Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

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Mazumdar, Tuhina; Gorgun, F Murat; Sha, Youbao

2010-01-01

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated ce......, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway....

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

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Hernández-Pérez, Santiago; Cabrera, Elisa; Amoedo, Hugo; Rodríguez-Acebes, Sara; Koundrioukoff, Stephane; Debatisse, Michelle; Méndez, Juan; Freire, Raimundo

2016-10-01

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

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Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

2010-04-30

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

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

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

2014-01-01

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

OTUD5 regulates p53 stability by deubiquitinating p53.

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Judong Luo

Full Text Available The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by its negative regulators ubiquitin ligase MDM2.In this study, we have identified OTUD5 as a DUB that interacts with and deubiquitinates p53. OTUD5 forms a direct complex with p53 and controls level of ubiquitination. The function of OTUD5 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent apoptosis in response to DNA damage stress.As a novel deubiquitinating enzyme for p53, OTUD5 is required for the stabilization and the activation of a p53 response.

Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

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Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

2012-10-01

Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

Involvement of proteasomal subunits zeta and iota in RNA degradation.

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Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

1997-01-01

We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

Quantum Mechanics and Molecular Mechanics Study of the Catalytic Mechanism of Human AMSH-LP Domain Deubiquitinating Enzymes.

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Zhu, Wenyou; Liu, Yongjun; Ling, Baoping

2015-08-25

Deubiquitinating enzymes (DUBs) catalyze the cleavage of the isopeptide bond in polyubiquitin chains to control and regulate the deubiquitination process in all known eukaryotic cells. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds in the Lys63-linked polyubiquitin chains. In this article, the catalytic mechanism of AMSH-LP has been studied using a combined quantum mechanics and molecular mechanics method. Two possible hydrolysis processes (Path 1 and Path 2) have been considered. Our calculation results reveal that the activation of Zn(2+)-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. In Path 1, the generated hydroxyl first attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated, which is calculated to be the rate limiting step with an energy barrier of 13.1 kcal/mol. The energy barrier of the rate limiting step and the structures of intermediate and product are in agreement with the experimental results. In Path 2, the protonation of amino group of Lys63 is prior to the nucleophilic attack of activated hydroxyl. The two proton transfer processes in Path 2 correspond to comparable overall barriers (33.4 and 36.1 kcal/mol), which are very high for an enzymatic reaction. Thus, Path 2 can be ruled out. During the reaction, Glu292 acts as a proton transfer mediator, and Ser357 mainly plays a role in stabilizing the negative charge of Gly76. Besides acting as a Lewis acid, Zn(2+) also influences the reaction by coordinating to the reaction substrates (W1 and Gly76).

Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor.

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Liao, Yuning; Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Xia, Xiaohong; Wang, Xuejun; Huang, Hongbiao; Liu, Jinbao

2017-02-02

Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer.

Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.

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Scott, Daniel; Layfield, Robert; Oldham, Neil J

2015-08-01

Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

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Stringer, Daniel K; Piper, Robert C

2011-09-15

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

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

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

2014-01-01

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

Association of proteasomal activity with metastasis in luminal breast cancer

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Shashova, E. E.; Fesik, E. A.; Doroshenko, A. V.

2017-09-01

Chimotrypsin-like (ChTL) and caspase-like (CL) proteasomal activities were investigated in different variants of the tumor progression of luminal breast cancer. Patients with primary luminal breast cancer (n = 123) in stage T1-3N0-2M0 who had not received neoadjuvant treatment were included in this study. Proteasome ChTL and CL activities were determined in the samples of tumor and adjacent tissues. The coefficients of chymotrypsin-like (kChTL) and caspase-like (kCL) proteasome activity were also calculated as the ratio of the corresponding activity in the tumor tissue to activity in the adjacent tissue. ChTL, CL, kChTL and kCL in the tissues of luminal A and B breast cancer with lymphogenic metastasis were compared, and their association with hematogenous metastasis was evaluated. On the one hand, CL activity of proteasomes increased in luminal A breast cancer with extensive lymphogenic metastasis (N2), on the other hand it decreased in the luminal B subtype of cancer. The ratio of proteasomal activity in the tumor and adjacent tissues plays a significant role in the hematogenic pathway of breast cancer progression and is associated with poor metastatic-free survival.

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

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Adi Ben Yehuda

Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors.

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Hui, Kwai Fung; Tam, Kam Pui; Chiang, Alan Kwok Shing

2017-11-21

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers.

Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

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Samanovic, Marie I.; Tu, Shengjiang; Novák, Ondřej; Iyer, Lakshminarayan M.; McAllister, Fiona E.; Aravind, L.; Gygi, Steven P.; Hubbard, Stevan R.; Strnad, Miroslav; Darwin, K. Heran

2015-01-01

Summary One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO. PMID:25728768

Ubiquitin-specific protease 11 (USP11) functions as a tumor suppressor through deubiquitinating and stabilizing VGLL4 protein

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Zhang, Encheng; Shen, Bing; Mu, Xingyu; Qin, Yan; Zhang, Fang; Liu, Yong; Xiao, Jiantao; Zhang, Pingzhao; Wang, Chenji; Tan, Mingyue; Fan, Yu

2016-01-01

VGLL4 is a transcriptional repressor that interacts with transcription factors TEADs and inhibits YAP-induced overgrowth and tumorigenesis. VGLL4 protein was dramatically reduced in various types of human cancers. But how VGLL4 protein is post-transcriptional regulated is poorly understood. In this study, we identify deubiquitinating enzyme USP11 as a novel VGLL4 interactor. We reveal that the USP domain of USP11 and the N-terminal region of VGLL4 are required for mutual binding. USP11 controls VGLL4 protein stability by promoting its deubiquitination. Furthermore, our results show that knockdown of USP11 promotes cell growth, migration, and invasion in a YAP-dependent manner. Together, our results suggest that USP11 may exert its tumor suppressor role by modulating VGLL4/YAP-TEADs regulatory loop. PMID:28042509

Impaired methylation as a novel mechanism for proteasome suppression in liver cells

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Osna, Natalia A., E-mail: nosna@UNMC.edu [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); White, Ronda L.; Donohue, Terrence M. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); Beard, Michael R. [Department of Molecular Biosciences, University of Adelaide (Australia); Tuma, Dean J.; Kharbanda, Kusum K. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States)

2010-01-08

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.

SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

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Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

2004-10-01

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

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Yedidi, Ravikiran S; Fatehi, Amatullah K; Enenkel, Cordula

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Identification of unstable network modules reveals disease modules associated with the progression of Alzheimer's disease.

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Masataka Kikuchi

Full Text Available Alzheimer's disease (AD, the most common cause of dementia, is associated with aging, and it leads to neuron death. Deposits of amyloid β and aberrantly phosphorylated tau protein are known as pathological hallmarks of AD, but the underlying mechanisms have not yet been revealed. A high-throughput gene expression analysis previously showed that differentially expressed genes accompanying the progression of AD were more down-regulated than up-regulated in the later stages of AD. This suggested that the molecular networks and their constituent modules collapsed along with AD progression. In this study, by using gene expression profiles and protein interaction networks (PINs, we identified the PINs expressed in three brain regions: the entorhinal cortex (EC, hippocampus (HIP and superior frontal gyrus (SFG. Dividing the expressed PINs into modules, we examined the stability of the modules with AD progression and with normal aging. We found that in the AD modules, the constituent proteins, interactions and cellular functions were not maintained between consecutive stages through all brain regions. Interestingly, the modules were collapsed with AD progression, specifically in the EC region. By identifying the modules that were affected by AD pathology, we found the transcriptional regulation-associated modules that interact with the proteasome-associated module via UCHL5 hub protein, which is a deubiquitinating enzyme. Considering PINs as a system made of network modules, we found that the modules relevant to the transcriptional regulation are disrupted in the EC region, which affects the ubiquitin-proteasome system.

The ubiquitin-proteasome system

Indian Academy of Sciences (India)

... the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first 'tagged' by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome.

Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.

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Mandy H Y Lam

Full Text Available Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui

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B. Franzetti

2002-01-01

Full Text Available A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome, obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

Changes in proteasome structure and function caused by HAMLET in tumor cells.

Science.gov (United States)

Gustafsson, Lotta; Aits, Sonja; Onnerfjord, Patrik; Trulsson, Maria; Storm, Petter; Svanborg, Catharina

2009-01-01

Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

Science.gov (United States)

Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

2014-01-01

Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

Bacterial proteasome activator bpa (rv3780) is a novel ring-shaped interactor of the mycobacterial proteasome.

Science.gov (United States)

Delley, Cyrille L; Laederach, Juerg; Ziemski, Michal; Bolten, Marcel; Boehringer, Daniel; Weber-Ban, Eilika

2014-01-01

The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins.

SPR imaging biosensor for the 20S proteasome: Sensor development and application to measurement of proteasomes in human blood plasma

International Nuclear Information System (INIS)

Gorodkiewicz, E.; Sankiewicz, A.; Ostrowska, H.

2011-01-01

The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome's catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng.mL -1 on average for control, and 42300 ng.mL -1 on average for leukemia patients). (author)

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

Science.gov (United States)

Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

2017-10-01

Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae ; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and

Modeling proteasome dynamics in Parkinson's disease

International Nuclear Information System (INIS)

Sneppen, Kim; Lizana, Ludvig; Jensen, Mogens H; Pigolotti, Simone; Otzen, Daniel

2009-01-01

In Parkinson's disease (PD), there is evidence that α-synuclein (αSN) aggregation is coupled to dysfunctional or overburdened protein quality control systems, in particular the ubiquitin–proteasome system. Here, we develop a simple dynamical model for the on-going conflict between αSN aggregation and the maintenance of a functional proteasome in the healthy cell, based on the premise that proteasomal activity can be titrated out by mature αSN fibrils and their protofilament precursors. In the presence of excess proteasomes the cell easily maintains homeostasis. However, when the ratio between the available proteasome and the αSN protofilaments is reduced below a threshold level, we predict a collapse of homeostasis and onset of oscillations in the proteasome concentration. Depleted proteasome opens for accumulation of oligomers. Our analysis suggests that the onset of PD is associated with a proteasome population that becomes occupied in periodic degradation of aggregates. This behavior is found to be the general state of a proteasome/chaperone system under pressure, and suggests new interpretations of other diseases where protein aggregation could stress elements of the protein quality control system

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

Science.gov (United States)

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

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.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

International Nuclear Information System (INIS)

Liu, Shuai; Lv, Jiaju; Han, Liping; Ichikawa, Tomonaga; Wang, Wenjuan; Li, Siying; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

2012-01-01

Highlights: ► Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. ► Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. ► CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-κB) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-κB activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-κB activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNFα)-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-κB transcriptional activity in RASMCs; however, did not affect the TNFα-induced NF-κB activity. Intriguingly, the TNFα-induced IκB phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of IκBα and IκBβ proteins, it did not alter the kinetics of TNFα-induced IκB protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-κB activity and TNFα-induced IκB kinase activation without affecting TNFα-induced NF-κB activity in VSMCs. In addition, knocking down of Cyld suppressed TNFα-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNFα-induced RASMC migration and monocyte adhesion to

Assessing Subunit Dependency of the Plasmodium Proteasome Using Small Molecule Inhibitors and Active Site Probes

NARCIS (Netherlands)

Li, H.; Linden, W.A. van der; Verdoes, M.; Florea, B.I.; McAllister, F.E.; Govindaswamy, K.; Elias, J.E.; Bhanot, P.; Overkleeft, H.S.; Bogyo, M.

