phenanthrene metabolic pathways by Stenotrophomonas maltophilia.

Gao, Shumei; Seo, Jong-Su; Wang, Jun; Keum, Young-Soo; Li, Jianqiang; Li, Qing X.

2013-01-01

239

Photo-fenton degradation of diclofenac: identification of main intermediates and degradation pathway.  

PubMed

In recent years, the presence of pharmaceuticals in the aquatic environment has been of growing interest. These new contaminants are important because many of them are not degraded under the typical biological treatments applied in the wastewater treatment plants and represent a continuous input into the environment. Thus, compounds such as diclofenac are present in surface waters in all Europe and a crucial need for more enhanced technologies that can reduce its presence in the environment has become evident. In this sense, advanced oxidation processes (AOPs) represent a good choice for the treatment of hazardous nonbiodegradable pollutants. This work deals with the solar photodegradation of diclofenac, an antiinflammatory drug, in aqueous solutions by photo-Fenton reaction. A pilot-scale facility using a compound parabolic collector (CPC) reactor was used for this study. Results obtained show rapid and complete oxidation of diclofenac after 60 min, and total mineralization (disappearance of dissolved organic carbon, DOC) after 100 min of exposure to sunlight. Although diclofenac precipitates during the process at low pH, its degradation takes place in the homogeneous phase governed by a precipitation-redissolution-degradation process. Establishment of the reaction pathway was made possible by a thorough analysis of the reaction mixture identifying the main intermediate products generated. Gas chromatography-mass spectrometry (GC/ MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOF-MS) were used to identify 18 intermediates, in two tentative degradation routes. The main one was based on the initial hydroxylation of the phenylacetic acid moiety in the C-4 position and subsequent formation of a quinone imine derivative that was the starting point for further multistep degradation involving hydroxylation, decarboxylation, and oxidation reactions. An alternative route was based on the transient preservation of the biphenyl amino moiety that underwent a similar oxidative process of C-N bond cleavage. The proposed degradation route differs from those previously reported involving alternative degradation processes (ozonization, UV/H2O2, or photolysis), indicating that diclofenac degradation follows different pathways, depending on the treatment applied. PMID:16294867

Pérez-Estrada, Leónidas A; Malato, Sixto; Gernjak, Wolfgang; Agüera, Ana; Thurman, E Michael; Ferrer, Imma; Fernández-Alba, Amadeo R

2005-11-01

240

Regulation of ethylene biosynthesis through protein degradation  

PubMed Central

The function of hormones during plant growth, development and response to environmental stresses relies heavily upon the actions of the ubiquitin proteasome system (UPS), which selectively degrades numerous proteins. Synthesis of ethylene, a growth and stress hormone, is regulated in part by the ubiquitin-dependent degradation of the rate-limiting enzymatic protein aminocyclopropane-1-carboxylic acid synthase (ACS). Regulation of ACS protein stability, and therefore ethylene production, is mediated by non-catalytic sequences within the C-terminal extension of many ACS proteins. In this review we provide a brief overview of the E3 ligases that target ACS proteins for degradation and discuss how post-translational modification of the C-terminal extensions influence protein stability.

Lyzenga, Wendy J.; Stone, Sophia L.

2012-01-01

241

An evolutionarily conserved function of proliferating cell nuclear antigen for Cdt1 degradation by the Cul4-Ddb1 ubiquitin ligase in response to DNA damage.  

PubMed

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF(Skp2), evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage. PMID:16407242

Hu, Jian; Xiong, Yue

2006-02-17

242

Degradation of Sulphonated Azo Dye Red HE7B by Bacillus sp. and Elucidation of Degradative Pathways.  

PubMed

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

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

2014-08-01

243

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways  

SciTech Connect

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. The authors have investigate the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of {sup 18}O{sub 2} indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1, 2-dihydroxy-3-nitrocyclohexa-3, 5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation.

Haigler, B.E.; Spain, J.C. (Air Force Civil Engineering Support Agency, Tyndall AFB, FL (United States))

1991-11-01

244

Necdin Promotes Ubiquitin-Dependent Degradation of PIAS1 SUMO E3 Ligase  

PubMed Central

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

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

2014-01-01

245

Necdin Promotes Ubiquitin-Dependent Degradation of PIAS1 SUMO E3 Ligase.  

PubMed

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

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

2014-01-01

246

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTR?F508  

PubMed Central

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR?F508, by specifically promoting ubiquitylation of CFTR?F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR?F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR?F508 and catalyzes further polyubiquitylation of CFTR?F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR?F508.

Morito, Daisuke; Hirao, Kazuyoshi; Oda, Yukako; Hosokawa, Nobuko; Tokunaga, Fuminori; Cyr, Douglas M.; Tanaka, Keiji; Iwai, Kazuhiro

2008-01-01

247

Extended Anti-inflammatory Action of a Degradation-resistant Mutant of Cell-penetrating Suppressor of Cytokine Signaling 3*  

PubMed Central

Suppressor of cytokine signaling 3 (SOCS3) regulates the proinflammatory cytokine signaling mediated by the JAK/STAT signaling pathway. SOCS3 is rapidly induced and then targeted to the ubiquitin-proteasome pathway via a mechanism that requires the C-terminal SOCS box. Due to its rapid turnover, the intracellular stores of SOCS3 seem insufficient to control acute or protracted inflammatory diseases. Previously, we developed an intracellular protein therapy that uses a recombinant cell-penetrating form of SOCS3 (CP-SOCS3) to inhibit the JAK/STAT pathway and prevent cytokine-mediated lethal inflammation and apoptosis of the liver (Jo, D., Liu, D., Yao, S., Collins, R. D., and Hawiger, J. (2005) Nat. Med. 11, 892–898). The potent anti-inflammatory and cytoprotective activity of CP-SOCS3 prompted us to analyze its intracellular turnover, as compared with that of endogenous SOCS3 protein induced in macrophages by the proinflammatory agonists, interferon-? and lipopolysaccharide. We found that the half-life (t½) of endogenous SOCS3 is 0.7 h in activated macrophages, compared with a t½ of 6.2 h for recombinant CP-SOCS3. Deletion of the SOCS box in CP-SOCS3 renders it more resistant to proteasomal degradation, extending its t½ to 29 h. Consequently, this SOCS box-deleted form of CP-SOCS3 displays persistent inhibitory activity for 24 h toward interferon-?- and lipopolysaccharide-induced cytokine and chemokine production. Compared with the wild-type suppressor, this gain-of-function CP-SOCS3 mutant provides a longer acting inhibitor of cytokine signaling, a feature that offers a clear advantage for the intracellular delivery of proteins to treat acute or protracted inflammatory diseases.

Fletcher, Tynetta C.; DiGiandomenico, Antonio; Hawiger, Jacek

2010-01-01

248

Degradation of diclofenac by ultrasonic irradiation: Kinetic studies and degradation pathways.  

PubMed

Diclofenac (DCF) is a widely used anti-inflammatory drug found in various water bodies, posing threats to human health. In this research, the effects of ultrasonic irradiation at 585kHz on the degradation of DCF were studied under the air, oxygen, argon, and nitrogen saturated conditions. First, the dechlorination efficiencies under the air, oxygen, argon, and nitrogen saturated conditions were calculated to be 67%, 60%, 53% and 59%. Second, there was full mineralization of nitrogen during DCF degradation under the air, oxygen, and argon saturated conditions, but no mineralization of nitrogen under the nitrogen-saturated condition. Different from nitrogen, only partial mineralization of carbon occurred under the four gas-saturated conditions. Third, OH scavengers were added to derive the rate constants in the three reaction zones: cavitation bubble, supercritical interface, and bulk solution. Comparison of the constants indicated that DCF degradation was not limited to the bulk solution as conventionally assumed. Oxidation in the supercritical interface played a dominant role under the air and oxygen saturated conditions, while OH reactions in the cavitation bubble and/or bulk solution were dominant under the nitrogen and argon saturated conditions. After the addition of H2O2, reactions in the cavitation bubble and bulk solution kept their dominant roles under the nitrogen and argon saturated conditions, while reaction in the supercritical interface decreased under the air and oxygen saturated conditions. Finally, LC-MS analysis was used to derive the by-products and propose the main pathways of DCF degradation by ultrasonic irradiation. PMID:25065805

Nie, Er; Yang, Mo; Wang, Dong; Yang, Xiaoying; Luo, Xingzhang; Zheng, Zheng

2014-10-01

249

Integrated control of protein degradation in C. elegans muscle.  

PubMed

Protein degradation is a fundamental cellular process, the genomic control of which is incompletely understood. The advent of transgene-coded reporter proteins has enabled the development of C. elegans into a model for studying this problem. The regulation of muscle protein degradation is surprisingly complex, integrating multiple signals from hypodermis, intestine, neurons and muscle itself. Within the muscle, degradation is executed by separately regulated autophagy-lysosomal, ubiquitin-proteasome and calpain-mediated systems. The signal-transduction mechanisms, in some instances, involve modules previously identified for their roles in developmental processes, repurposed in terminally differentiated muscle to regulate the activities of pre-formed proteins. Here we review the genes, and mechanisms, which appear to coordinately control protein degradation within C. elegans muscle. We also consider these mechanisms in the context of development, physiology, pathophysiology and disease models. PMID:23457662

Lehmann, Susann; Shephard, Freya; Jacobson, Lewis A; Szewczyk, Nathaniel J

2012-07-01

250

Hepatitis C Virus Activates the mTOR/S6K1 Signaling Pathway in Inhibiting IRS-1 Function for Insulin Resistance  

PubMed Central

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.

Bose, Sandip K.; Shrivastava, Shubham; Meyer, Keith; Ray, Ratna B.

2012-01-01

251

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.  

PubMed

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes. PMID:1781679

Haigler, B E; Spain, J C

1991-11-01

252

Proteasome Inhibitors Block a Late Step in Lysosomal Transport of Selected Membrane but not Soluble Proteins  

PubMed Central

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.

van Kerkhof, Peter; dos Santos, Cristina M. Alves; Sachse, Martin; Klumperman, Judith; Bu, Guojun; Strous, Ger J.

2001-01-01

253

A New 4-Nitrotoluene Degradation Pathway in a Mycobacterium Strain  

PubMed Central

Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (?max, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage.