2014-01-01

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to

Role of the Ubiquitin Proteasome System in Regulating Skin Pigmentation

Directory of Open Access Journals (Sweden)

Hideya Ando

2009-10-01

Full Text Available Pigmentation of the skin, hair and eyes is regulated by tyrosinase, the critical rate-limiting enzyme in melanin synthesis by melanocytes. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin proteasome system (UPS. Several types of inherited hypopigmentary diseases, such as oculocutaneous albinism and Hermansky-Pudlak syndrome, involve the aberrant processing and/or trafficking of tyrosinase and its subsequent degradation which can occur due to the quality-control machinery. Studies on carbohydrate modifications have revealed that tyrosinase in the endoplasmic reticulum (ER is proteolyzed via ER-associated protein degradation and that tyrosinase degradation can also occur following its complete maturation in the Golgi. Among intrinsic factors that regulate the UPS, fatty acids have been shown to modulate tyrosinase degradation in contrasting manners through increased or decreased amounts of ubiquitinated tyrosinase that leads to its accelerated or decelerated degradation by proteasomes.

Changes in expression of proteasome in rats at different stages of atherosclerosis.

Science.gov (United States)

Ismawati; Oenzil, Fadil; Yanwirasti; Yerizel, Eti

2016-06-01

It has been suggested that proteasome system has a role in initiation, progression, and complication stages of atherosclerosis. Although there is still controversy, there has been no research that compares the expression of proteasome in tissue and serum at each of these stages. This study aimed to investigated the expression of proteasome at different stages of atherosclerosis using rat model. We measured the expression of aortic proteasome by immunohistochemical analyses and were then analyzed using ImageJ software for percentage of area and integrated density. We used Photoshop version 3.0 to analyze aortic proteasome expression as a comparison. We measured serum proteasome expression by enzyme linked immunosorbents assays. Kruskal-Wallis test was used to compare mean value of percentage of area and serum proteasome. Analysis of variance test was used to compare mean value of integrated density. Correlation test between vascular proteasome expression and serum proteasome expression was made using Spearman test. A P-value of 0.05 was considered statistically significant. Compared with normal, percentage of area was higher in initiation, progression, and complication. Compared with normal, integrated density was higher in initiation and further higher in progression and complication. Data from Image J is similar with data from Photoshop. Serum proteasome expression was higher in initiation compared with normal, and further higher in progression and complication. It was concluded that there were different vascular proteasome expression and serum proteasome expression at the stages of atherosclerosis. These results may be used in research into new marker and therapeutic target in atherosclerosis.

Features of proteasome functioning in malignant tumors

Science.gov (United States)

Kondakova, I. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Slonimskaya, E. M.; Kolomiets, L. A.; Afanas'ev, S. G.; Choinzonov, Y. L.

2017-09-01

Proteasome ubiquitin system is the important system of intracellular proteolysis. The activity of the proteasomes may undergo changes during cancer development. We studied the chymotrypsin-like activity of proteasomes, their subunit composition, and their association with tumor stage in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer, ovarian cancer, and colorectal cancer. The increase in chymotrypsin-like activity of proteasomes and decrease in total proteasome pool compared with adjacent tissues were shown in all malignant tumors excluding kidney cancer. The increase in chymotrypsin-like activity of proteasomes was found in primary tumors with all types of metastasis: lymphogenous of head and neck squamous cell carcinoma, intraperitoneal metastasis of ovarian cancer, hematogenous metastasis colorectal cancer. The exception was kidney cancer, in which there was a decrease in chymotrypsin-like activity with distant metastasis.

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

Science.gov (United States)

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

DEFF Research Database (Denmark)

Dutaud, Dominique; Aubry, Laurent; Henry, Laurent

2002-01-01

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination...

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features

DEFF Research Database (Denmark)

Santiago-Sim, Teresa; Burrage, Lindsay C; Ebstein, Frédéric

2017-01-01

regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated...

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Shuai [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Lv, Jiaju [Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan 250021 (China); Han, Liping; Ichikawa, Tomonaga; Wang, Wenjuan; Li, Siying [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Wang, Xing Li [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@pathology.ufl.edu [Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0275 (United States); Cui, Taixing, E-mail: taixing.cui@uscmed.sc.edu [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States)

2012-03-30

Highlights: Black-Right-Pointing-Pointer Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. Black-Right-Pointing-Pointer Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. Black-Right-Pointing-Pointer CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-{kappa}B) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-{kappa}B activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-{kappa}B activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNF{alpha})-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-{kappa}B transcriptional activity in RASMCs; however, did not affect the TNF{alpha}-induced NF-{kappa}B activity. Intriguingly, the TNF{alpha}-induced I{kappa}B phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of I{kappa}B{alpha} and I{kappa}B{beta} proteins, it did not alter the kinetics of TNF{alpha}-induced I{kappa}B protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-{kappa}B activity and TNF{alpha}-induced I{kappa}B kinase activation without affecting TNF{alpha}-induced NF-{kappa}B activity in VSMCs. In addition, knocking down of Cyld suppressed TNF{alpha}-induced activation of mitogen activated protein

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

Science.gov (United States)

Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Proteasomal and Lysosomal Protein Degradation and Heart Disease

OpenAIRE

Wang, Xuejun; Robbins, Jeffrey

2013-01-01

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 ...

Proteasomal and lysosomal protein degradation and heart disease.

Science.gov (United States)

Wang, Xuejun; Robbins, Jeffrey

2014-06-01

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". Copyright © 2013 Elsevier Ltd. All rights reserved.

Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity*

Science.gov (United States)

Chen, Ying; Yang, Li-Na; Cheng, Li; Tu, Shun; Guo, Shu-Juan; Le, Huang-Ying; Xiong, Qian; Mo, Ran; Li, Chong-Yang; Jeong, Jun-Seop; Jiang, Lizhi; Blackshaw, Seth; Bi, Li-Jun; Zhu, Heng; Tao, Sheng-Ce; Ge, Feng

2013-01-01

Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. PMID:23824909

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice.

Science.gov (United States)

Yamada, Yosuke; Tomaru, Utano; Ishizu, Akihiro; Ito, Tomoki; Kiuchi, Takayuki; Ono, Ayako; Miyajima, Syota; Nagai, Katsura; Higashi, Tsunehito; Matsuno, Yoshihiro; Dosaka-Akita, Hirotoshi; Nishimura, Masaharu; Miwa, Soichi; Kasahara, Masanori

2015-06-01

Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

Structural analysis of the 26S proteasome by cryoelectron tomography

International Nuclear Information System (INIS)

Nickell, Stephan; Mihalache, Oana; Beck, Florian; Hegerl, Reiner; Korinek, Andreas; Baumeister, Wolfgang

2007-01-01

The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

Science.gov (United States)

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

2014-01-01

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

Proteasomes in the archaea: from structure to function.

Science.gov (United States)

Maupin-Furlow, J A; Wilson, H L; Kaczowka, S J; Ou, M S

2000-09-01

Survival of cells is critically dependent on their ability to rapidly adapt to changes in the natural environment no matter how 'extreme'the habitat. An interplay between protein folding and hydrolysis is emerging as a central mechanism for stress survival and proper cell function. In eucaryotic cells, most proteins destined for destruction are covalently modified by the ubiquitin-system and then degraded in an energy-dependent mechanism by the 26S proteasome, a multicatalytic protease. The 26S proteasome is composed of a 20S proteolytic core and 19S cap (PA700) regulator which includes six AAA+ ATPase subunits. Related AAA+ proteins and 20S proteasomes are found in the archaea and Gram positive actinomycetes. In general, 20S proteasomes form a barrel-shaped nanocompartment with narrow openings which isolate rather non-specific proteolytic active-sites to the interior of the cylinder and away from interaction with cytosolic proteins. The proteasome-associated AAA+ proteins are predicted to form ring-like structures which unfold substrate proteins for entry into the central proteolytic 20S chamber resulting in an energy-dependent and processive destruction of the protein. Detailed biochemical and biophysical analysis as well as identification of proteasomes in archaea with developed genetic tools are providing a foundation for understanding the biological role of the proteasome in these unusual organisms.

Proteins interacting with the 26S proteasome

DEFF Research Database (Denmark)

Hartmann-Petersen, R; Gordon, C

2004-01-01

The 26S proteasome is the multi-protein protease that recognizes and degrades ubiquitinylated substrates targeted for destruction by the ubiquitin pathway. In addition to the well-documented subunit organization of the 26S holoenzyme, it is clear that a number of other proteins transiently...... associate with the 26S complex. These transiently associated proteins confer a number of different roles such as substrate presentation, cleavage of the multi-ubiquitin chain from the protein substrate and turnover of misfolded proteins. Such activities are essential for the 26S proteasome to efficiently...... fulfill its intracellular function in protein degradation....

Detection of antibodies to the 20s proteasome by ELISA

DEFF Research Database (Denmark)

Jørgensen, Karin Meinike; Frederiksen, Jette Lautrup; Nielsen, Christoffer Tandrup

2013-01-01

The presence of antibodies against the 20S proteasome has been correlated with diseases like multiple sclerosis (MS) and systemic lupus erythematosus (SLE) but no definite association has been established. In order to investigate this further, we optimized an ELISA for proteasome antibodies...

MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function

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Hofmann Kay

2002-09-01

Full Text Available Abstract Background Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. Results We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. Conclusions The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity.

Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition

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Seung-Wook Shin

2012-11-01

During the maternal-to-zygotic transition (MZT, maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS, and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

WIDE AND THICK GRAIN 1, which encodes an otubain-like protease with deubiquitination activity, influences grain size and shape in rice.

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Huang, Ke; Wang, Dekai; Duan, Penggen; Zhang, Baolan; Xu, Ran; Li, Na; Li, Yunhai

2017-09-01

Grain size and shape are two crucial traits that influence grain yield and grain appearance in rice. Although several factors that affect grain size have been described in rice, the molecular mechanisms underlying the determination of grain size and shape are still elusive. In this study we report that WIDE AND THICK GRAIN 1 (WTG1) functions as an important factor determining grain size and shape in rice. The wtg1-1 mutant exhibits wide, thick, short and heavy grains and also shows an increased number of grains per panicle. WTG1 determines grain size and shape mainly by influencing cell expansion. WTG1 encodes an otubain-like protease, which shares similarity with human OTUB1. Biochemical analyses indicate that WTG1 is a functional deubiquitinating enzyme, and the mutant protein (wtg1-1) loses this deubiquitinating activity. WTG1 is expressed in developing grains and panicles, and the GFP-WTG1 fusion protein is present in the nucleus and cytoplasm. Overexpression of WTG1 results in narrow, thin, long grains due to narrow and long cells, further supporting the role of WTG1 in determining grain size and shape. Thus, our findings identify the otubain-like protease WTG1 to be an important factor that determines grain size and shape, suggesting that WTG1 has the potential to improve grain size and shape in rice. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

ρ0 Cells Feature De-Ubiquitination of SLC Transporters and Increased Levels and Fluxes of Amino Acids

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André Bordinassi Medina

2017-04-01

Full Text Available Solute carrier (SLC transporters are a diverse group of membrane transporter proteins that regulate the cellular flux and distribution of endogenous and xenobiotic compounds. Post-translational modifications (PTMs, such as ubiquitination, have recently emerged as one of the major regulatory mechanisms in protein function and localization. Previously, we showed that SLC amino acid transporters were on average 6-fold de-ubiquitinated and increased amino acid levels were detected in ρ0 cells (lacking mitochondrial DNA, mtDNA compared to parental cells. Here, we elucidated the altered functionality of SLC transporters and their dynamic ubiquitination status by measuring the uptake of several isotopically labeled amino acids in both human osteosarcoma 143B.TK- and ρ0 cells. Our pulse chase analysis indicated that de-ubiquitinated amino acid transporters in ρ0 cells were accompanied by an increased transport rate, which leads to higher levels of amino acids in the cell. Finding SLC transport enhancers is an aim of the pharmaceutical industry in order to compensate for loss of function mutations in these genes. Thus, the ubiquitination status of SLC transporters could be an indicator for their functionality, but evidence for a direct connection between de-ubiquitination and transporter activity has to be further elucidated.

Proteasome impairment by α-synuclein.

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Lisa Zondler

Full Text Available Parkinson's disease (PD is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V-GFP cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

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Amanda C. Stacer

2015-08-01

Full Text Available Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

Trial Watch: Proteasomal inhibitors for anticancer therapy.

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Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

2015-01-01

The so-called "ubiquitin-proteasome system" (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients.

Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

Energy Technology Data Exchange (ETDEWEB)

Saji, Chiaki [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Higashi, Chizuka; Niinaka, Yasufumi [Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Yamada, Koji [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Noguchi, Kohji [Faculty of Pharmacy, Keio University, 1-5-30 Shiba-koen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

2011-12-02

Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch.

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Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena

2014-09-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the "BAG-instructed proteasomal to autophagosomal switch and sorting" (BIPASS).

Fulltext PDF

Indian Academy of Sciences (India)

'Ubiquitous' Quality Control of life. C S Karigar and K R ..... Salt precipitation: A laboratory method for precipitation of proteins. Deubiquitination: Release of the ... already in clinical trials is the proteasome inhibitor Ve1cade. (PS341), that is ...

Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

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Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

2016-02-11

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a

Structure and function based design of Plasmodium-selective proteasome inhibitors

Science.gov (United States)

Li, Hao; O'Donoghue, Anthony J.; van der Linden, Wouter A.; Xie, Stanley C.; Yoo, Euna; Foe, Ian T.; Tilley, Leann; Craik, Charles S.; da Fonseca, Paula C. A.; Bogyo, Matthew

2016-01-01

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the

Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe

DEFF Research Database (Denmark)

Cabrera, Rodrigo; Sha, Zhe; Vadakkan, Tegy J.

2010-01-01

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasom...

Transcriptional upregulation of BAG3 upon proteasome inhibition

International Nuclear Information System (INIS)

Wang Huaqin; Liu Haimei; Zhang Haiyan; Guan Yifu; Du Zhenxian

2008-01-01

Proteasome inhibitors exhibit antitumoral activity against malignancies of different histology. Emerging evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, thus it seems plausible that activation of survival signaling cascades might compromise their antitumoral effects. Bcl-2-associated athanogene (BAG) family proteins are characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance, including heat shock proteins (HSP), Bcl-2, Raf-1. In this report, we demonstrated that BAG3 is a novel antiapoptotic molecule induced by proteasome inhibitors in various cancer cells at the transcriptional level. Moreover, we demonstrated that BAG3 knockdown by siRNA sensitized cancer cells to MG132-induced apoptosis. Taken together, our results suggest that BAG3 induction might represents as an unwanted molecular consequence of utilizing proteasome inhibitors to combat tumors

Targeting proteasomes in infectious organisms to combat disease.

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Bibo-Verdugo, Betsaida; Jiang, Zhenze; Caffrey, Conor R; O'Donoghue, Anthony J

2017-05-01

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents. © 2017 Federation of European Biochemical Societies.

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

Science.gov (United States)

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

2001-01-01

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

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta : Implications for a proteasome-to-autophagy switch

NARCIS (Netherlands)

Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F.; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch

NARCIS (Netherlands)

Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F.; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena

2014-01-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested

Regulation of G Protein-Coupled Receptors by Ubiquitination

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Kamila Skieterska

2017-04-01

Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

Science.gov (United States)

Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

2017-01-01

In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

Proteasome phosphorylation regulates cocaine-induced sensitization.

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Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

The ubiquitin-proteasome system and chromosome 17 in cerebellar granule cells and medulloblastoma subgroups.

Science.gov (United States)

Vriend, Jerry; Marzban, Hassan

2017-02-01

Chromosome 17 abnormalities are often observed in medulloblastomas (MBs), particularly those classified in the consensus Groups 3 and 4. Herein we review MB signature genes associated with chromosome 17 and the relationship of these signature genes to the ubiquitin-proteasome system. While clinical investigators have not focused on the ubiquitin-proteasome system in relation to MB, a substantial amount of data on the topic has been hidden in the form of supplemental datasets of gene expression. A supplemental dataset associated with the Thompson classification of MBs shows that a subgroup of MB with 17p deletions is characterized by reduced expression of genes for several core particle subunits of the beta ring of the proteasome (β1, β4, β5, β7). One of these genes (PSMB6, the gene for the β1 subunit) is located on chromosome 17, near the telomeric end of 17p. By comparison, in the WNT group of MBs only one core proteasome subunit, β6, associated with loss of a gene (PSMB1) on chromosome 6, was down-regulated in this dataset. The MB subgroups with the worst prognosis have a significant association with chromosome 17 abnormalities and irregularities of APC/C cyclosome genes. We conclude that the expression of proteasome subunit genes and genes for ubiquitin ligases can contribute to prognostic classification of MBs. The therapeutic value of targeting proteasome subunits and ubiquitin ligases in the various subgroups of MB remains to be determined separately for each classification of MB.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluster

Directory of Open Access Journals (Sweden)

Scott Christopher J

2010-04-01

Full Text Available Abstract Background The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A. Subsequently we have identified a number of human family members and shown that one of these (DUB-3 is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1. Results Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable beta-defensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating. Conclusions The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells.

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Noolu, Bindu; Ajumeera, Rajanna; Chauhan, Anitha; Nagalla, Balakrishna; Manchala, Raghunath; Ismail, Ayesha

2013-01-09

Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau's method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification of active component(s) from the leaf

Use of proteasome inhibitors in anticancer therapy

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Sara M. Schmitt

2011-10-01

Full Text Available The importance of the ubiquitin-proteasome pathway to cellular function has brought it to the forefront in the search for new anticancer therapies. The ubiquitin-proteasome pathway has proven promising in targeting various human cancers. The approval of the proteasome inhibitor bortezomib for clinical treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma has validated the ubiquitin-proteasome as a rational target. Bortezomib has shown positive results in clinical use but some toxicity and side effects, as well as resistance, have been observed, indicating that further development of novel, less toxic drugs is necessary. Because less toxic drugs are necessary and drug development can be expensive and time-consuming, using existing drugs that can target the ubiquitin-proteasome pathway in new applications, such as cancer therapy, may be effective in expediting the regulatory process and bringing new drugs to the clinic. Toward this goal, previously approved drugs, such as disulfiram, as well as natural compounds found in common foods, such as green tea polyphenol (--EGCG and the flavonoid apigenin, have been investigated for their possible proteasome inhibitory and cell death inducing abilities. These compounds proved quite promising in preclinical studies and have now moved into clinical trials, with preliminary results that are encouraging. In addition to targeting the catalytic activity of the proteasome pathway, upstream regulators, such as the 19S regulatory cap, as well as E1, E2, and E3, are now being investigated as potential drug targets. This review outlines the development of novel proteasome inhibitors from preclinical to clinical studies, highlighting their abilities to inhibit the tumor proteasome and induce apoptosis in several human cancers.

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

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Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

2017-01-10

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

HIV-1 replication through hHR23A-mediated interaction of Vpr with 26S proteasome.