Spiess, Tilmann; Desiere, Frank; Fischer, Peter; Spain, Jim C.; Knackmuss, Hans-Joachim; Lenke, Hiltrud

1998-01-01

254

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

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

255

Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy  

PubMed Central

The perihydroxylated perylene quinone hypericin has been reported to possess potent anti-metastatic and antiangiogenic activities, generated by targeting diverse crossroads of cancer-promoting processes via unique mechanisms. Hypericin is the only known exogenous reagent that can induce forced poly-ubiquitination and accelerated degradation of heat shock protein 90 (Hsp90) in cancer cells. Hsp90 client proteins are thereby destabilized and rapidly degraded. Hsp70 client proteins may potentially be also affected via preventing formation of hsp90-hsp70 intermediate complexes. We show here that hypericin also induces enhanced degradation of hypoxia-inducible factor 1? (HIF-1?) in two human tumor cell lines, U87-MG glioblastoma and RCC-C2VHL?/? renal cell carcinoma and in the non-malignant ARPE19 retinal pigment epithelial cell line. The hypericin-accelerated turnover of HIF-1?, the regulatory precursor of the HIF-1 transcription factor which promotes hypoxic stress and angiogenic responses, overcomes the physiologic HIF-1? protein stabilization which occurs in hypoxic cells. The hypericin effect also eliminates the high HIF-1? levels expressed constitutively in the von-Hippel Lindau protein (pVHL)-deficient RCC-C2VHL?/? renal cell carcinoma cell line. Unlike the normal ubiquitin-proteasome pathway-dependent turnover of HIF-? proteins which occurs in normoxia, the hypericin-induced HIF-1? catabolism can occur independently of cellular oxygen levels or pVHL-promoted ubiquitin ligation of HIF-1?. It is mediated by lysosomal cathepsin-B enzymes with cathepsin-B activity being optimized in the cells through hypericin-mediated reduction in intracellular pH. Our findings suggest that hypericin may potentially be useful in preventing growth of tumors in which HIF-1? plays pivotal roles, and in pVHL ablated tumor cells such as renal cell carcinoma through elimination of elevated HIF-1? contents in these cells, scaling down the excessive angiogenesis which characterizes these tumors.

Livnat, Tami; Weinberger, Dov; Lavie, Gad

2011-01-01

256

Acetylation of Drosha on the N-Terminus Inhibits Its Degradation by Ubiquitination  

PubMed Central

The RNase III enzyme Drosha initiates microRNA (miRNA) biogenesis in the nucleus by cleaving primary miRNA transcripts into shorter precursor molecules that are subsequently exported into the cytoplasm for further processing. While numerous disease states appear to be associated with aberrant expression of Drosha, the molecular mechanisms that regulate its protein levels are largely unknown. Here, we report that ubiquitination and acetylation regulate Drosha protein levels oppositely. Deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NIA) increase Drosha protein level as measured by western blot but have no effects on its mRNA level in HEK293T cells. TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells. Treatment of AGS and HEK293T cells with proteasome inhibitors MG132 or Omuralide increases Drosha protein levels. Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5. Acetylation of Drosha competes with its ubquitination, inhibiting the degradation induced by the ubiquitin-proteasome pathway, thereby increasing Drosha protein levels. Infection of the gastric mucosa AGS cells by H. pylori, the gastric cancer associated carcinogen, leads to the ubiquitination and reduction of Drosha protein levels. H. pylori infection of AGS cells has no significant effects on Drosha mRNA levels. Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.

Tang, Xiaoli; Wen, Sicheng; Zheng, Dong; Tucker, Lynne; Cao, Lulu; Pantazatos, Dennis; Moss, Steven F.; Ramratnam, Bharat

2013-01-01

257

Acetylation of drosha on the N-terminus inhibits its degradation by ubiquitination.  

PubMed

The RNase III enzyme Drosha initiates microRNA (miRNA) biogenesis in the nucleus by cleaving primary miRNA transcripts into shorter precursor molecules that are subsequently exported into the cytoplasm for further processing. While numerous disease states appear to be associated with aberrant expression of Drosha, the molecular mechanisms that regulate its protein levels are largely unknown. Here, we report that ubiquitination and acetylation regulate Drosha protein levels oppositely. Deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NIA) increase Drosha protein level as measured by western blot but have no effects on its mRNA level in HEK293T cells. TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells. Treatment of AGS and HEK293T cells with proteasome inhibitors MG132 or Omuralide increases Drosha protein levels. Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5. Acetylation of Drosha competes with its ubquitination, inhibiting the degradation induced by the ubiquitin-proteasome pathway, thereby increasing Drosha protein levels. Infection of the gastric mucosa AGS cells by H. pylori, the gastric cancer associated carcinogen, leads to the ubiquitination and reduction of Drosha protein levels. H. pylori infection of AGS cells has no significant effects on Drosha mRNA levels. Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis. PMID:24009686

Tang, Xiaoli; Wen, Sicheng; Zheng, Dong; Tucker, Lynne; Cao, Lulu; Pantazatos, Dennis; Moss, Steven F; Ramratnam, Bharat

2013-01-01

258

Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells.  

PubMed

Arsenic trioxide (As(2)O(3)), used to treat promyelocytic leukemia, triggers cell death through unknown mechanisms. To further our understanding of As(2)O(3)-induced death, we analyzed its effects on transforming growth factor-? (TGF?) signaling mediators in ovarian cells. Dysregulated TGF? signaling is a characteristic of ovarian cancers. As(2)O(3) reduced the protein expression of EVI1, TAK1, SMAD2/3, and TGF?RII while increasing SnoN/SkiL. EVI1 protein was modulated by treatment with the proteasome inhibitors, MG132 and PS-341/Velcade, suggesting that degradation occurs through the ubiquitin-proteasome pathway. The sensitivity of ovarian cells to As(2)O(3)-induced apoptosis correlated with expression of multidrug resistance protein 1. Interestingly, expression of SnoN was similar to LC3-II (autophagy marker), which increased with induction of cytoplasmic vacuolation preceding apoptosis. These vesicles were identified as autophagosomes based on transmission electron microscopy and immunofluorescence staining with EGFP-LC3. The addition of N-acetyl-L-cysteine (ROS scavenger) to As(2)O(3)-treated cells reversed changes in SnoN protein and the autophagic/apoptotic response. In contrast to beclin-1 knockdown, siRNA targeting ATG5, ATG7, and hVps34 markedly reduced autophagy in As(2)O(3)-treated ovarian carcinoma cells. Further, treatment with SnoN siRNA markedly decreased LC3-II levels and increased PARP degradation (an apoptosis marker). Collectively, these findings suggest that As(2)O(3) induces a beclin-1-independent autophagic pathway in ovarian carcinoma cells and implicates SnoN in promoting As(2)O(3)-mediated autophagic cell survival. PMID:20508647

Smith, D M; Patel, S; Raffoul, F; Haller, E; Mills, G B; Nanjundan, M

2010-12-01

259

Chromium-Insulin Reduces Insulin Clearance and Enhances Insulin Signaling by Suppressing Hepatic Insulin-Degrading Enzyme and Proteasome Protein Expression in KKAy Mice  

PubMed Central

JDS–chromium–insulin (CRI)-003 is a novel form of insulin that has been directly conjugated with chromium (Cr) instead of zinc. Our hypothesis was that CRI enhances insulin’s effects by altering insulin-degrading enzyme (IDE) and proteasome enzymes. To test this hypothesis, we measured hepatic IDE content and proteasome parameters in a diabetic animal model. Male KKAy mice were randomly divided into three groups (n?=?8/group); Sham (saline), human regular insulin (Reg-In), and chromium conjugated human insulin (CRI), respectively. Interventions were initiated at doses of 2?U insulin/kg body weight daily for 8-weeks. Plasma glucose and insulin were measured. Hepatic IDE, proteasome, and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments significantly reduced glucose concentrations and increased insulin levels compared with the Sham group, CRI significantly reduced glucose at 4 and 6?h relative to Reg-In (P?degradation of IRS-1 and IRS-2 by suppressing ubiquitin-proteasome pathway in diabetic mice.

Wang, Zhong Q.; Yu, Yongmei; Zhang, Xian H.; Komorowski, James

2014-01-01

260

Insulin-degrading enzyme is exported via an unconventional protein secretion pathway  

Microsoft Academic Search

Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid ?-protein (A?), and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD). Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s)

Ji Zhao; Lilin Li; Malcolm A Leissring

2009-01-01

261

Fenretinide Induces Ubiquitin-Dependent Proteasomal Degradation of Stearoyl-CoA Desaturase in Human Retinal Pigment Epithelial Cells.  

PubMed

Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases. J. Cell. Physiol. 229: 1028-1038, 2014. Published 2013. This article is a U.S. Government work and is in the public domain in the USA. PMID:24357007

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

2014-08-01

262

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway  

Microsoft Academic Search

Background: In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase

Laurence Serre; Alain Sailland; Denise Sy; Philippe Boudec; Anne Rolland; Eva Pebay-Peyroula; Claudine Cohen-Addad

1999-01-01

263

Characterization of a New Pathway for Epichlorohydrin Degradation by Whole Cells of Xanthobacter Strain Py2  

PubMed Central

The degradation of epichlorohydrin (3-chloropropylene oxide or 1-chloro-2,3-epoxypropane) by whole-cell suspensions of Xanthobacter strain Py2 was investigated. Cell suspensions prepared from cultures grown with propylene as the carbon source readily degraded epichlorohydrin. The ability to degrade epichlorohydrin correlated with the expression of enzymes involved in alkene and epoxide metabolism, since cell suspensions prepared from cultures grown with glucose or acetone, in which the enzymes of alkene and epoxide oxidation are not expressed, did not degrade epichlorohydrin. The alkene monooxygenase-specific inhibitor propyne had no effect on the degradation of epichlorohydrin, demonstrating that alkene monooxygenase is not involved in epichlorohydrin conversion. The interaction of epichlorohydrin and epibromohydrin with the epoxidase which catalyzes aliphatic epoxide conversions was established by showing that the epihalohydrins were specific and potent inhibitors of propylene oxide-dependent O(inf2) consumption by cell suspensions. The rates of degradation of epoxides in whole-cell suspensions decreased in the series propylene oxide > epifluorohydrin > epichlorohydrin > epibromohydrin. The pathway of epichlorohydrin degradation was investigated and found to proceed with stoichiometric dechlorination of epichlorohydrin. The first detectable product of epichlorohydrin degradation was chloroacetone. Chloroacetone was further degraded by the cell suspensions, and in the process, acetone was formed as a nonstoichiometric product. Acetone was further degraded by the cell suspensions with enzymes apparently induced by the accumulation of acetone. The metabolism of allyl chloride (3-chloropropylene) by propylene-grown cells was initiated by alkene monooxygenase and proceeded through epichlorohydrin, chloroacetone, and acetone as intermediate degradation products. These studies reveal a new pathway for halogenated epoxide degradation which involves halogenated and aliphatic ketones as well as other unidentified intermediates and which is unique from previously characterized hydrolytic degradative pathways.

Small, F. J.; Tilley, J. K.; Ensign, S. A.

1995-01-01

264

Characterization of a new pathway for epichlorohydrin degradation by whole cells of xanthobacter strain py2.  

PubMed

The degradation of epichlorohydrin (3-chloropropylene oxide or 1-chloro-2,3-epoxypropane) by whole-cell suspensions of Xanthobacter strain Py2 was investigated. Cell suspensions prepared from cultures grown with propylene as the carbon source readily degraded epichlorohydrin. The ability to degrade epichlorohydrin correlated with the expression of enzymes involved in alkene and epoxide metabolism, since cell suspensions prepared from cultures grown with glucose or acetone, in which the enzymes of alkene and epoxide oxidation are not expressed, did not degrade epichlorohydrin. The alkene monooxygenase-specific inhibitor propyne had no effect on the degradation of epichlorohydrin, demonstrating that alkene monooxygenase is not involved in epichlorohydrin conversion. The interaction of epichlorohydrin and epibromohydrin with the epoxidase which catalyzes aliphatic epoxide conversions was established by showing that the epihalohydrins were specific and potent inhibitors of propylene oxide-dependent O(inf2) consumption by cell suspensions. The rates of degradation of epoxides in whole-cell suspensions decreased in the series propylene oxide > epifluorohydrin > epichlorohydrin > epibromohydrin. The pathway of epichlorohydrin degradation was investigated and found to proceed with stoichiometric dechlorination of epichlorohydrin. The first detectable product of epichlorohydrin degradation was chloroacetone. Chloroacetone was further degraded by the cell suspensions, and in the process, acetone was formed as a nonstoichiometric product. Acetone was further degraded by the cell suspensions with enzymes apparently induced by the accumulation of acetone. The metabolism of allyl chloride (3-chloropropylene) by propylene-grown cells was initiated by alkene monooxygenase and proceeded through epichlorohydrin, chloroacetone, and acetone as intermediate degradation products. These studies reveal a new pathway for halogenated epoxide degradation which involves halogenated and aliphatic ketones as well as other unidentified intermediates and which is unique from previously characterized hydrolytic degradative pathways. PMID:16535000

Small, F J; Tilley, J K; Ensign, S A

1995-04-01

265

Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation  

PubMed Central

Unilateral denervation (DNV) of rat diaphragm muscle increases protein synthesis at 3 days after DNV (DNV-3D) and degradation at DNV-5D, such that net protein breakdown is evident by DNV-5D. On the basis of existing models of protein balance, we examined DNV-induced changes in Akt, AMP-activated protein kinase (AMPK), and ERK½ activation, which can lead to increased protein synthesis via mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K), glycogen synthase kinase-3? (GSK3?), or eukaryotic initiation factor 4E (eIF4E), and increased protein degradation via forkhead box protein O (FoxO). Protein phosphorylation was measured using Western analyses through DNV-5D. Akt phosphorylation decreased at 1 h and 6 h after DNV compared with sham despite decreased AMPK phosphorylation. Both Akt and AMPK phosphorylation returned to sham levels by DNV-1D. Phosphorylation of their downstream effector mTOR (Ser2481) did not change at any time point after DNV, and phosphorylated p70S6K and eIF4E-binding protein 1 (4EBP1) increased only by DNV-5D. In contrast, ERK½ phosphorylation and its downstream effector eIF4E increased 1.7-fold at DNV-1D and phosphorylated GSK3? increased 1.5-fold at DNV-3D (P < 0.05 for both comparisons). Thus, following DNV there are differential effects on protein synthetic pathways with preferential activation of GSK3? and eIF4E over p70S6K. FoxO1 nuclear translocation occurred by DNV-1D, consistent with its role in increasing expression of atrogenes necessary for subsequent ubiquitin-proteasome activation evident by DNV-5D. On the basis of our results, increased protein synthesis following DNV is associated with changes in ERK½-dependent pathways, but protein degradation results from downregulation of Akt and nuclear translocation of FoxO1. No single trigger is responsible for protein balance following DNV. Protein balance in skeletal muscle depends on multiple synthetic/degradation pathways that should be studied in concert.

Argadine, Heather M.; Mantilla, Carlos B.; Zhan, Wen-Zhi

2011-01-01

266

Degradation of aromatic compounds and degradative pathway of 4-nitrocatechol by Ochrobactrum sp. B2  

Microsoft Academic Search

The potential capacity of a soil methyl parathion-degrading bacterium strain, Ochrobactrum sp. B2, for degrading various aromatic compounds were investigated. The results showed B2 was capable of degrading diverse aromatic compounds, but amino-substituted benzene compounds, at a concentration up to 100 mg L in 4 days. B2 could use 4-nitrocatechol (4-NC) as a sole carbon and energy source with release

Qiuzan Zhong; Haiyan Zhang; Wenqin Bai; Mei Li; Baotong Li; Xinghui Qiu

2007-01-01

267

Proteasome Inhibitors Activate Autophagy Involving Inhibition of PI3K-Akt-mTOR Pathway as an Anti-Oxidation Defense in Human RPE Cells  

PubMed Central

The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE), ARPE-19 cells, proteasome inhibitors, clasto-lactacystin?-lactone (LA) or epoxomicin (Epo), at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3) from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf). Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt), a downstream target of phosphatidylinositol-3-kinases (PI3K), and mammalian target of rapamycin (mTOR) in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3) or 4-hydroxynonenal (4-HNE). Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.

Sun, Lin; Li, Yiping; Qu, Jia; Snider, Barbara Joy; Wu, Shengzhou

2014-01-01

268

Novel pathway for degradation of protocatechuic acid in Bacillus species.  

PubMed Central

A species of Bacillus, tentatively identified as B. circulans, degrades protocatechuic acid by a novel reaction involving meta-fission between C2 and C3 of the benzene nucleus. 2-Hydroxymuconic semialdehyde is then degraded to pyruvate and acetaldehyde by enzymatic reactions described in previous work. Protocatechuate 2,3-oxygenase exhibits a rather narrow substrate specificity; the methyl and ethyl esters of protocatechuic acid are oxidized, but other substrates for ring-fission oxygenases, notably catechol, gallic acid, and homoprotocatechuic acid, are not attached.

Crawford, R L

1975-01-01

269

Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas  

PubMed Central

Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

Lwin, Tint; Hazlehurst, Lori A.; Dessureault, Sophie; Lai, Raymond; Bai, Wenlong; Sotomayor, Eduardo; Moscinski, Lynn C.; Dalton, William S.

2007-01-01

270

Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin\\/proteasome pathway and epidermal growth factor receptor tyrosine kinase  

Microsoft Academic Search

Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis.

Markus Braun-Falco; Angelika Eisenried; Hildegard Büning; Johannes Ring

2005-01-01

271

Evolution of efficient pathways for degradation of anthropogenic chemicals  

Microsoft Academic Search

Anthropogenic compounds used as pesticides, solvents and explosives often persist in the environment and can cause toxicity to humans and wildlife. The persistence of anthropogenic compounds is due to their recent introduction into the environment; microbes in soil and water have had relatively little time to evolve efficient mechanisms for degradation of these new compounds. Some anthropogenic compounds are easily

Shelley D Copley

2009-01-01

272

Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease  

Microsoft Academic Search

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin–proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been

Brian Bauereis; William E. Haskins; Richard G. LeBaron; Robert Renthal

2011-01-01

273

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene  

Microsoft Academic Search

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. The authors report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii

SHIRLEY F. NISHINO; GEORGE C. PAOLI; JIM C. SPAIN

2000-01-01

274

Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1  

Microsoft Academic Search

The ubiquitin\\/proteasome pathway has been implicated in a wide variety of cellular processes and the number of substrates degraded by the proteasome is impressive. Most prominently, the stability of a large number of transcription factors is regulated by ubiquitination. To elucidate pathways regulated by the proteasome, gene expression profiles were generated, comparing changes of mRNA expression of 7900 genes from

Johann Zimmermann; Dirk Erdmann; Isabelle Lalande; Rita Grossenbacher; Maria Noorani; Peter Fürst

2000-01-01

275

Microbial degradation of fomesafen by a newly isolated strain Pseudomonas zeshuii BY-1 and the biochemical degradation pathway.  

PubMed

Fomesafen is a diphenyl ether herbicide used to control the growth of broadleaf weeds in bean fields. Although the degradation of fomesafen in soils was thought to occur primarily by microbial activity, little was known about the kinetic and metabolic behaviors of this herbicide. This paper reported the capability of the newly isolated strain Pseudomonas zeshuii BY-1 to use fomesafen as the sole source of carbon in pure culture for its growth. Up to 88.7% of 50 mg of L(-1) fomesafen was degraded by this bacterium in mineral medium within 3 days. Strain BY-1 could also degrade other diphenyl ethers, including lactofen, acifluorfen, and fluoroglycofen. During the fomesafen degradation, five metabolites were detected and identified by liquid chromatography-mass spectrometry and tandem mass spectrometry. The primary degradation pathway of fomesafen might be the reduction of the nitro group to an amino group, followed by the acetylation of the amino derivative, dechlorination, and cleavage of the S-N bond. The addition of the BY-1 stain into soils treated with fomesafen resulted in a higher degradation rate than that observed in uninoculated soils, and the bacteria community in contaminated soil recovered after inoculation of the BY-1 stain. On the basis of these results, strain P. zeshuii BY-1 has the potential to be used in the bioremediation of fomesafen-contaminated soils. PMID:22757645

Feng, Zhao-zhong; Li, Qin-fen; Zhang, Jun; Zhang, Jing; Huang, Xing; Lu, Peng; Li, Shun-peng

2012-07-25

276

High-resolution live-cell imaging reveals novel cyclin A2 degradation foci involving autophagy.  

PubMed

Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis relies on the ubiquitin-proteasome system (UPS). Using high-resolution microscopic imaging, we find that cyclin A2 persists beyond metaphase. Indeed, we identify a novel cyclin-A2-containing compartment that forms dynamic foci. Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) analyses show that cyclin A2 ubiquitylation takes place predominantly in these foci before spreading throughout the cell. Moreover, inhibition of autophagy in proliferating cells induces the stabilisation of a subset of cyclin A2, whereas induction of autophagy accelerates the degradation of cyclin A2, thus showing that autophagy is a novel regulator of cyclin A2 degradation. PMID:24634511

Loukil, Abdelhalim; Zonca, Manuela; Rebouissou, Cosette; Baldin, Véronique; Coux, Olivier; Biard-Piechaczyk, Martine; Blanchard, Jean-Marie; Peter, Marion

2014-05-15

277

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

EPA Science Inventory

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

278

Degradation of methamidophos by Hyphomicrobium species MAP1 and the biochemical degradation pathway  

Microsoft Academic Search

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative\\u000a methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It\\u000a was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of\\u000a methamidophos. Further, this strain could utilize

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

2010-01-01

279

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

Microsoft Academic Search

The purpose of this study was to perform proteome analysis of Acinetobacter sp. KS-1, a bacterium capable of degrading benzoate as a sole carbon source. In order to understand the benzoate degradation pathway used by strain KS-1, proteomes of benzoate-cultured and succinate-cultured KS-1 were comparatively analyzed by two dimensional gel electrophoresis (2-DE). Eighteen protein spots proteins were exclusively induced from

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

2003-01-01

280

Degradation of nitroaromatic compounds: a model to study evolution of metabolic pathways.  

PubMed

Although many nitroaromatic compounds have been in nature for only a few decades, bacteria have already evolved the ability to metabolize them. Both horizontal transfer of genes and mutagenesis induced under stressful conditions might facilitate evolution of new catabolic pathways. Nitrotoluene degradation pathways are supposedly derived from an ancestral naphthalene degradation pathway. The 2-nitrotoluene degradation genes in Acidovorax sp. strain JS42 are controlled by the transcriptional activator NtdR, which differs from NagR, the activator of the naphthalene degradation operon in Ralstonia sp. strain U2, by only five amino acids. Both regulators respond to salicylate, an intermediate of naphthalene degradation, but NtdR also recognizes a wide range of nitroaromatic compounds. In this issue of Molecular Microbiology, Ju et al. present results of site-directed mutagenesis of NtdR and NagR and show how the nitrotoluene-responsive regulator NtdR can be generated from a NagR-like ancestor by only a few mutations. The reconstructed hypothetical pathway for the evolution of NtdR from NagR demonstrates stepwise broadening of the effector range of the evolving protein without loss of the original activity. These results provide strong evidence for the idea that promiscuity of proteins is an important step in the evolution of new functions. PMID:19818019

Kivisaar, Maia

2009-11-01

281

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

PubMed

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

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

2014-04-01

282

Photolysis of Chlorantraniliprole and Cyantraniliprole in Water and Soil: Verification of Degradation Pathways via Kinetics Modeling.  