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Ge Li

2010-06-01

Full Text Available HIV-1 Vpr is a virion-associated protein. Its activities link to viral pathogenesis and disease progression of HIV-infected patients. In vitro, Vpr moderately activates HIV-1 replication in proliferating T cells, but it is required for efficient viral infection and replication in vivo in non-dividing cells such as macrophages. How exactly Vpr contributes to viral replication remains elusive. We show here that Vpr stimulates HIV-1 replication at least in part through its interaction with hHR23A, a protein that binds to 19S subunit of the 26S proteasome and shuttles ubiquitinated proteins to the proteasome for degradation. The Vpr-proteasome interaction was initially discovered in fission yeast, where Vpr was shown to associate with Mts4 and Mts2, two 19S-associated proteins. The interaction of Vpr with the 19S subunit of the proteasome was further confirmed in mammalian cells where Vpr associates with the mammalian orthologues of fission yeast Mts4 and S5a. Consistently, depletion of hHR23A interrupts interaction of Vpr with proteasome in mammalian cells. Furthermore, Vpr promotes hHR23A-mediated protein-ubiquitination, and down-regulation of hHR23A using RNAi significantly reduced viral replication in non-proliferating MAGI-CCR5 cells and primary macrophages. These findings suggest that Vpr-proteasome interaction might counteract certain host restriction factor(s to stimulate viral replication in non-dividing cells.

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

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

2012-01-01

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-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β 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-β. In addition, each TGF-β 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-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β 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.

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

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Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

2015-01-01

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

Impaired proteasome function in sporadic amyotrophic lateral sclerosis.

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Kabashi, Edor; Agar, Jeffrey N; Strong, Michael J; Durham, Heather D

2012-06-01

Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS.

On the role of proteasomes in cell biology and proteasome inhibition as a novel frontier in the development of immunosuppressants.

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Wu, Jiangping

2002-11-01

The proteasome, a large protease complex in cells, is the major machinery for protein degradation. It was previously considered a humble garbage collector, performing housekeeping duties to remove misfolded or spent proteins. Until recently, the interests of immunologists in proteasomes were focused largely on its role in antigen processing. Its real importance in cell biology has only been revealed contemporarily due to the availability of relatively specific inhibitors. It has now become increasingly clear that many aspects of immune responses highly depend on proper proteasome activity. Recently, a proteasome inhibitor has been successfully used to prevent acute as well as ongoing heart allograft rejection in mice. Such inhibitors are also efficacious in treating several autoimmune diseases, such as arthritis, psoriasis, and probably type I diabetes, in animal models. Phase II and III clinical trials of proteasome inhibitors in treating various tumors have shown promising results, and the side-effects of these drugs are tolerable. Therefore, proteasome inhibition represents a new and promising frontier in immunosuppressant development.

UBE2S associated with OSCC proliferation by promotion of P21 degradation via the ubiquitin-proteasome system

International Nuclear Information System (INIS)

Yoshimura, Shusaku; Kasamatsu, Atsushi; Nakashima, Dai; Iyoda, Manabu; Kasama, Hiroki; Saito, Tomoaki; Takahara, Toshikazu; Endo-Sakamoto, Yosuke; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2017-01-01

Ubiquitin-conjugating enzyme E2S (UBE2S), a family of E2 protein in the ubiquitin-proteasome system, is highly expressed in several types of cancers; however, its roles in oral squamous cell carcinoma (OSCC) have not yet been well elucidated. The purpose of this study was to clarify the functional activities of UBE2S in OSCCs. We analyzed the expression levels of UBE2S in nine OSCC cell lines and primary OSCC tissues by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry (IHC). The correlations between UBE2S expression and clinical classifications of OSCCs were analyzed using the IHC scoring system. We also used UBE2S knockdown OSCC cells for functional assays (proliferation assay, flow cytometry, and Western blotting). UBE2S was overexpressed in OSCCs in vitro and in vivo and was correlated significantly (P < 0.05) with the primary tumoral size. The cellular growth was decreased and the cell-cycle was arrested in the G2/M phase in the UBE2S knockdown (shUBE2S) cells. The expression level of P21, a target of the ubiquitin-proteasome system, was increased in the shUBE2S cells because of lower anaphase activity that promotes complex subunit 3 (APC3), an E3 ubiquitin ligase, compared with shMock cells. These findings might promote the understanding of the relationship between UBE2S overexpression and oral cancer proliferation, indicating that UBE2S would be a potential biomarker of and therapeutic target in OSCCs. - Highlights: • UBE2S contributes to tumor progression in OSCCs. • UBE2S regulated the cell-cycle arrest at G2/M phase in OSCC cells. • UBE2S and APC3 co-regulate the expression level of P21 at G2/M check point via the ubiquitin-proteasome system. • P21 is one of the proliferation-regulating factors in OSCC. • UBE2S would be a potential therapeutic target for OSCCs.

Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

DEFF Research Database (Denmark)

Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

2003-01-01

Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

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Candida Vannini

Full Text Available Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

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Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-07-05

Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome.

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Bolten, Marcel; Delley, Cyrille L; Leibundgut, Marc; Boehringer, Daniel; Ban, Nenad; Weber-Ban, Eilika

2016-12-06

Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex. Copyright © 2016 Elsevier Ltd. All rights reserved.

Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells

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Noolu Bindu

2013-01-01

Full Text Available Abstract Background Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves, a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Methods Hydro-methanolic extract of curry leaves (CLE was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau’s method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. Results CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Conclusions Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

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Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

2015-03-01

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

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Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L; Hink, Mark A; Bates, Gillian P; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

2014-01-03

Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease. Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

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Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

2014-01-01

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

OpenAIRE

Lomonosova, Elena; Ryerse, Jan; Chinnadurai, G.

2009-01-01

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds...

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

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Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

2013-09-01

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

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

International Nuclear Information System (INIS)

Kwak, Mi-Kyoung; Kensler, Thomas W.

2006-01-01

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

Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

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Gili Ben-Nissan

2014-09-01

Full Text Available For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by the core 20S proteasome itself. Degradation by the 20S proteasome does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the inherent structural disorder of the protein being degraded. Thus, proteins that contain unstructured regions due to oxidation, mutation, or aging, as well as naturally, intrinsically unfolded proteins, are susceptible to 20S degradation. Unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome, relatively little is known about the means by which 20S-mediated proteolysis is controlled. Here, we describe our current understanding of the regulatory mechanisms that coordinate 20S proteasome-mediated degradation, and highlight the gaps in knowledge that remain to be bridged.

Insulin-like Growth Factor-I Mediates Neuroprotection in Proteasome Inhibition-Induced Cytotoxicity in SH-SY5Y Cells

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Cheng, Benxu; Maffi, Shivani Kaushal; Martinez, Alex Anthony; Acosta, Yolanda P Villarreal; Morales, Liza D; Roberts, James L

2011-01-01

The proteasome is an enzyme complex responsible for targeted intracellular proteolysis. Alterations in proteasome-mediated protein clearance have been implicated in the pathogenesis of aging, Alzheimer's disease (AD) and Parkinson's disease (PD). In such diseases, proteasome inhibition may contribute to formation of abnormal protein aggregates, which in turn activate intracellular unfolded protein responses that cause oxidative stress and apoptosis. In this study, we investigated the protective effect of Insulin-like Growth Factor-I (IGF-1) for neural SH-SY5Y cells treated with the proteasomal inhibitor, Epoxomicin, In SH-SY5Y cells, Epoxomicin treatment results in accumulation of intracellular ubiquitinated proteins and cytochrome c release from damaged mitochondria, leading to cell death, in Epoxomicin time- and dose-dependent manner. In cells treated with small amounts of IGF-1, the same dosages of Epoxomicin reduced both mitochondrial damage (cytochrome c release) and reduced caspase-3 activation and PARP cleavage, both of which are markers of apoptosis. Notably, however, IGF-1-treated SH-SY5Y cells still contained ubiquitinated protein aggregates. This result indicates that IGF-1 blocks the downstream apoptotic consequences of Epoxomicin treatment leading to decreased proteasome function. Clues as to the mechanism for this protective effect come from (a) increased AKT phosphorylation observed in IGF-1-protected cells, vs. cells exposed to Epoxomicin without IGF-1, and (b) reduction of IGF-1 protection by pretreatment of the cells with LY294002 (an inhibitor of PI3-kinase). Together these findings suggest that activation of PI3/AKT pathways by IGF-1 is involved in IGF-1 neuroprotection against apoptosis following proteasome inhibition. PMID:21545837

p53 mutations promote proteasomal activity.

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Oren, Moshe; Kotler, Eran

2016-07-27

p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

Targeting proteasomes in infectious organisms to combat disease

OpenAIRE

Bibo-Verdugo, B; Jiang, Z; Caffrey, CR; O'Donoghue, AJ

2017-01-01

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein...

Mechanisms promoting and inhibiting the process of proteasomal degradation of cells

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Pedrycz Agnieszka

2016-03-01

Full Text Available Defects in the process of degradation of unneeded cellular proteins underlie many diseases. This article discusses one of the most important systems of removal of abnormal proteins. It describes the process of ubiquitination of proteins for proteasome degradation. It also describes the structure of the 26S and 20S proteasomes and the mechanism of ubiquitin-proteasome system. Proteasome proteolytic system is highly specialized and organized. Protease-proteasome 26S is particularly important for proper cell functioning. It recognizes and degrades marked proteins. Inhibition of proteasome pathway leads to cell cycle arrest and apoptosis.

Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system.

Science.gov (United States)

Cerqueira, Paula G; Passos-Silva, Danielle G; Vieira-da-Rocha, João P; Mendes, Isabela Cecilia; de Oliveira, Karla A; Oliveira, Camila F B; Vilela, Liza F F; Nagem, Ronaldo A P; Cardoso, Joseane; Nardelli, Sheila C; Krieger, Marco A; Franco, Glória R; Macedo, Andrea M; Pena, Sérgio D J; Schenkman, Sérgio; Gomes, Dawidson A; Guerra-Sá, Renata; Machado, Carlos R

2017-03-01

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and Characterisation of a Proteasome -

DEFF Research Database (Denmark)

Andersen, Katrine Mølgaard

this domain. A txl1 yeast knockout mutant displays a synthetic growth defect with a cut8 knockout, whereas the txc1 knockout does not. In fission yeast, Cut8 is a nuclear protein that tethers the 26S proteasome to the nuclear membrane. In both wild type cells and in a cut8 null mutant Txl1 co......-down of Txnl1 in HeLa cells, whereas no stabilisation was detected in txl1¿ cells in S. pombe. In human cells, an active site Txnl1 mutant formed a mixed disulfide intermediate with eEF1A1, a reported substrate-recruiting factor of the 26S proteasome. Fission yeast Txl1 can reduce an oxidised proteasomal model...