PubMed

Photodegradation of [(14)C]-chlorantraniliprole (CLAP) and [(14)C]-cyantraniliprole (CNAP) was investigated in sterile buffer solutions, in natural water, and on soil surfaces. Both compounds displayed rapid degradation in aqueous buffers when exposed to light at concentrations which could result from direct overspray to a shallow water body. While the main products observed had analogous structures, a substantial difference was noted in the rate of degradation of the two compounds despite minimal differences in their structures. Transformations observed were primarily intramolecular rearrangements and degradations resulting from addition of hydroxyl radicals leading to molecular cleavage. Some of the degradation products were transient, and several degradates had isomeric molecular compositions. The sequence of transformations was established definitively with the help of kinetics modeling. Utility of kinetics analysis in verification of the proposed pathways is illustrated. PMID:24971760

Sharma, Ashok K; Zimmerman, William T; Singles, Suzanne K; Malekani, Kalumbu; Swain, Scott; Ryan, David; Mcquorcodale, Gordon; Wardrope, Laura

2014-07-16

283

Mechanochemical degradation of tetrabromobisphenol A: performance, products and pathway.  

PubMed

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant (BFR), which has received more and more concerns due to its high lipophilicity, persistency and endocrine disrupting property in the environment. Considering the possible need for the safe disposal of TBBPA containing wastes in the future, the potential of mechanochemical (MC) destruction as a promising non-combustion technology was investigated in this study. TBBPA was co-ground with calcium oxide (CaO) or the mixture of iron powder and quartz sand (Fe+SiO(2)) in a planetary ball mill at room temperature. The method of Fe+SiO(2) destructed over 98% of initial TBBPA after 3h and acquired 95% debromination rate after 5h, which showed a better performance than the CaO method. Raman spectra and Fourier transform infrared spectroscopy (FTIR) demonstrated the generation of inorganic carbon with the disappearance of benzene ring and CBr bond, indicating the carbonization and debromination process during mechanochemical reaction. LC-MS-MS screening showed that the intermediates of the treatment with Fe+SiO(2) were tri-, bi-, mono-brominated BPA, BPA and other fragments. Finally all the intermediates were also destroyed after 5h grinding. The bromine balance was calculated and a possible reaction pathway was proposed. PMID:23158692

Zhang, Kunlun; Huang, Jun; Zhang, Wang; Yu, Yunfei; Deng, Shubo; Yu, Gang

2012-12-01

284

Specific small molecule inhibitors of Skp2-mediated p27 degradation.  

PubMed

In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein, Cks1, promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type-specific blocks in the G1 or G2/M phases. Designing SCF-Skp2-specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis. PMID:23261596

Wu, Lily; Grigoryan, Arsen V; Li, Yunfeng; Hao, Bing; Pagano, Michele; Cardozo, Timothy J

2012-12-21

285

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

PubMed Central

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

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

2011-01-01

286

Specific Small Molecule Inhibitors of Skp2-Mediated p27 Degradation  

PubMed Central

SUMMARY In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein Cks1 promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type specific blocks in the G1 or G2/M phases. Designing SCF-Skp2 specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.

Wu, Lily; Grigoryan, Arsen V.; Li, Yunfeng; Hao, Bing; Pagano, Michele; Cardozo, Timothy

2012-01-01

287

The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein  

PubMed Central

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.

Kwon, Deborah Y.; Dimitriadi, Maria; Terzic, Barbara; Cable, Casey; Hart, Anne C.; Chitnis, Ajay; Fischbeck, Kenneth H.; Burnett, Barrington G.

2013-01-01

288

New hydrocarbon degradation pathways in the microbial metagenome from Brazilian petroleum reservoirs.  

PubMed

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

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

2014-01-01

289

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

PubMed Central

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

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

2014-01-01

290

Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis.  

PubMed

Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases. PMID:12692559

Ditzel, Mark; Wilson, Rebecca; Tenev, Tencho; Zachariou, Anna; Paul, Angela; Deas, Emma; Meier, Pascal

2003-05-01

291

Cooperative catabolic pathways within an atrazine-degrading enrichment culture isolated from soil.  

PubMed

Atrazine degradation previously has been shown to be carried out by individual bacterial species or by relatively simple consortia that have been isolated using enrichment cultures. Here, the degradative pathway for atrazine was examined for a complex 8-membered enrichment culture. The species composition of the culture was determined by PCR-DGGE. The bacterial species included Agrobacterium tumefaciens, Caulobacter crescentus, Pseudomonas putida, Sphingomonas yaniokuyae, Nocardia sp., Rhizobium sp., Flavobacterium oryzihabitans, and Variovorax paradoxus. All of the isolates were screened for the presence of known genes that function for atrazine degradation including atzA,-B,-C,-D,-E,-F and trzD,-N. Dechlorination of atrazine, which was obligatory for complete mineralization, was carried out exclusively by Nocardia sp., which contained the trzN gene. Following dechlorination, the resulting product, hydroxyatrazine was further degraded via two separate pathways. In one pathway Nocardia converted hydroxyatrazine to N-ethylammelide via an unidentified gene product. In the second pathway, hydroxyatrazine generated by Nocardia sp. was hydrolyzed to N-isopropylammelide by Rhizobium sp., which contained the atzB gene. Each member of the enrichment culture contained atzC, which is responsible for ring cleavage, but none of the isolates carried the atzD,-E, or -F genes. Each member further contained either trzD or exhibited urease activity. The enrichment culture was destabilized by loss of Nocardia sp. when grown on ethylamine, ethylammelide, and cyanuric acid, after which the consortium was no longer able to degrade atrazine. The analysis of this enrichment culture highlights the broad level bacterial community interactions that may be involved in atrazine degradation in nature. PMID:16329946

Smith, Daniel; Alvey, Sam; Crowley, David E

2005-07-01

292

Proteasome inhibition: a new anti-inflammatory strategy  

Microsoft Academic Search

The ubiquitin-proteasome pathway has a central role in the selective degradation of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. Consequently proteasome inhibition is a potential treatment option for cancer and inflammatory conditions. Thus far, proof of principle has been obtained from studies

Peter J. Elliott; Thomas Matthias Zollner; Wolf-Henning Boehncke

2003-01-01

293

Ubiquitin domain proteins in disease  

Microsoft Academic Search

The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable

Louise Madsen; Andrea Schulze; Michael Seeger; Rasmus Hartmann-Petersen

2007-01-01

294

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

ERIC Educational Resources Information Center

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

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

2008-01-01

295

Roles of Protein Ubiquitination and Degradation Kinetics in Biological Oscillations  

PubMed Central

Protein ubiquitination and degradation play important roles in many biological functions and are associated with many human diseases. It is well known that for biochemical oscillations to occur, proper degradation rates of the participating proteins are needed. In most mathematical models of biochemical reactions, linear degradation kinetics has been used. However, the degradation kinetics in real systems may be nonlinear, and how nonlinear degradation kinetics affects biological oscillations are not well understood. In this study, we first develop a biochemical reaction model of protein ubiquitination and degradation and calculate the degradation rate against the concentration of the free substrate. We show that the protein degradation kinetics mainly follows the Michaelis-Menten formulation with a time delay caused by ubiquitination and deubiquitination. We then study analytically how the Michaelis-Menten degradation kinetics affects the instabilities that lead to oscillations using three generic oscillation models: 1) a positive feedback mediated oscillator; 2) a positive-plus-negative feedback mediated oscillator; and 3) a negative feedback mediated oscillator. In all three cases, nonlinear degradation kinetics promotes oscillations, especially for the negative feedback mediated oscillator, resulting in much larger oscillation amplitudes and slower frequencies than those observed with linear kinetics. However, the time delay due to protein ubiquitination and deubiquitination generally suppresses oscillations, reducing the amplitude and increasing the frequency of the oscillations. These theoretical analyses provide mechanistic insights into the effects of specific proteins in the ubiquitination-proteasome system on biological oscillations.

Xu, Lida; Qu, Zhilin

2012-01-01

296

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

PubMed

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

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

2014-02-01

297

Degradation of diclofenac by advanced oxidation and reduction processes: kinetic studies, degradation pathways and toxicity assessments.  

PubMed

Many pharmaceutical compounds and metabolites are found in surface and ground waters suggesting their ineffective removal by conventional wastewater treatment technologies. Advanced oxidation/reduction processes (AO/RPs), which utilize free radical reactions to directly degrade chemical contaminants, are alternatives to traditional water treatment. This study reports the absolute rate constants for reaction of diclofenac sodium and model compound (2, 6-dichloraniline) with the two major AO/RP radicals: the hydroxyl radical (•OH) and hydrated electron (e(aq)(-)). The bimolecular reaction rate constants (M(-1) s(-1)) for diclofenac for •OH was (9.29 ± 0.11) × 10(9), and for e(-)(aq) was (1.53 ± 0.03) ×10(9). To provide a better understanding of the decomposition of the intermediate radicals produced by hydroxyl radical reactions, transient absorption spectra are observed from 1 - 250 ?s. In addition, preliminary degradation mechanisms and major products were elucidated using (60)Co ?-irradiation and LC-MS. The toxicity of products was evaluated using luminescent bacteria. These data are required for both evaluating the potential use of AO/RPs for the destruction of these compounds and for studies of their fate and transport in surface waters where radical chemistry may be important in assessing their lifetime. PMID:23384514

Yu, Hui; Nie, Er; Xu, Jun; Yan, Shuwen; Cooper, William J; Song, Weihua

2013-04-01

298

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways  

PubMed Central

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death.

Delgado, M E; Dyck, L; Laussmann, M A; Rehm, M

2014-01-01

299

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways.  

PubMed

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death. PMID:24457955

Delgado, M E; Dyck, L; Laussmann, M A; Rehm, M

2014-01-01

300

Kinetics and pathways of cyanide degradation at high temperatures and pressures.  

PubMed

The degradation of cyanide was performed in a 1-L semibatch reactor at temperatures between 393 and 473 K and at total pressures in the range of 2.0-8.0 MPa. The initial pH of the solution was set at 11, whereas initial concentrations ranged from 3.85 to 25 mM, which resemble the typical concentrations of cyanide-containing wastewater. The change with time of cyanide concentration, intermediates, and final products was analyzed in order to elucidate the reaction pathways. The experimental results suggest two parallel pathways of alkaline hydrolysis for the degradation of the pollutant. Formate and ammonia were identified as the final reaction products for one of the pathways, whereas carbon dioxide, nitrogen, and hydrogen were considered to be the final products for the other one. The degradation reaction results were fitted to first-order kinetic equations with respect to cyanide, giving respectively activation energies of 108.2 ± 3.3 and 77.6 ± 3.0 kJ/mol. Consequently, the formation of formate and ammonia is favored at high temperatures, whereas low temperatures favored the pathway leading to the formation of carbon dioxide and nitrogen. PMID:23249361

Oulego, Paula; Laca, Adriana; Diaz, Mario

2013-02-01

301

Metagenomic Identification of Bacterioplankton Taxa and Pathways Involved in Microcystin Degradation in Lake Erie  

PubMed Central

Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems.

Mou, Xiaozhen; Lu, Xinxin; Jacob, Jisha; Sun, Shulei; Heath, Robert

2013-01-01

302

Multiple degradation pathways for misfolded mutants of the yeast plasma membrane ATPase, Pma1.  

PubMed

To understand protein sorting and quality control in the secretory pathway, we have analyzed intracellular trafficking of the yeast plasma membrane ATPase, Pma1. Pma1 is ideal for such studies because it is a very abundant polytopic membrane protein, and its localization and activity at the plasma membrane are essential for cell viability and growth. We have tested whether the cytoplasmic amino- and carboxyl-terminal domains of Pma1 carry sorting information. As the sole copy of Pma1, mutants truncated at either NH2 or COOH termini are targeted at least partially to the plasma membrane and have catalytic activity to sustain cell viability. The mutants are also delivered to degradative pathways. Strikingly, NH2- and COOH-terminal Pma1 mutants are differentially recognized for degradation at distinct cellular locales. COOH-terminal mutants are recognized for destruction by endoplasmic reticulum-associated degradation. By contrast, NH2-terminal mutants escape detection by endoplasmic reticulum-associated degradation entirely, and undergo endocytosis for vacuolar degradation after apparently normal cell surface targeting. Both NH2- and COOH-terminal mutants are conformationally abnormal, as revealed by increased sensitivity to tryptic cleavage, but are able to assemble to form oligomers. We propose that different quality control mechanisms may assess discrete domains of Pma1 rather than a global conformational state. PMID:16928681

Liu, Yu; Sitaraman, Sujatha; Chang, Amy

2006-10-20

303

A novel degradation pathway of chloroaniline in Diaphorobacter sp. PCA039 entails initial hydroxylation  

Microsoft Academic Search

A novel degradation pathway of chloroaniline in Diaphorobacter sp. PCA039 entails initial hydroxylation. A novel gene cluster (pca), involved in the degradation of p-chloroaniline, was identified from Diaphorobacter sp. PCA039 capable of utilizing p-chloroaniline as sole carbon, nitrogen and energy source for growth. There were 29 ORFs in the pca cluster, in the order of pcaTRKLMNOPR\\u000a \\u000a 1\\u000a \\u000a D\\u000a \\u000a 1\\u000a \\u000a C

Tao ZhangHua-Feng; Hua-Feng Ren; Ying Liu; Bao-Li Zhu; Zhi-Pei Liu

2010-01-01

304

Degradation of 2,3-Dihydroxybenzoate by a Novel meta-Cleavage Pathway  

PubMed Central

2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate.

Marin, Macarena; Plumeier, Iris

2012-01-01

305

Buthionine sulfoximine diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.  

PubMed

Buthionine sulfoximine (BSO) is a specific inhibitor of ?-glutamylcysteine synthetase, thus blocking the synthesis of glutathione (GSH). It is known that this makes that BSO affects melanin synthesis because of the role of thiols in melanogenesis. However, BSO may also react with the intermediate oxidation products of melanogenesis, a possibility that has not been investigated from the initial steps of the pathway. We created in vitro conditions simulating eumelanogenesis (oxidation of l-DOPA in the absence of GSH) and pheomelanogenesis (oxidation of l-DOPA in the presence of GSH) under presence or absence of BSO. BSO made that eumelanogenesis results in pigments more soluble and less resistant to degradation by hydrogen peroxide than pigments obtained without BSO. A similar but less marked effect was observed for pheomelanogenesis only at subsaturating concentrations of GSH. These results suggest that BSO diverts the melanogenesis pathway toward the production of more soluble and degradable pigments. PMID:24703231

Galván, Ismael; Wakamatsu, Kazumasa; Alonso-Alvarez, Carlos; Solano, Francisco

2014-05-01

306

Disease-associated variants of microsomal retinol dehydrogenase 12 (RDH12) are degraded at mutant-specific rates.  

PubMed

Mutations in retinol dehydrogenase 12 (RDH12) cause severe retinal degeneration. However, some of the disease-associated RDH12 mutants retain significant catalytic activity, indicating the existence of additional pathophysiological mechanisms. This study demonstrates that the catalytically active T49M and I51N mutants undergo accelerated degradation, which results in their reduced cellular levels. Inhibition of proteasome leads to significant accumulation of ubiquitylated T49M and I51N. Furthermore, the degree of ubiquitylation strongly correlates with the half-lives of the proteins. These results suggest that the accelerated degradation of RDH12 mutants by the ubiquitin-proteasome system contributes to the pathophysiology and phenotypic variability associated with mutations in the RDH12 gene. PMID:20006610

Lee, Seung-Ah; Belyaeva, Olga V; Kedishvili, Natalia Y

2010-02-01

307

Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1  

Microsoft Academic Search

Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in

Robin L. Stingley; Ashraf A. Khan; Carl E. Cerniglia

2004-01-01

308

Placing a Disrupted Degradation Motif at the C Terminus of Proteasome Substrates Attenuates Degradation without Impairing Ubiquitylation*  

PubMed Central

Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.

Alfassy, Omri S.; Cohen, Itamar; Reiss, Yuval; Tirosh, Boaz; Ravid, Tommer

2013-01-01

309

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation  

PubMed Central

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

Costaglioli, Patricia; Barthe, Christophe; Claverol, Stephane; Brozel, Volker S; Perrot, Michel; Crouzet, Marc; Bonneu, Marc; Garbay, Bertrand; Vilain, Sebastien

2012-01-01

310

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

PubMed

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

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

2014-10-01

311

Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in Aspergillus fumigatus?  

PubMed Central

Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from l-tyrosine. Proteome analysis indicated that the l-tyrosine degradation enzymes are synthesized when the fungus is grown with l-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (hmgA) and 4-hydroxyphenylpyruvate dioxygenase (hppD). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by l-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus. Homogentisic acid is the major intermediate, and the l-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.

Schmaler-Ripcke, Jeannette; Sugareva, Venelina; Gebhardt, Peter; Winkler, Robert; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A.

2009-01-01

312

Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus.  

PubMed

Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from L-tyrosine. Proteome analysis indicated that the l-tyrosine degradation enzymes are synthesized when the fungus is grown with L-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (hmgA) and 4-hydroxyphenylpyruvate dioxygenase (hppD). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by L-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus. Homogentisic acid is the major intermediate, and the L-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin. PMID:19028908

Schmaler-Ripcke, Jeannette; Sugareva, Venelina; Gebhardt, Peter; Winkler, Robert; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

2009-01-01

313

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene  

PubMed Central

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (?)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the ?-oxidation pathway.

van der Werf, Mariet J.; Swarts, Henk J.; de Bont, Jan A. M.

1999-01-01

314

Effects of reforesting degraded grassland on hydrological flow pathways on Leyte, the Philippines  

NASA Astrophysics Data System (ADS)

Reforestation of degraded land in the tropics is promoted for a wide range of expected benefits, including carbon sequestration and streamflow regulation. However, how reforestation of degraded land affects runoff generation mechanisms and catchment water yield is still poorly understood as most experimental work pertains to non-degraded terrain. We set out to study the differences in hydrological functioning of a small degraded grassland catchment and a similar catchment that was reforested 15 years ago. Both catchments are located near Tacloban, Leyte, the Philippines. Stream stage, EC and temperature are measured continuously since June 2013. Precipitation, soil moisture content, and groundwater levels are monitored as well. Samples are taken from streamflow, precipitation, groundwater, and soil water prior to and during rainfall events for geochemical and stable isotope analysis to elucidate source contributions to storm runoff. Streamflow and event water contributions increase rapidly during almost every rainfall event in the grassland. In the reforested catchment, event water contributions to streamflow are much smaller and only increase during large events. These tracer results suggest that overland flow occurs much less frequently and is much less widespread in the reforested catchment compared to the grassland catchment. Our results thus indicate that the dominant flow pathways have changed as a result of reforestation and suggest that reforestation can largely restore the hydrological functioning of degraded sites if the forest is allowed to develop over a sufficiently long period without subsequent disturbance.

van Meerveld, Ilja; Zhang, Jun; Bruijnzeel, Sampurno

2014-05-01

315

Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway.  

PubMed

The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 androgens. By using a (13)C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (?500 ?M) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19 androgens. PMID:24657867

Wang, Po-Hsiang; Yu, Chang-Ping; Lee, Tzong-Huei; Lin, Ching-Wen; Ismail, Wael; Wey, Shiaw-Pyng; Kuo, An-Ti; Chiang, Yin-Ru

2014-06-01

316

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

SciTech Connect

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

Shen Jianying; Hisaeda, Hajime; Chou Bin [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yu Qingsheng [Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tu Liping [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)], E-mail: himeno@parasite.med.kyushu-u.ac.jp

2008-01-25

317

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix.  

PubMed

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria. PMID:24874806

Thomas, Ruth E; Andrews, Laurie A; Burman, Jonathon L; Lin, Wen-Yang; Pallanck, Leo J

2014-05-01

318

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix  

PubMed Central

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.

Thomas, Ruth E.; Andrews, Laurie A.; Burman, Jonathon L.; Lin, Wen-Yang; Pallanck, Leo J.

2014-01-01

319

Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2  

PubMed Central

The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search.

Hiessl, Sebastian; Schuldes, Jorg; Thurmer, Andrea; Halbsguth, Tobias; Broker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf

2012-01-01

320

Targeting proteasomal pathways by dietary curcumin for cancer prevention and treatment.  

PubMed

Curcumin, a major component of the golden spice turmeric (Curcuma longa), has been linked with the prevention and treatment of a wide variety of cancers through modulation of multiple cell signaling pathways. Since the first report from our laboratory in 1995 that curcumin can inhibit activation of the proinflammatory transcription factor NF-?B by inhibiting the 26S proteasomal degradation of I?B?, an inhibitor of NF-?B, this yellow pigment has been shown to inhibit the protease activities of the proteasome. The carbonyl carbons of the curcumin molecule directly interact with the hydroxyl group of the amino-terminal threonine residue of the proteasomal CT-L subunit of 20S proteasome and cellular 26S proteasome. Curcumin is also a potent inhibitor of COP9 signalosome and associated kinases, casein kinase 2 and protein kinase D, all linked to the ubiquitin-proteasomal system (UPS). Curcumin can also directly inhibit ubiquitin isopeptidases, a family of deubiquitinases (DUBs) that salvage ubiquitin for reuse by the 26S proteasome system. The inhibition of this enzyme by curcumin is mediated through ?,?-unsaturated ketone and two sterically accessible ?-carbons. Regulation of the UPS pathway by curcumin has been linked to regulation of cancer-linked inflammatory proteins (such as COX-2 and iNOS), transcription factors (NF-?B, STAT3, Sp, AP-1, GADD153/CHOP, HIF-1?), growth factors (VEGF, HER2), apoptotic proteins (p53, Bcl-2, survivin, DNA topoisomerase II, HDAC2, p300, hTERT) and cell cycle proteins (cyclin D1, cyclin E, cyclin B, p21, p27) associated with the prevention and therapy of cancer. Interestingly, the effect of curcumin on 26S proteasome appears to be dose-dependent, as low doses (?1 µM) increase proteasome activity whereas high doses (?10 µM) inhibit the proteasome activity. In this review, we discuss in detail how modulation of these targets by curcumin is linked to prevention and treatment of cancer. PMID:23834173

Hasima, Noor; Aggarwal, Bharat B

2014-01-01

321

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

PubMed Central

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

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

2009-01-01

322

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

PubMed Central

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

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

2013-01-01

323

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol.  