Proteasome function is not impaired in healthy aging of the lung.

Science.gov (United States)

Caniard, Anne; Ballweg, Korbinian; Lukas, Christina; Yildirim, Ali Ö; Eickelberg, Oliver; Meiners, Silke

2015-10-01

Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

Txl1 and Txc1 are co-factors of the 26S proteasome in fission yeast

DEFF Research Database (Denmark)

Andersen, Katrine M; Jensen, Camilla; Kriegenburg, Franziska

2011-01-01

and thereby equips proteasomes with redox capabilities. Here, we characterize the fission yeast orthologue of Txnl1, called Txl1. Txl1 associates with the 26S proteasome via its C-terminal domain. This domain is also found in the uncharacterized protein, Txc1, which was also found to interact with 26S...

Proteasome- and Ethanol-Dependent Regulation of HCV-Infection Pathogenesis

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Natalia A. Osna

2014-09-01

Full Text Available This paper reviews the role of the catabolism of HCV and signaling proteins in HCV protection and the involvement of ethanol in HCV-proteasome interactions. HCV specifically infects hepatocytes, and intracellularly expressed HCV proteins generate oxidative stress, which is further exacerbated by heavy drinking. The proteasome is the principal proteolytic system in cells, and its activity is sensitive to the level of cellular oxidative stress. Not only host proteins, but some HCV proteins are degraded by the proteasome, which, in turn, controls HCV propagation and is crucial for the elimination of the virus. Ubiquitylation of HCV proteins usually leads to the prevention of HCV propagation, while accumulation of undegraded viral proteins in the nuclear compartment exacerbates infection pathogenesis. Proteasome activity also regulates both innate and adaptive immunity in HCV-infected cells. In addition, the proteasome/immunoproteasome is activated by interferons, which also induce “early” and “late” interferon-sensitive genes (ISGs with anti-viral properties. Cleaving viral proteins to peptides in professional immune antigen presenting cells and infected (“target” hepatocytes that express the MHC class I-antigenic peptide complex, the proteasome regulates the clearance of infected hepatocytes by the immune system. Alcohol exposure prevents peptide cleavage by generating metabolites that impair proteasome activity, thereby providing escape mechanisms that interfere with efficient viral clearance to promote the persistence of HCV-infection.

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta

Science.gov (United States)

Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena

2014-01-01

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS). PMID:25046115

Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice.

Science.gov (United States)

Le Pecheur, Marie; Bourdon, Emmanuel; Paly, Evelyne; Farout, Luc; Friguet, Bertrand; London, Jacqueline

2005-07-04

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.

E1AF degradation by a ubiquitin-proteasome pathway

International Nuclear Information System (INIS)

Takahashi, Akiko; Higashino, Fumihiro; Aoyagi, Mariko; Yoshida, Koichi; Itoh, Miyuki; Kobayashi, Masanobu; Totsuka, Yasunori; Kohgo, Takao; Shindoh, Masanobu

2005-01-01

E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function

A new structural class of proteasome inhibitors that prevent NF-kappa B activation.

Science.gov (United States)

Lum, R T; Kerwar, S S; Meyer, S M; Nelson, M G; Schow, S R; Shiffman, D; Wick, M M; Joly, A

1998-05-01

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit IC50 values ranging from 0.1 to 0.5 microgram/mL (0.1 to 1 microM). In cell proliferation assays, these compounds inhibit growth with an IC50 ranging from 5 to 10 micrograms/mL (10-20 microM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu-D-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor kappa B (NF-kappa B) in vitro by preventing signal-induced degradation of I kappa B-alpha. In these studies, the I kappa B-alpha that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit I kappa B-alpha kinase, the enzyme responsible for signal-induced phosphorylation of I kappa B-alpha. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 +/- 59 and 83 +/- 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 +/- 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-kappa B activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-kappa B.

Altered composition of liver proteasome assemblies contributes to enhanced proteasome activity in the exceptionally long-lived naked mole-rat.

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Rodriguez, Karl A; Edrey, Yael H; Osmulski, Pawel; Gaczynska, Maria; Buffenstein, Rochelle

2012-01-01

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

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Przemysław Karpowicz

Full Text Available The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

Science.gov (United States)

Murakawa, Yasuhiro; Sonoda, Eiichiro; Barber, Louise J; Zeng, Weihua; Yokomori, Kyoko; Kimura, Hiroshi; Niimi, Atsuko; Lehmann, Alan; Zhao, Guang Yu; Hochegger, Helfrid; Boulton, Simon J; Takeda, Shunichi

2007-09-15

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

Science.gov (United States)

Hyer, Marc L; Milhollen, Michael A; Ciavarri, Jeff; Fleming, Paul; Traore, Tary; Sappal, Darshan; Huck, Jessica; Shi, Judy; Gavin, James; Brownell, Jim; Yang, Yu; Stringer, Bradley; Griffin, Robert; Bruzzese, Frank; Soucy, Teresa; Duffy, Jennifer; Rabino, Claudia; Riceberg, Jessica; Hoar, Kara; Lublinsky, Anya; Menon, Saurabh; Sintchak, Michael; Bump, Nancy; Pulukuri, Sai M; Langston, Steve; Tirrell, Stephen; Kuranda, Mike; Veiby, Petter; Newcomb, John; Li, Ping; Wu, Jing Tao; Powe, Josh; Dick, Lawrence R; Greenspan, Paul; Galvin, Katherine; Manfredi, Mark; Claiborne, Chris; Amidon, Benjamin S; Bence, Neil F

2018-02-01

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Altered composition of liver proteasome assemblies contributes to enhanced proteasome activity in the exceptionally long-lived naked mole-rat.

Directory of Open Access Journals (Sweden)

Karl A Rodriguez

Full Text Available The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber, maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L activity and a two-fold increase in trypsin-like (T-L in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.

26 S proteasomes function as stable entities

DEFF Research Database (Denmark)

Hendil, Klavs B; Hartmann-Petersen, Rasmus; Tanaka, Keiji

2002-01-01

, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during...

Antiatherogenic effect of quercetin is mediated by proteasome inhibition in the aorta and circulating leukocytes.

Science.gov (United States)

Pashevin, Denis A; Tumanovska, Lesya V; Dosenko, Victor E; Nagibin, Vasyl S; Gurianova, Veronika L; Moibenko, Alexey A

2011-01-01

Quercetin, a plant-derived flavonoid, has attracted considerable attention as promising compound for heart disease prevention and therapy. It has been linked to decreased mortality from heart disease and decreased incidence of stroke. Here, we report new data showing the angioprotective properties of quercetin mediated by its effect on proteasomal proteolysis. This study was designed to investigate the ability of quercetin to modulate proteasomal activity in a rabbit model of cholesterol-induced atherosclerosis. First, we show proteasomal trypsin-like (TL) activity increased up to 2.4-fold, chymotrypsin-like (CTL) activity increased by up to 43% and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activity increased by up to 10% after 8 weeks of a cholesterol-rich diet. A single intravenous injection of the water-soluble form of quercetin (Corvitin) significantly decreased proteasomal TL activity 1.85-fold in monocytes, and decreased the CTL and PGPH activities more than 2-fold in polymorphonuclear leukocytes (PMNL) after 2 h. Prolonged administration (1 month) of Corvitin to animals following a cholesterol-rich diet significantly decreased all types of proteolytic proteasome activities both in tissues and in circulating leukocytes and was associated with the reduction of atherosclerotic lesion areas in the aorta. Additionally, the pharmacological form of quercetin (Quertin) was shown to have an antiatherogenic effect and an ability to inhibit proteasome activities.

Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

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Hannes C A Drexler

Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4.

Science.gov (United States)

Tsubouchi, Kazuya; Araya, Jun; Minagawa, Shunsuke; Hara, Hiromichi; Ichikawa, Akihiro; Saito, Nayuta; Kadota, Tsukasa; Sato, Nahoko; Yoshida, Masahiro; Kurita, Yusuke; Kobayashi, Kenji; Ito, Saburo; Fujita, Yu; Utsumi, Hirofumi; Yanagisawa, Haruhiko; Hashimoto, Mitsuo; Wakui, Hiroshi; Yoshii, Yutaka; Ishikawa, Takeo; Numata, Takanori; Kaneko, Yumi; Asano, Hisatoshi; Yamashita, Makoto; Odaka, Makoto; Morikawa, Toshiaki; Nakayama, Katsutoshi; Nakanishi, Yoichi; Kuwano, Kazuyoshi

2017-08-03

Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells.

Science.gov (United States)

Dron, Michel; Dandoy-Dron, Françoise; Farooq Salamat, Muhammad Khalid; Laude, Hubert

2009-08-01

Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

Antitumorigenic effect of proteasome inhibitors on insulinoma cells

DEFF Research Database (Denmark)

Størling, Joachim; Allaman-Pillet, Nathalie; Karlsen, Allan E

2004-01-01

inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also...

Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

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Lomax Michael A

2007-03-01

Full Text Available Abstract Background The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis Results Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA, relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p Conclusion In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

Science.gov (United States)

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

2013-01-01

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

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Panagiotis D Velentzas

Full Text Available Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6 or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4. Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18 autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue

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Nanduri, Bindu; Shack, Leslie A.; Rai, Aswathy N.; Epperson, William B.; Baumgartner, Wes; Schmidt, Ty B.; Edelmann, Mariola J.

2016-01-01

To develop a reproducible tissue-lysis method that retains enzyme function for activity-based protein profiling, we compared four different tissue lysis methods of bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue and focused ultrasonication had also the fastest pr...

Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

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Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F

2010-02-04

Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

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Koues Olivia I

2010-02-01

Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

Studies on terrein as a new class of proteasome inhibitors

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Demasi, M.; Felicio, A.L.; Lima, C.; Pacheco, A.O.; Leite, H.G.; Andrade, L.H.

2010-01-01

The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (muM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore, the present results point out for the possibility of exploring terrein as a new molecular fragment for the development of synthetic proteasome inhibitors. (author)

Denervation-Induced Activation of the Ubiquitin-Proteasome System Reduces Skeletal Muscle Quantity Not Quality.

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Baumann, Cory W; Liu, Haiming M; Thompson, LaDora V

2016-01-01

It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle's intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6-8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling.

Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

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Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

2015-05-07

The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21.

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

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Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

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Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

2013-06-01

Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

SOCS-1 Localizes to the Microtubule Organizing Complex-Associated 20S Proteasome

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Vuong, Bao Q.; Arenzana, Teresita L.; Showalter, Brian M.; Losman, Julie; Chen, X. Peter; Mostecki, Justin; Banks, Alexander S.; Limnander, Andre; Fernandes, Neil; Rothman, Paul B.

2005-01-01

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unkno...

Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

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Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

2015-01-01

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

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Barbara Hämmerle

Full Text Available Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2 neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

The 20S proteasome splicing activity discovered by SpliceMet.

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Juliane Liepe

2010-06-01

Full Text Available The identification of proteasome-generated spliced peptides (PSP revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS. For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

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

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Christian Muñoz

2015-01-01

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

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties.

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Fuchs, Adrian C D; Maldoner, Lorena; Hipp, Katharina; Hartmann, Marcus D; Martin, Jörg

2018-01-19

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct temporal requirements for autophagy and the proteasome in yeast meiosis.

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Wen, Fu-ping; Guo, Yue-shuai; Hu, Yang; Liu, Wei-xiao; Wang, Qian; Wang, Yuan-ting; Yu, Hai-Yan; Tang, Chao-ming; Yang, Jun; Zhou, Tao; Xie, Zhi-ping; Sha, Jia-hao; Guo, Xuejiang; Li, Wei

2016-01-01

Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.

Bladder cancer detection using a peptide substrate of the 20S proteasome.

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Gruba, Natalia; Wysocka, Magdalena; Brzezińska, Magdalena; Dębowski, Dawid; Sieńczyk, Marcin; Gorodkiewicz, Ewa; Guszcz, Tomasz; Czaplewski, Cezary; Rolka, Krzysztof; Lesner, Adam

2016-08-01

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods. © 2016 Federation of European Biochemical Societies.

Implication of altered ubiquitin-proteasome system and ER stress in the muscle atrophy of diabetic rats.

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Reddy, S Sreenivasa; Shruthi, Karnam; Prabhakar, Y Konda; Sailaja, Gummadi; Reddy, G Bhanuprakash

2018-02-01

Skeletal muscle is adversely affected in type-1 diabetes, and excessively stimulated ubiquitin-proteasome system (UPS) was found to be a leading cause of muscle wasting or atrophy. The role of endoplasmic reticulum (ER) stress in muscle atrophy of type-1 diabetes is not known. Hence, we investigated the role of UPS and ER stress in the muscle atrophy of chronic diabetes rat model. Diabetes was induced with streptozotocin (STZ) in male Sprague-Dawley rats and were sacrificed 2- and 4-months thereafter to collect gastrocnemius muscle. In another experiment, 2-months post-STZ-injection diabetic rats were treated with MG132, a proteasome inhibitor, for the next 2-months and gastrocnemius muscle was collected. The muscle fiber cross-sectional area was diminished in diabetic rats. The expression of UPS components: E1, MURF1, TRIM72, UCHL1, UCHL5, ubiquitinated proteins, and proteasome activity were elevated in the diabetic rats indicating activated UPS. Altered expression of ER-associated degradation (ERAD) components and increased ER stress markers were detected in 4-months diabetic rats. Proteasome inhibition by MG132 alleviated alterations in the UPS and ER stress in diabetic rat muscle. Increased UPS activity and ER stress were implicated in the muscle atrophy of diabetic rats and proteasome inhibition exhibited beneficiary outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

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Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.

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van Twest, Sylvie; Murphy, Vincent J; Hodson, Charlotte; Tan, Winnie; Swuec, Paolo; O'Rourke, Julienne J; Heierhorst, Jörg; Crismani, Wayne; Deans, Andrew J

2017-01-19

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

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Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

2010-01-15

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Insights into the Mechanism of Deubiquitination by JAMM Deubiquitinases from Cocrystal Structures of the Enzyme with the Substrate and Product

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2015-01-01

comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (∼20 μM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (∼20 μM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect. PMID:24787148

Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

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Milly M Choy

2015-11-01

Full Text Available The mosquito-borne dengue virus (DENV is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro.

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Nandita Sahana

Full Text Available The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV accumulation in its natural host papaya (Carica papaya. We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome, but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome, associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54, which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

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Qingxi Yue

Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

The use of activity based protein profiling to study proteasome biology

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Paniagua Soriano, Guillem

2016-01-01

The work described in this thesis focuses on the characterization of proteasome directed activity-based probes (ABPs) as well as on the adaptation mechanisms that make multiple myeloma derived cell lines resistant against proteasome inhibitors (PIs).

Peptide-based proteasome inhibitors in anticancer drug design.

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Micale, Nicola; Scarbaci, Kety; Troiano, Valeria; Ettari, Roberta; Grasso, Silvana; Zappalà, Maria

2014-09-01

The identification of the key role of the eukaryotic 26S proteasome in regulated intracellular proteolysis and its importance as a target in many pathological conditions wherein the proteasomal activity is defective (e.g., malignancies, autoimmune diseases, neurodegenerative diseases, etc.) prompted several research groups to the development of specific inhibitors of this multicatalytic complex with the aim of obtaining valid drug candidates. In regard to the anticancer therapy, the peptide boronate bortezomib (Velcade®) represents the first molecule approved by FDA for the treatment of multiple myeloma in 2003 and mantle cell lymphoma in 2006. Since then, a plethora of molecules targeting the proteasome have been identified as potential anticancer agents and a few of them reached clinical trials or are already in the market (i.e., carfilzomib; Kyprolis®). In most cases, the design of new proteasome inhibitors (PIs) takes into account a proven peptide or pseudopeptide motif as a base structure and places other chemical entities throughout the peptide skeleton in such a way to create an efficacious network of interactions within the catalytic sites. The purpose of this review is to provide an in-depth look at the current state of the research in the field of peptide-based PIs, specifically those ones that might find an application as anticancer agents. © 2014 Wiley Periodicals, Inc.

The 11S Proteasomal Activator REGγ Impacts Polyglutamine-Expanded Androgen Receptor Aggregation and Motor Neuron Viability through Distinct Mechanisms

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Jill M. Yersak

2017-05-01

Full Text Available Spinal and bulbar muscular atrophy (SBMA is caused by expression of a polyglutamine (polyQ-expanded androgen receptor (AR. The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28β counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation and contributed to polyQ-expanded AR toxicity in the presence of dihydrotestosterone (DHT. These effects of REGγ were independent of its association with the proteasome and may be due, in part, to the decreased binding of polyQ-expanded AR by the E3 ubiquitin-ligase MDM2. Unlike its effects in PC12 cells, REGγ overexpression rescued transgenic SBMA motor neurons from DHT-induced toxicity in a proteasome binding-dependent manner, suggesting that the degradation of a specific 11S proteasome substrate or substrates promotes motor neuron viability. One potential substrate that we found to play a role in mutant AR toxicity is the splicing factor SC35. These studies reveal that, depending on the cellular context, two biological roles for REGγ impact cell viability in the face of polyQ-expanded AR; a proteasome binding-independent mechanism directly promotes mutant AR aggregation while a proteasome binding-dependent mechanism promotes cell viability. The balance between these functions likely determines REGγ effects on polyQ-expanded AR-expressing cells.

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

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

2014-01-01

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

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

International Nuclear Information System (INIS)

Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon

2007-01-01

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

Apoptosis and radiosensitization of Hodgkin cells by proteasome inhibition

International Nuclear Information System (INIS)

Pajonk, Frank; Pajonk, Katja; McBride, William H.

2000-01-01

Purpose: Malignant cells from Hodgkin's disease have been reported to be defective in regulation of NF-κB activity. Ionizing radiation is known to activate NF-κB, and it has been suggested that this pathway may protect cells from apoptosis following exposure to radiation and other therapeutic agents. Defective NF-κB regulation in Hodgkin cells could therefore dictate the response of this disease to therapy, as well as be responsible for maintaining the malignant phenotype. The purpose of this study was to explore whether NF-κB activity could be modulated in Hodgkin cells and whether it determines the response of these cells to treatment with ionizing radiation and/or dexamethasone. Methods and Materials: Activation of NF-κB in cells is accomplished in large part by degradation of its inhibitor IκB through the 26s proteasome. HD-My-Z Hodgkin cells were treated with the proteasome inhibitor MG-132 or transduced with a dominant negative super-repressor IκBα. Clonogenic survival, apoptosis, proteasome activity, and NF-κB binding activity were monitored in response to ionizing radiation and/or dexamethasone treatment. Results: HD-My-Z Hodgkin cells had modest NF-κB levels but, unlike other cell types, did not decrease their level of constitutively active NF-κB in response to proteasome inhibition with MG-132. In contrast, transduction with a non-phosphorable IκBα construct abolished expression. MG-132 did, however, induce apoptosis in HD-My-Z cells and sensitized them to ionizing radiation. Dexamethasone treatment had no effect on NF-κB activity or clonogenic survival of Hodgkin cells, but protected them from irradiation. Conclusion: We conclude that inhibition of 26s proteasome activity can induce apoptosis in HD-My-Z Hodgkin cells and radiosensitize them, in spite of the fact that their constitutively active NF-κB levels are unaltered. The proteasome may be a promising new therapeutic target for intervention in this disease. In contrast, the use of

BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein.

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Chiappetta, Gennaro; Basile, Anna; Arra, Claudio; Califano, Daniela; Pasquinelli, Rosa; Barbieri, Antonio; De Simone, Veronica; Rea, Domenica; Giudice, Aldo; Pezzullo, Luciano; De Laurenzi, Vincenzo; Botti, Gerardo; Losito, Simona; Conforti, Daniela; Turco, Maria Caterina

2012-01-01

Anaplastic thyroid tumors (ATC) express high levels of BAG3, a member of the BAG family of cochaperone proteins that is involved in regulating cell apoptosis through multiple mechanisms. The objective of the study was the investigation of the influence of B-cell lymphoma-2-associated athanogene 3 (BAG3) on ATC growth. We investigated the effects of BAG3 down-modulation, obtained by using a specific small interfering RNA, on in vitro and in vivo growth of the human ATC cell line 8505C. Because BRAF protein plays an important role in ATC cell growth, we analyzed the effects of BAG3 down-modulation on BRAF protein levels. Furthermore, by using a proteasome inhibitor, we verified whether BAG3-mediated regulation of BRAF levels involved a proteasome-dependent mechanism. BAG3 down-modulation significantly inhibits ATC growth in vitro and in vivo. BAG3 coimmunoprecipitates with BRAF protein, and its down-modulation results in a significant reduction of BRAF protein levels, which can be reverted by incubation with the proteasome inhibitor MG132. BAG3 protein sustains ATC growth in vitro and in vivo. The underlying molecular mechanism appears to rely on BAG3 binding to BRAF, thus protecting it from proteasome-dependent degradation. These results are in line with the reported ability of BAG3 to interfere with the proteasomal delivery of a number of other client proteins.

Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

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Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

2010-02-01

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

FoxM1 is a general target for proteasome inhibitors.

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Uppoor G Bhat

2009-08-01

Full Text Available Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

Proteasome inhibitors block DNA repair and radiosensitize non-small cell lung cancer.

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Kyle R Cron

Full Text Available Despite optimal radiation therapy (RT, chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80-90% decrease in homologous recombination (HR, a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.

Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

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Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

2013-01-01

SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly

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Martín Hugo

2018-06-01

Full Text Available Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.

Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

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Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

2008-06-01

In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

High-throughput siRNA screening applied to the ubiquitin-proteasome system

DEFF Research Database (Denmark)

Poulsen, Esben Guldahl; Nielsen, Sofie V.; Pietras, Elin J.

2016-01-01

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Due to the large number of genes dedicated to the ubiquitin-proteasome system, mapping degradation pathways for short lived proteins is a daunting task, in particular in mammalian cells...

The Ubiquitin-associated (UBA) 1 Domain of Schizosaccharomyces pombe Rhp23 Is Essential for the Recognition of Ubiquitin-proteasome System Substrates Both in Vitro and in Vivo*

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Medina, Bethan; Paraskevopoulos, Konstantinos; Boehringer, Jonas; Sznajder, Anna; Robertson, Morag; Endicott, Jane; Gordon, Colin

2012-01-01

The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys48-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2. PMID:23038266

Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

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Ghosh, Rajeshwary; Goswami, Sumanta K; Feitoza, Luis Felipe B B; Hammock, Bruce; Gomes, Aldrin V

2016-11-15

One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors

NARCIS (Netherlands)

Hiroi, Toyoko; Deming, Clayton B.; Zhao, Haige; Hansen, Baranda S.; Arkenbout, Elisabeth K.; Myers, Thomas J.; McDevitt, Michael A.; Rade, Jeffrey J.

2009-01-01

OBJECTIVE: Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathological thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We

Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations.

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Lydia Chang

Full Text Available Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS. However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

Proteasome (Prosome Subunit Variations during the Differentiation of Myeloid U937 Cells

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Laurent Henry

1997-01-01

Full Text Available 20S proteasomes (prosomes/multicatalytic proteinase are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3, p27K (IOTA and p30/33K (C2 subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance

International Nuclear Information System (INIS)

Kraus, M; Bader, J; Overkleeft, H; Driessen, C

2013-01-01

HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC 50 near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μℳ. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro

Proteasome activity related with the daily physical activity of COPD patients

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Lee KY

2017-05-01

Full Text Available Kang-Yun Lee,1,2,* Tzu-Tao Chen,1,* Ling-Ling Chiang,1,3 Hsiao-Chi Chuang,1,3 Po-Hao Feng,1,2 Wen-Te Liu,1–3 Kuan-Yuan Chen,1 Shu-Chuan Ho1,3 1Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, 2Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, 3School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan *These authors contributed equally to this work Background: COPD is a debilitating disease that affects patients’ daily lives. One’s daily physical activity (DPA decreases due to multifactorial causes, and this decrease is correlated with a poor prognosis in COPD patients. Muscle wasting may at least be partly due to increased activity of the ubiquitin proteasome pathway and apoptosis.Methods: This study investigated the relationships among DPA, circulating proteasome activity, and protein carbonyl in COPD patients and healthy subjects (HSs. This study included 57 participants (42 patients and 15 healthy subjects. Ambulatory DPA was measured using actigraphy, and oxygen saturation was measured with a pulse oximeter.Results: COPD patients had lower DPA, lower 6 min walking distance (6MWD, lower delta saturation pulse oxygenation (SpO2 during the 6MWT, and lower delta SpO2 during DPA than HSs. COPD patients had higher proteasome activity and protein carbonyl than HSs. Circulating proteasome activity was significantly negatively correlated with DPA (r=−0.568, P<0.05 in COPD patients, whereas delta SpO2 during the 6MWT was significantly positively correlated with proteasome activity (r=0.685, P<0.05 in HSs. Protein carbonyl was significantly negatively correlated with the body mass index (r=−0.318, P<0.05, mid-arm circumference (r=0.350, P<0.05, calf circumference (r=0.322, P<0.05, forced expiratory volume in the first second (r=−0.441, P<0

VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8.

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Smith, Gina A; Fearnley, Gareth W; Abdul-Zani, Izma; Wheatcroft, Stephen B; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

2016-01-01

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology. © 2015 The Authors. Traffic published by John Wiley & Sons Ltd.

Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

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Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

2005-03-01

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

Prediction of proteasome cleavage motifs by neural networks

DEFF Research Database (Denmark)

Kesimir, C.; Nussbaum, A.K.; Schild, H.

2002-01-01

physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

DEFF Research Database (Denmark)

Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas

2015-01-01

Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM...

The 20S proteasome as an assembly platform for the 19S regulatory complex

DEFF Research Database (Denmark)

Hendil, Klaus Aksel Bjørner; Kriegenburg, Franziska; Tanaka, Keiji

2009-01-01

26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which...... is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly...... complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base....

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis.

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Kabashi, Edor; Agar, Jeffrey N; Taylor, David M; Minotti, Sandra; Durham, Heather D

2004-06-01

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex)

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Weitman, D.; Etlinger, J. D.

1992-01-01

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

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Chiu, Rex Shun; Pan, Shiyue; Zhao, Rongmin; Gazzarrini, Sonia

2016-12-01

During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing.

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Snoberger, Aaron; Brettrager, Evan J; Smith, David M

2018-06-18

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN's symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases' coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells.

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Ramos de Carvalho, J Emanuel; Verwoert, Milan T; Vogels, Ilse M C; Schipper-Krom, Sabine; Van Noorden, Cornelis J F; Reits, Eric A; Klaassen, Ingeborg; Schlingemann, Reinier O

2018-01-01

Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β2 and β5i/β1 and reduced activity of β5/β1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells. The Author(s). Published by S. Karger AG, Basel.

Effects of Radiation on Proteasome Function in Prostate Cancer Cells

Science.gov (United States)

2009-02-01

precursor [ Homo sapiens ] BCL2-associated athanogene 3 adhesion regulating molecule 1 precursor CD44 antigen heat shock 27kDa protein 1 DnaJ... feature of CICs that can easily be exploited to identify , track, and target them in vitro and in vivo . J Natl Cancer Inst 2009;101:1–10 Q1 Q5 JNCI...P = .055; Supplementary Figure 1, D and E, available online), indicating that reduced proteasome activity in CICs is a feature found across tumor

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

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Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

2011-02-01

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

KAUST Repository

Magnacca, A.

2012-07-17

Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

Poly-Ub-substrate-degradative activity of 26S proteasome is not impaired in the aging rat brain.

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Carolin Giannini

Full Text Available Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young and 24 month old (aged rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process.

Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

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Karl Andrew Rodriguez

2014-11-01

Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.

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Boehringer, Jonas; Riedinger, Christiane; Paraskevopoulos, Konstantinos; Johnson, Eachan O D; Lowe, Edward D; Khoudian, Christina; Smith, Dominique; Noble, Martin E M; Gordon, Colin; Endicott, Jane A

2012-11-15

The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

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Lafuente Esther M

2010-09-01

Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

An extract of Artemisia dracunculus L. inhibits ubiquitin-proteasome activity and preserves skeletal muscle mass in a murine model of diabetes.

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Heather Kirk-Ballard

Full Text Available 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-A(y 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-A(y 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.

Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

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Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

2013-09-01

Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

OpenAIRE

Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

2010-01-01

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degra...

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

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Scaife Jes R

2008-02-01

Full Text Available Abstract 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 of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of

The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence.

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Suayib Üstün

Full Text Available The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv requires type III effector proteins (T3Es for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

Discovery of an Inhibitor of the Proteasome Subunit Rpn11.

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Perez, Christian; Li, Jing; Parlati, Francesco; Rouffet, Matthieu; Ma, Yuyong; Mackinnon, Andrew L; Chou, Tsui-Fen; Deshaies, Raymond J; Cohen, Seth M

2017-02-23

The proteasome plays a crucial role in degradation of normal proteins that happen to be constitutively or inducibly unstable, and in this capacity it plays a regulatory role. Additionally, it degrades abnormal/damaged/mutant/misfolded proteins, which serves a quality-control function. Inhibitors of the proteasome have been validated in the treatment of multiple myeloma, with several FDA-approved therapeutics. Rpn11 is a Zn 2+ -dependent metalloisopeptidase that hydrolyzes ubiquitin from tagged proteins that are trafficked to the proteasome for degradation. A fragment-based drug discovery (FBDD) approach was utilized to identify fragments with activity against Rpn11. Screening of a library of metal-binding pharmacophores (MBPs) revealed that 8-thioquinoline (8TQ, IC 50 value ∼2.5 μM) displayed strong inhibition of Rpn11. Further synthetic elaboration of 8TQ yielded a small molecule compound (35, IC 50 value ∼400 nM) that is a potent and selective inhibitor of Rpn11 that blocks proliferation of tumor cells in culture.

Exploiting nature's rich source of proteasome inhibitors as starting points in drug development.