PubMed

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s(-1)) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB. PMID:23676275

Yadid, Itamar; Rudolph, Johannes; Hlouchova, Klara; Copley, Shelley D

2013-06-11

324

Formation and Operation of the Histidine-degrading Pathway in Pseudomonas aeruginosa  

PubMed Central

Histidine ammonia lyase (histidase), urocanase, and the capacity to degrade formiminoglutamate, which are respectively involved in steps I, II, and IV in the catabolism of histidine, were induced during growth of Pseudomonas aeruginosa on histidine or urocanate, and were formed gratuitously in the presence of dihydro-urocanate. Urocanase-deficient bacteria formed enzymes I and IV constitutively; presumably they accumulate enough urocanate from the breakdown of endogenous histidine to induce formation of the pathway. Urocanate did not satisfy the histidine requirement of a histidine auxotroph, indicating that it probably acted as an inducer without being converted to histidine. The results imply that urocanate is the physiological inducer of the histidine-degrading enzymes in P. aeruginosa. Enzymes of the pathway were extremely sensitive to catabolite repression; enzymes I and II, but not IV, were coordinately repressed. Our results suggest a specific involvement of nitrogenous metabolites in the repression. Mutant bacteria with altered sensitivity to repression were obtained. The molecular weight of partially purified histidase was estimated at 210,000 by sucrose gradient centrifugation. Its Km for histidine was 2 × 10?3 m in tris(hydroxymethyl)aminomethane chloride buffer. Sigmoid saturation curves were obtained in pyrophosphate buffer, indicating that the enzyme might have multiple binding sites for histidine. Under certain conditions, histidase appeared to be partially inactive in vivo. These findings suggest that some sort of allosteric interaction involving histidase may play a role in governing the operation of the pathway of histidine catabolism.

Lessie, Thomas G.; Neidhardt, Frederick C.

1967-01-01

325

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol  

PubMed Central

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s?1) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Yadid, Itamar; Rudolph, Johannes; Hlouchova, Klara; Copley, Shelley D.

2013-01-01

326

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

SciTech Connect

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

Chou, Bin [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan) [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Hiromatsu, Kenji, E-mail: khiromatsu@fukuoka-u.ac.jp [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan)] [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)] [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Murata, Shigeo; Tanaka, Keiji [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan)] [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)] [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)

2010-02-12

327

A Co-Translational Ubiquitination Pathway For Quality Control of Misfolded Proteins  

PubMed Central

Previous studies have indicated that 6–30% of all newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system, however the relationship of ubiquitination to translation for these proteins has been unclear. We report that co-translational ubiquitination (CTU) is a robust process, with ~12–15% of nascent polypeptides being ubiquitinated in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. While nascent chains have been shown previously to be ubiquitinated within stalled complexes (CTUS), the majority of nascent chain ubiquitination occurred within active translation complexes (CTUA). CTUA was increased in response to agents that induce protein misfolding, while CTUS was increased in response to agents that lead to translational errors or stalling. These results indicate that ubiquitination of nascent polypeptides occurs in two contexts, and define CTUA as a component of a quality control system that marks proteins for destruction while they are being synthesized.

Wang, Feng; Durfee, Larissa A.; Huibregtse, Jon M.

2013-01-01

328

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains  

PubMed

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc. PMID:10099396

Van Dien SJ; Keasling

1998-09-20

329

Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.  

PubMed Central

An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.

Bourne, D G; Jones, G J; Blakeley, R L; Jones, A; Negri, A P; Riddles, P

1996-01-01

330

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

PubMed

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

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

2014-01-01

331

Deubiquitination of Tip60 by USP7 Determines the Activity of the p53-Dependent Apoptotic Pathway  

PubMed Central

Tip60 is an essential acetyltransferase required for acetylation of nucleosomal histones and other nonhistone proteins. Tip60 acetylates the p53 tumor suppressor at lysine 120 (K120), a modification essential for p53-dependent induction of PUMA and apoptosis. It is known that Tip60 is turned over in cells by the ubiquitin-proteasome system. However, the deubiquitinase activity for stabilizing Tip60 is unknown. Here we show that USP7 interacts with and deubiquitinates Tip60 both in vitro and in vivo. USP7 deubiquitinase activity is required for the stabilization of Tip60 in order to operate an effective p53-dependent apoptotic pathway in response to genotoxic stress. Inhibiting USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60.

Dar, Ashraf; Shibata, Etsuko

2013-01-01

332

Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway.  

PubMed

Tip60 is an essential acetyltransferase required for acetylation of nucleosomal histones and other nonhistone proteins. Tip60 acetylates the p53 tumor suppressor at lysine 120 (K120), a modification essential for p53-dependent induction of PUMA and apoptosis. It is known that Tip60 is turned over in cells by the ubiquitin-proteasome system. However, the deubiquitinase activity for stabilizing Tip60 is unknown. Here we show that USP7 interacts with and deubiquitinates Tip60 both in vitro and in vivo. USP7 deubiquitinase activity is required for the stabilization of Tip60 in order to operate an effective p53-dependent apoptotic pathway in response to genotoxic stress. Inhibiting USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60. PMID:23775119

Dar, Ashraf; Shibata, Etsuko; Dutta, Anindya

2013-08-01

333

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum-associated degradation requires Atg19 and the Cvt/autophagy pathway.  

PubMed

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H(+)-ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild-type allele, both proteins are retained in the ER. We have used a pma1-D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1-pma1-D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1-D378T. ATG19 accelerates the degradation of pma1-D378T thus allowing the co-retained wild-type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1. PMID:17238920

Mazón, María J; Eraso, Pilar; Portillo, Francisco

2007-02-01

334

Degradation pathway of pentachlorophenol by Mucor plumbeus involves phase II conjugation and oxidation-reduction reactions.  

PubMed

Environmental pollution by pentachlorophenol (PCP) is a critical concern worldwide and fungal bioremediation constitutes an elegant and environment-friendly solution. Mucorales from the Zygomycota phylum are often observed to be competitive in field conditions and Mucor plumbeus, in particular, can efficiently deplete PCP from media. The pathway for PCP degradation used by this fungus has not been investigated. In this study, PCP-derived metabolites were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, including tetra- and tri-chlorohydroquinones and phase II-conjugated metabolites. Amongst the latter are the previously reported glucose, sulfate and ribose conjugates, and identified for the first time in fungi sulfate-glucose conjugates. A PCP transformation pathway for M. plumbeus is proposed, which excludes the involvement of cytochrome P-450 and extracellular ligninolytic enzymes. PMID:22074894

Carvalho, Mariana B; Tavares, Sandra; Medeiros, Joana; Núñez, Oscar; Gallart-Ayala, Héctor; Leitão, Maria Cristina; Galceran, Maria Teresa; Hursthouse, Andrew; Pereira, Cristina Silva

2011-12-30

335

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

USGS Publications Warehouse

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

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

2000-01-01

336

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

SciTech Connect

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

Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2012-06-01

337

A polyomic approach to elucidate the fluoranthene-degradative pathway in Mycobacterium vanbaalenii PYR-1.  

PubMed

Mycobacterium vanbaalenii PYR-1 is capable of degrading a wide range of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs), including fluoranthene. We used a combination of metabolomic, genomic, and proteomic technologies to investigate fluoranthene degradation in this strain. Thirty-seven fluoranthene metabolites including potential isomers were isolated from the culture medium and analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and UV-visible absorption. Total proteins were separated by one-dimensional gel and analyzed by liquid chromatography-tandem mass spectrometry in conjunction with the M. vanbaalenii PYR-1 genome sequence (http://jgi.doe.gov), which resulted in the identification of 1,122 proteins. Among them, 53 enzymes were determined to be likely involved in fluoranthene degradation. We integrated the metabolic information with the genomic and proteomic results and proposed pathways for the degradation of fluoranthene. According to our hypothesis, the oxidation of fluoranthene is initiated by dioxygenation at the C-1,2, C-2,3, and C-7,8 positions. The C-1,2 and C-2,3 dioxygenation routes degrade fluoranthene via fluorene-type metabolites, whereas the C-7,8 routes oxidize fluoranthene via acenaphthylene-type metabolites. The major site of dioxygenation is the C-2,3 dioxygenation route, which consists of 18 enzymatic steps via 9-fluorenone-1-carboxylic acid and phthalate with the initial ring-hydroxylating oxygenase, NidA3B3, oxidizing fluoranthene to fluoranthene cis-2,3-dihydrodiol. Nonspecific monooxygenation of fluoranthene with subsequent O methylation of dihydroxyfluoranthene also occurs as a detoxification reaction. PMID:17449607

Kweon, Ohgew; Kim, Seong-Jae; Jones, Richard C; Freeman, James P; Adjei, Michael D; Edmondson, Ricky D; Cerniglia, Carl E

2007-07-01

338

Assessment of degradation pathways in an aquifer with mixed chlorinated hydrocarbon contamination using stable isotope analysis.  

PubMed

The demonstration of monitored natural attenuation (MNA) of chlorinated hydrocarbons in groundwater is typically conducted through the evaluation of concentration trends and parent-daughter product relationships along prevailing groundwater flow paths. Unfortunately, at sites contaminated by mixtures of chlorinated ethenes, ethanes, and methanes, the evaluation of MNA by using solely concentration data and parent-daughter relationships can result in erroneous conclusions regarding the degradation mechanisms that are truly active at the site, since many of the daughter products can be derived from multiple parent compounds. Stable carbon isotope analysis was used, in conjunction with concentration data, to clarify and confirm the active degradation pathways at a former waste solvent disposal site where at least 14 different chlorinated hydrocarbons have been detected in the groundwater. The isotope data indicate that TCE, initially believed to be present as a disposed product and/or a PCE dechlorination intermediate, is attributable to dehydrochlorination of 1,1,2,2-PCA. The isotope data further support that vinyl chloride and ethene in the site groundwater result from dichloroelimination of 1,1,2-trichlorethane and 1,2-dichloroethane, respectively, rather than from reductive dechlorination of the chlorinated ethenes PCE, TCE, or 1,2-DCE. The isotope data confirm that the chlorinated ethanes and chlorinated methanes are undergoing significant intrinsic degradation, whereas degradation of the chlorinated ethenes may be limited. In addition to the classical trend of enriched isotope values of the parent compounds with increasing distance associated to biodegradation, shifts of isotope ratios of degradation byproduct in the opposite direction due to mixing of isotopically light byproducts of biodegradation with compounds from the source are shown to be of high diagnostic value. These data underline the value of stable isotope analysis in confirming transformation processes at sites with complex mixtures of chlorinated compounds. PMID:16173553

Hunkeler, Daniel; Aravena, Ramon; Berry-Spark, Karen; Cox, Evan

2005-08-15

339

19F NMR metabolomics for the elucidation of microbial degradation pathways of fluorophenols.  

PubMed

Of all NMR-observable isotopes 19F is the one most convenient for studies on the biodegradation of environmental pollutants and especially for fast initial metabolic screening of newly isolated organisms. In the past decade we have identified the 19F NMR characteristics of many fluorinated intermediates in the microbial degradation of fluoroaromatics including especially fluorophenols. In the present paper we give an overview of results obtained for the initial steps in the aerobic microbial degradation of fluorophenols, i.e. the aromatic hydroxylation to di -, tri - or even tetrahydroxybenzenes ultimately suitable as substrates for the second step, ring cleavage by dioxygenases. In addition we present new results from studies on the identification of metabolites resulting from reaction steps following aromatic ring cleavage, i.e. resulting from the conversion of fluoromuconates by chloromuconate cycloisomerase. Together the presented data illustrate the potential of the 19F NMR technique for (1) fast initial screening of biodegradative pathways, i.e. for studies on metabolomics in newly isolated microorganisms, and (2) identification of relatively unstable pathway intermediates like fluoromuconolactones and fluoromaleylacetates. PMID:11548746

Boersma, M G; Solyanikova, I P; Van Berkel, W J; Vervoort, J; Golovleva, L A; Rietjens, I M

2001-01-01

340

Pathways and determinants of early spontaneous vegetation succession in degraded lowland of South China.  

PubMed

Continuous and prolonged human disturbances have caused severe degradation of a large portion of lowland in South China, and how to restore such degraded ecosystems becomes an increasing concern. The process and mechanisms of spontaneous succession, which plays an important role in vegetation restoration, have not been adequately examined. To identify the pathways of early spontaneous vegetation succession, 41 plots representing plant communities abandoned over different times were established and investigated. The communities and indicator species of the vegetation were classified by analyzing the important values of plant species using multivariate analyses. The results indicated that the plant species could be classified into nine plant communities representing six succession stages. The pathway and species composition also changed in the process of succession. We also measured 13 environmental variables of microtopography, soil structure and soil nutrition in each plot to examine the driving forces of succession and the vegetation-environment relationships. Our results showed that the environmental variables changed in diverse directions, and that soil bulk density, soil water capacity and soil acidity were the most important factors. PMID:18713436

Duan, Wen-Jun; Ren, Hai; Fu, Sheng-Lei; Guo, Qin-Feng; Wang, Jun

2008-02-01

341

Kinetics and reaction pathways of formaldehyde degradation using the UV-fenton method.  

PubMed

This study was based on the purpose of investigating the reaction rules of formaldehyde (HCHO) as an intermediate product in the degradation of many other organic wastewaters. The process conditions of UV-Fenton method for the degradation of the low concentrations of HCHO were studied in a batch photochemical reactor. The results showed that, when the original HCHO concentration was 30 mg/L, at an operating temperature of 23 degrees C, pH = 3, an H202 dosage of 68 mg/L, and an H2O2-to-Fe2+ mole ratio (H2O2:Fe2+) of 5, 91.89% of the HCHO was removed after 30 minutes. The degradation of HCHO in the UV-Fenton system was basically in accordance with the exponential decay. The kinetic study results showed that the reaction orders of HCHO, Fe2+, and H2O2 in the system were 1.054, 0.510, and 0.728, respectively, and the activation energy (Ea) was 9.85 kJ/mol. The comparison of UV/H2O2, Fenton, and UV-Fenton systems for the degradation of HCHO, and the results of iron catalyst tests showed that the mechanism of UV-Fenton on the degradation of HCHO was through a synergistic effect of Fe2+ and UV light to catalyze the decomposition of H2O2. The introduction of UV irradiation to the Fenton system largely increased the degradation rate of HCHO, mainly as a result of the accelerating effect on the formation of the Fe2+/Fe3+ cycle. The reaction products were analyzed by gas chromatography-mass spectrometry and a chemical oxygen demand (COD) analyzer. The effluent gases also were analyzed by gas chromatography. Based on those results, the reaction pathways of HCHO in the UV-Fenton system were proposed. The qualitative and quantitative analysis of the reaction products and the COD showed that the main intermediate product of the reaction was formic acid, and the further oxidation of it was the rate-limiting step for the degradation of HCHO. PMID:21657193

Liu, Xiangxuan; Liang, Jiantao; Wang, Xuanjun

2011-05-01

342

Degradation and Pathway of Tetracycline Hydrochloride in Aqueous Solution by Potassium Ferrate  

PubMed Central

Abstract In the context of water treatment, the ferrate ([FeO4]2?) ion has long been known for its strong oxidizing power and for producing a coagulant from its reduced form [i.e., Fe(III)]. However, it has not been widely applied in water treatment, because of preparation difficulties and high cost. This article describes a low-cost procedure for producing solid potassium ferrate. In this synthetic procedure, NaClO was used in place of chlorine generation; and 10?M KOH was used in place of saturated KOH in the previous procedures. In addition, this study investigated the reactions of potassium ferrate with tetracycline hydrochloride (TC) at different pH and molar ratios. Results showed that the optimal pH range for TC degradation was pH 9–10, and TC could be mostly removed by Fe(VI) in 60?s. However, results showed >70% of TC degraded and <15% of dissolved organic carbon (DOC) reduction at molar ratio of 1:20. The main degradation pathway of TC is proposed based on the experimental data.

Ma, Yan; Gao, Naiyun; Li, Cong

2012-01-01

343

Degradation and Pathway of Tetracycline Hydrochloride in Aqueous Solution by Potassium Ferrate.  

PubMed

In the context of water treatment, the ferrate ([FeO(4)](2-)) ion has long been known for its strong oxidizing power and for producing a coagulant from its reduced form [i.e., Fe(III)]. However, it has not been widely applied in water treatment, because of preparation difficulties and high cost. This article describes a low-cost procedure for producing solid potassium ferrate. In this synthetic procedure, NaClO was used in place of chlorine generation; and 10?M KOH was used in place of saturated KOH in the previous procedures. In addition, this study investigated the reactions of potassium ferrate with tetracycline hydrochloride (TC) at different pH and molar ratios. Results showed that the optimal pH range for TC degradation was pH 9-10, and TC could be mostly removed by Fe(VI) in 60?s. However, results showed >70% of TC degraded and <15% of dissolved organic carbon (DOC) reduction at molar ratio of 1:20. The main degradation pathway of TC is proposed based on the experimental data. PMID:22566741

Ma, Yan; Gao, Naiyun; Li, Cong

2012-05-01

344

Photocatalytic transformation of sixteen substituted phenylurea herbicides in aqueous semiconductor suspensions: intermediates and degradation pathways.  

PubMed

The photocatalytic degradation of sixteen substituted phenylurea herbicides (PUHs) in pure water has been studied using zinc oxide (ZnO) and titanium dioxide (TiO(2)) as photocatalyst under artificial light irradiation. Photocatalytic experiments showed that the addition of these chalcogenide oxides in tandem with the oxidant (Na(2)S(2)O(8)) strongly enhances the degradation rate of these compounds in comparison with those carried out with ZnO and TiO(2) alone and photolytic tests. Comparison of catalysts showed that ZnO is the most efficient for the removal of such herbicides in optimal conditions and at constant volumetric rate of photon absorption in the photoreactor. Thus, the complete disappearance of all the studied compounds was achieved after 20 min of illumination in the ZnO/Na(2)S(2)O(8) system. The main photocatalytic intermediates detected during the degradation of PUHs were identified. The probable photodegradation pathways were proposed and discussed. The main steps involved: N-demethylation of the N,N-dimethylurea-substituted compounds followed of N-demethylation and N-demethoxylation of the N-methoxy-N-methyl-substituted ureas and hydroxylation of aromatic rings and their aliphatic side-chains of both, parent compounds and intermediates. PMID:23270960

Fenoll, José; Sabater, Paula; Navarro, Gines; Pérez-Lucas, Gabriel; Navarro, Simón

2013-01-15

345

Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway  

PubMed Central

The key gluconeogenic enzyme, fructose1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30–40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

Huang, Pei-Hsin; Chiang, Hui-Ling

1997-01-01

346

Degradation pathway of quinolines in a biofilm system under denitrifying conditions  

SciTech Connect

This article reports for the first time the degradation pathways of quinoline, isoquinoline, and methylquinolines by a mixed culture in a biofilm under nitrate-reducing conditions. A simple reverse-phase high-performance liquid chromatography method using ultraviolet detection at 223 nm for determination of seven quinoline analogues and 15 metabolites was developed, and gas chromatography--mass spectrometry and thin-layer chromatography analyses were used for identification. The inhibition of nitrification by the parent compounds and their degradation products was assessed by a nitrification toxicity test called MINNTOX. Quinoline and 3-, 4-, 6-, and 8-methylquinoline were all transformed by hydroxylation into their 2-hydroxyquinoline analogues (2-quinolinones), and isoquinoline was transformed into 1-hydroxyisoquinoline. 2-Methylquinoline was not transformed by this microcosm, likely due to the blockage at position 2 by the methyl group. The hydroxylated metabolites of isoquinoline and quinolines methylated at the heterocyclic ring were not transformed further, whereas metabolites of quinoline and quinolines methylated at the homocyclic ring were hydrogenated at position 3 and 4, and the resulting 3,4-dihydro-2-quinolinone analogues accumulated. Of these metabolites, only 3,4-dihydro-2-quinolinone from the degradation of quinoline was further transformed into unidentified products. All quinolines and their metabolites had inhibiting effects on the nitrifying bacteria at the same level (ppm) in the applied bioassay, indicating that the inhibition of the compounds was not influenced by the initial transformation reactions.

Johansen, S.S.; Arvin, E.; Mosbaek, H. [Technical Univ. of Denmark, Lyngby (Denmark). Dept. of Environmental Science and Engineering; Hansen, A.B. [National Environmental Research Inst., Roskilde (Denmark). Dept. of Environmental Chemistry

1997-09-01

347

Alteration of Dynein Function Affects ?-Synuclein Degradation via the Autophagosome-Lysosome Pathway  

PubMed Central

Growing evidence suggests that dynein dysfunction may be implicated in the pathogenesis of neurodegeneration. It plays a central role in aggresome formation, the delivery of autophagosome to lysosome for fusion and degradation, which is a pro-survival mechanism essential for the bulk degradation of misfolded proteins and damaged organells. Previous studies reported that dynein dysfuntion was associated with aberrant aggregation of ?-synuclein, which is a major component of inclusion bodies in Parkinson’s disease (PD). However, it remains unclear what roles dynein plays in ?-synuclein degradation. Our study demonstrated a decrease of dynein expression in neurotoxin-induced PD models in vitro and in vivo, accompanied by an increase of ?-synuclein protein level. Dynein down-regulation induced by siRNA resulted in a prolonged half-life of ?-synuclein and its over-accumulation in A53T overexpressing PC12 cells. Dynein knockdown also prompted the increase of microtubule-associated protein 1 light chain 3 (LC3-II) and sequestosome 1 (SQSTM1, p62) expression, and the accumulation of autophagic vacuoles. Moreover, dynein suppression impaired the autophagosome fusion with lysosome. In summary, our findings indicate that dynein is critical for the clearance of aberrant ?-synuclein via autophagosome-lysosome pathway.

Li, Da; Shi, Ji-Jun; Mao, Cheng-Jie; Liu, Sha; Wang, Jian-Da; Chen, Jing; Wang, Fen; Yang, Ya-Ping; Hu, Wei-Dong; Hu, Li-Fang; Liu, Chun-Feng

2013-01-01

348

Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.  

PubMed

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern. PMID:22480422

Skop, V; Cahová, M; Papá?ková, Z; Pálení?ková, E; Da?ková, H; Baranowski, M; Zabielski, P; Zdychová, J; Zídková, J; Kazdová, L

2012-07-20

349

Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.  

PubMed

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system. PMID:23637911

Silva, Cynthia C; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; De Paula, Sérgio O; Silva, Lívia C F; Vidigal, Pedro M P; Vicentini, Renato; Sousa, Maíra P; Torres, Ana Paula R; Santiago, Vânia M J; Oliveira, Valéria M

2013-01-01

350

Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater  

PubMed Central

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

Silva, Cynthia C.; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; De Paula, Sergio O.; Silva, Livia C. F.; Vidigal, Pedro M. P.; Vicentini, Renato; Sousa, Maira P.; Torres, Ana Paula R.; Santiago, Vania M. J.; Oliveira, Valeria M.

2013-01-01

351

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

SciTech Connect

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

Lee, Sanghwan; Hur, Eu-gene [Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712?749 (Korea, Republic of)] [Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712?749 (Korea, Republic of); Ryoo, In-geun; Jung, Kyeong-Ah [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)] [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of); Kwak, Jiyeon [Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402?751 (Korea, Republic of)] [Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402?751 (Korea, Republic of); Kwak, Mi-Kyoung, E-mail: mkwak@catholic.ac.kr [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)] [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)

2012-11-01

352

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.  

PubMed Central

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.

Grifoll, M; Selifonov, S A; Chapman, P J

1994-01-01

353

The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo-Lysosomal Pathway  

PubMed Central

Background Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. Methodology/Principal Findings By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. Conclusions/Significance Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs.

Ramos, Irene; Herrera, Monica; Stamatakis, Konstantinos

2009-01-01

354

Physiology of deletion mutants in the anaerobic ?-myrcene degradation pathway in Castellaniella defragrans  

PubMed Central

Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic ?-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of ?-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans ?ldi and C. defragrans ?geoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic ?-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the ?-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons.

2012-01-01

355

Degradation of the synthetic dye amaranth by the fungus Bjerkandera adusta Dec 1: inference of the degradation pathway from an analysis of decolorized products.  

PubMed

We examined the degradation of amaranth, a representative azo dye, by Bjerkandera adusta Dec 1. The degradation products were analyzed by high performance liquid chromatography (HPLC), visible absorbance, and electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS). At the primary culture stage (3 days), the probable reaction intermediates were 1-aminonaphthalene-2,3,6-triol, 4-(hydroxyamino) naphthalene-1-ol, and 2-hydroxy-3-[2-(4-sulfophenyl) hydrazinyl] benzenesulfonic acid. After 10 days, the reaction products detected were 4-nitrophenol, phenol, 2-hydroxy-3-nitrobenzenesulfonic acid, 4-nitrobenzene sulfonic acid, and 3,4'-disulfonyl azo benzene, suggesting that no aromatic amines were created. Manganese-dependent peroxidase activity increased sharply after 3 days culture. Based on these results, we herein propose, for the first time, a degradation pathway for amaranth. Our results suggest that Dec 1 degrades amaranth via the combined activities of peroxidase and hydrolase and reductase action. PMID:21526388

Gomi, Nichina; Yoshida, Shuji; Matsumoto, Kazutsugu; Okudomi, Masayuki; Konno, Hiroki; Hisabori, Toru; Sugano, Yasushi

2011-11-01

356

Human THP1 Monocytic Leukemic Cells Induced to Undergo Monocytic Differentiation by Bryostatin 1 Are Refractory to Proteasome Inhibitor-induced Apoptosis1  

Microsoft Academic Search

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. We demonstrated that treatment of THP-1 human monocytic leukemia cells with Z-LLL- CHO, a reversible proteasome inhibitor, induced cell death through an apoptotic pathway. Apoptosis in THP-1 cells induced by Z-LLL-CHO involved a cytochrome c-dependent pathway, which included the release of mitochondrial cytochrome c,

Catheryne Chen; Hong Lin; Chachata Karanes; George R. Pettit; Ben D. Chen

2000-01-01

357

Fluoranthene metabolism in Mycobacterium sp. strain KR20: identity of pathway intermediates during degradation and growth.  

PubMed

Mycobacterium sp. strain KR20, which was isolated from a polycyclic aromatic hydrocarbon (PAH) contaminated soil of a former gaswork plant site, metabolized about 60% of the fluoranthene added (0.5 mg ml(-1)) to batch cultures in mineral salts medium within 10 d at 20 degrees C. It thereby increased its cell number about 30-fold and produced at least seven metabolites. Five metabolites, namely cis-2,3-fluoranthene dihydrodiol, Z-9-carboxymethylene-fluorene-1-carboxylic acid, cis-1,9a-dihydroxy-1-hydro-fluorene-9-one-8-carboxylic acid, 4-hydroxybenzochromene-6-one-7-carboxylic acid and benzene-1,2,3-tricarboxylic acid, could be identified by NMR and MS spectroscopic techniques and ascribed to an alternative fluoranthene degradation pathway. Besides fluoranthene, the isolate could not use any of the PAHs tested as a sole source of carbon and energy. PMID:11577157

Rehmann, K; Hertkorn, N; Kettrup, A A

2001-10-01

358

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways  

PubMed Central

Objectives To investigate the role that oxidative stress plays in the development of diabetic cystopathy. Materials and methods Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month-old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using western blot analysis. Results Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10?10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. Conclusions Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.

Kanika, Nirmala; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J.; Melman, Arnold; Davies, Kelvin

2010-01-01

359

Characterization of an aldolase-dehydrogenase complex from the cholesterol degradation pathway of Mycobacterium tuberculosis.  

PubMed

HsaF and HsaG are an aldolase and dehydrogenase from the cholesterol degradation pathway of Mycobacterium tuberculosis. HsaF could be heterologously expressed and purified as a soluble dimer, but the enzyme was inactive in the absence of HsaG. HsaF catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. The enzyme requires divalent metals for activity, with a preference for Mn(2+). The Km values for 4-hydroxy-2-oxoacids were about 20-fold lower than observed for the aldolase homologue, BphI from the polychlorinated biphenyl degradation pathway. Acetaldehyde and propionaldehyde were channeled directly to the dehydrogenase, HsaG, without export to the bulk solvent where they were transformed to acyl-CoA in an NAD(+) and coenzyme A dependent reaction. HsaG is able to utilize aldehydes up to five carbons in length as substrates, with similar catalytic efficiencies. The HsaF-HsaG complex was crystallized and its structure was determined to a resolution of 1.93 Å. Substitution of serine 41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km dephospho-CoA, suggesting that this residue interacts with the 3'-ribose phosphate of CoA. A second protein annotated as a 4-hydroxy-2-oxopentanoic acid aldolase in M. tuberculosis (MhpE, Rv3469c) was expressed and purified, but was found to lack aldolase activity. Instead this enzyme was found to possess oxaloacetate decarboxylase activity, consistent with the conservation (with the 4-hydroxy-2-oxoacid aldolases) of residues involved in pyruvate enolate stabilization. PMID:23614353

Carere, Jason; McKenna, Sarah E; Kimber, Matthew S; Seah, Stephen Y K

2013-05-21

360

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

PubMed Central

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

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

1998-01-01

361

Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway.  

PubMed

Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2h by T. gondii, and after 6h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway. PMID:24845536

Padrão, Juliana da Cruz; Cabral, Gabriel Rabello de Abreu; da Silva, Maria de Fátima Sarro; Seabra, Sergio Henrique; DaMatta, Renato Augusto

2014-10-01

362

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6.  

PubMed

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into 'upper' (carbaryl to salicylate), 'middle' (salicylate to gentisate) and 'lower' (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s). PMID:23728496

Singh, Randhir; Trivedi, Vikas D; Phale, Prashant S

2013-08-01

363

Genetic Evidence for a Defective Xylan Degradation Pathway in Lactococcus lactis  

PubMed Central

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974–3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a ?-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.

Erlandson, Karn A.; Delamarre, Soazig C.; Batt, Carl A.

2001-01-01

364

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation.  

PubMed

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation. PMID:24743736

Yamamoto-Tanaka, M; Makino, T; Motoyama, A; Miyai, M; Tsuboi, R; Hibino, T

2014-01-01

365

Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8.  

PubMed Central

Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser(174) within the connecting region between the second and third Kunitz domains ( k (cat)/ K (m) approximately 75 M(-1).s(-1)) as well as following Lys(20) within the NH(2)-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. (125)I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, (125)I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser(174), which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

Cunningham, Anna C; Hasty, Karen A; Enghild, Jan J; Mast, Alan E

2002-01-01

366

Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9  

PubMed Central

We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the ? and ? subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Kmr mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.

Martin, Vincent J. J.; Mohn, William W.

2000-01-01

367

Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9.  

PubMed

We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the alpha and beta subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8, 13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12, 14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Km(r) mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation. PMID:10850995

Martin, V J; Mohn, W W

2000-07-01

368

Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans  

PubMed Central

Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.

Lee, I. Russel; Yang, Liting; Sebetso, Gaseene; Allen, Rebecca; Doan, Thi H. N.; Blundell, Ross; Lui, Edmund Y. L.; Morrow, Carl A.; Fraser, James A.

2013-01-01

369

Pathways for degrading TNT by Thu-Z: a Pantoea sp. strain.  

PubMed

2,4,6-Trinitrotoluene (TNT), an extensively used and versatile explosive, is harmful in soil and water. In the present study, four bacterial strains capable of degrading TNT have been isolated from contaminated sites and named as Thu-A, Thu-B, Thu-C, and Thu-Z. Thu-Z, which gave the highest degradation efficiency compared to the others, was assigned to the genus Pantoea according to its 16S rRNA gene. Similarities in both biochemical properties and morphology suggested that Thu-Z was a Pantoea sp. strain. Thu-Z was proved to be capable of using TNT as a sole nitrogen source by cleaving NO(2) from the nitroaromatic ring by direct aromatic ring reduction. Under nitrogen-limited conditions, 96.6 %?N of TNT was consumed by Thu-Z for growth, which was determined in terms of NaNO(2). Trace nitro reduction metabolites such as 2,4-diamino-6-nitrotoluene (24D