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Gräwert, Melissa Ann; Groll, Michael

2012-02-01

Cancer is the No. 2 cause of death in the Western world and one of the most expensive diseases to treat. Thus, it is not surprising, that every major pharmaceutical and biotechnology company has a blockbuster oncology product. In 2003, Millennium Pharmaceuticals entered the race with Velcade®, a first-in-class proteasome inhibitor that has been approved by the FDA for treatment of multiple myeloma and its sales have passed the billion dollar mark. Velcade®'s extremely toxic boronic acid pharmacophore, however, contributes to a number of severe side effects. Nevertheless, the launching of this product has validated the proteasome as a target in fighting cancer and further proteasome inhibitors have entered the market as anti-cancer drugs. Additionally, proteasome inhibitors have found application as crop protection agents, anti-parasitics, immunosuppressives, as well as in new therapies for muscular dystrophies and inflammation. Many of these compounds are based on microbial metabolites. In this review, we emphasize the important role of the structural elucidation of the various unique binding mechanisms of these compounds that have been optimized throughout evolution to target the proteasome. Based on this knowledge, medicinal chemists have further optimized these natural products, resulting in potential drugs with reduced off-target activities. This journal is © The Royal Society of Chemistry 2012

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

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Nora Semren

Full Text Available Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ, provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ. OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

Overexpression of the E2 ubiquitin-conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation.

NARCIS (Netherlands)

Ree, J.H.; Jeganathan, K.B.; Malureanu, L.; Deursen, J.M.A. van

2010-01-01

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and

Discovery and development of inhibitors selective for human constitutive proteasome and immunoproteasome active sites

NARCIS (Netherlands)

Xin, B.

2017-01-01

This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally

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

Science.gov (United States)

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

2009-04-17

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

Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients

DEFF Research Database (Denmark)

Anvar, Seyed Yahya; hoen, Peter Ac; Venema, Andrea

2011-01-01

molecular pathways that are consistently associated with OPMD, we performed an integrated high-throughput transcriptome study in affected muscles of OPMD animal models and patients. The ubiquitin-proteasome system (UPS) was found to be the most consistently and significantly OPMD-deregulated pathway across...

HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

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Hongbiao Huang

Full Text Available Combinations of proteasome inhibitors and histone deacetylases (HDAC inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like activity assay. Here we report that (i the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii the combination also synergistically inhibits tumor growth in vivo; (iii two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle.

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Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A

2015-06-12

Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics.

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

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Zhang, Fang Fang; Morioka, Norimitsu; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

2015-09-04

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state. Copyright © 2015 Elsevier Inc. All rights reserved.

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function

DEFF Research Database (Denmark)

Kristensen, Line V.; Oppermann, Felix S.; Rauen, Matthias J.

2018-01-01

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3...

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

Science.gov (United States)

Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

2017-11-15

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Compensatory role of the Nrf2–ARE pathway against paraquat toxicity: Relevance of 26S proteasome activity

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Yasuhiko Izumi

2015-11-01

Full Text Available Oxidative stress and the ubiquitin–proteasome system play a key role in the pathogenesis of Parkinson disease. Although the herbicide paraquat is an environmental factor that is involved in the etiology of Parkinson disease, the role of 26S proteasome in paraquat toxicity remains to be determined. Using PC12 cells overexpressing a fluorescent protein fused to the proteasome degradation signal, we report here that paraquat yielded an inhibitory effect on 26S proteasome activity without an obvious decline in 20S proteasome activity. Relative low concentrations of proteasome inhibitors caused the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2, which is targeted to the ubiquitin–proteasome system, and activated the antioxidant response element (ARE-dependent transcription. Paraquat also upregulated the protein level of Nrf2 without increased expression of Nrf2 mRNA, and activated the Nrf2–ARE pathway. Consequently, paraquat induced expression of Nrf2-dependent ARE-driven genes, such as γ-glutamylcysteine synthetase, catalase, and hemeoxygenase-1. Knockdown of Nrf2 or inhibition of γ-glutamylcysteine synthetase and catalase exacerbated paraquat-induced toxicity, whereas suppression of hemeoxygenase-1 did not. These data indicate that the compensatory activation of the Nrf2–ARE pathway via inhibition of 26S proteasome serves as part of a cellular defense mechanism to protect against paraquat toxicity.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

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Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

2003-01-01

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

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

Science.gov (United States)

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

2014-01-01

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

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

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Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

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Antonio Pereira-Neves

Full Text Available Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF, also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin

The putative roles of the ubiquitin/proteasome pathway in resistance to anticancer therapy.

Science.gov (United States)

Smith, Laura; Lind, Michael J; Drew, Philip J; Cawkwell, Lynn

2007-11-01

The ubiquitin/proteasome (UP) pathway plays a significant role in many important biological functions and alterations in this pathway have been shown to contribute to the pathology of many human diseases, including cancer. Proteasome inhibition has been well established as a rational strategy for the treatment of multiple myeloma and is currently under investigation for the treatment of other haematological malignancies and solid tumours. Recent evidence suggests that proteasome inhibition may also sensitise tumour cells to the actions of both conventional chemotherapy and radiotherapy, suggesting that this pathway may modify clinical response to anticancer therapy. However, conflicting evidence exists as to the roles of the UP pathway in resistance to treatment. This review endeavours to discuss such roles.

Dipeptidyl Peptidase IV in Angiotensin-Converting Enzyme Inhibitor–Associated Angioedema

OpenAIRE

Byrd, James Brian; Touzin, Karine; Sile, Saba; Gainer, James V.; Yu, Chang; Nadeau, John; Adam, Albert; Brown, Nancy J.

2007-01-01

Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor–associated angioedema. This case-control study tested the hy...

Ubiquitin-conjugating enzyme E2-like gene associated to pathogen response in Concholepas concholepas: SNP identification and transcription expression.

Science.gov (United States)

Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

2012-10-01

Ubiquitin-conjugated E2 enzyme (UBE2) is one of the main components of the proteasome degradation cascade. Previous studies have shown an increase of expression levels in individuals challenged to some pathogen organism such as virus and bacteria. The study was to characterize the immune response of UBE2 gene in the gastropod Concholepas concholepas through expression analysis and single nucleotide polymorphisms (SNP) discovery. Hence, UBE2 was identified from a cDNA library by 454 pyrosequencing, while SNP identification and validation were performed using De novo assembly and high resolution melting analysis. Challenge trials with Vibrio anguillarum was carried out to evaluate the relative transcript abundance of UBE2 gene from two to thirty-three hours post-treatment. The results showed a partial UBE2 sequence of 889 base pair (bp) with a partial coding region of 291 bp. SNP variation (A/C) was observed at the 546th position. Individuals challenged by V. anguillarum showed an overexpression of the UBE2 gene, the expression being significantly higher in homozygous individuals (AA) than (CC) or heterozygous individuals (A/C). This study contributes useful information relating to the UBE2 gene and its association with innate immune response in marine invertebrates. Copyright © 2012 Elsevier Ltd. All rights reserved.

A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition

Science.gov (United States)

Rodriguez, Karl A.; Osmulski, Pawel A.; Pierce, Anson; Weintraub, Susan T.; Gaczynska, Maria; Buffenstein, Rochelle

2015-01-01

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31y) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although HSP72 and HSP40 (Hdj1) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging. PMID:25018089

Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

Science.gov (United States)

Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M. Maral; Lee, Gwang

2016-01-01

The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles. PMID:27378605

Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

Science.gov (United States)

Tawa, N E; Odessey, R; Goldberg, A L

1997-07-01

Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

International Nuclear Information System (INIS)

Matsumoto, Hotaru; Saitoh, Hisato

2016-01-01

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

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

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

2016-07-29

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

Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells.

Science.gov (United States)

Bomsel, Morgane; Ganor, Yonatan

2017-12-01

The neuroimmune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans -infect CD4 + T cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LC-mediated HIV-1 trans -infection. In this study, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans -infection. We first show that HIV-1 degradation occurs in endolysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans -infection. We demonstrate that CGRP acts via its cognate receptor and at a viral postentry step to induce faster HIV-1 degradation, but without affecting the kinetics of endolysosomal degradation. We reveal that unexpectedly, CGRP shifts HIV-1 degradation from endolysosomes toward the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans -infection, and proteasomal, but not endolysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls the HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response, to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in vivo Hence, proteasomal, rather than endolysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans -infection. IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet during trans -infection, infectious virions escaping degradation are transferred

Proteomic Profiling of Radiation-Induced Skin Fibrosis in Rats: Targeting the Ubiquitin-Proteasome System

Energy Technology Data Exchange (ETDEWEB)

Wang, Wenjie [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Cyrus Tang Hematology Center, Soochow University, Suzhou (China); Luo, Judong [Department of Radiotherapy, Changzhou Tumor Hospital, Soochow University, Changzhou (China); Sheng, Wenjiong; Xue, Jiao; Li, Ming [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Ji, Jiang [Department of Dermatology, the Second Affiliated Hospital of Soochow University, Suzhou (China); Liu, Pengfei [Department of Gastroenterology, the Affiliated Jiangyin Hospital of Southeast University, Jiangyin (China); Zhang, Xueguang [Institute of Medical Biotechnology and Jiangsu Stem Cell Key Laboratory, Medical College of Soochow University, Suzhou (China); Cao, Jianping [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Zhang, Shuyu, E-mail: zhang.shuyu@hotmail.com [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Cyrus Tang Hematology Center, Soochow University, Suzhou (China)

2016-06-01

Purpose: To investigate the molecular changes underlying the pathogenesis of radiation-induced skin fibrosis. Methods and Materials: Rat skin was irradiated to 30 or 45 Gy with an electron beam. Protein expression in fibrotic rat skin and adjacent normal tissues was quantified by label-free protein quantitation. Human skin cells HaCaT and WS-1 were treated by x-ray irradiation, and the proteasome activity was determined with a fluorescent probe. The effect of proteasome inhibitors on Transforming growth factor Beta (TGF-B) signaling was measured by Western blot and immunofluorescence. The efficacy of bortezomib in wound healing of rat skin was assessed by the skin injury scale. Results: We found that irradiation induced epidermal and dermal hyperplasia in rat and human skin. One hundred ninety-six preferentially expressed and 80 unique proteins in the irradiated fibrotic skin were identified. Through bioinformatic analysis, the ubiquitin-proteasome pathway showed a significant fold change and was investigated in greater detail. In vitro experiments demonstrated that irradiation resulted in a decline in the activity of the proteasome in human skin cells. The proteasome inhibitor bortezomib suppressed profibrotic TGF-β downstream signaling but not TGF-β secretion stimulated by irradiation in HaCaT and WS-1 cells. Moreover, bortezomib ameliorated radiation-induced skin injury and attenuated epidermal hyperplasia. Conclusion: Our findings illustrate the molecular changes during radiation-induced skin fibrosis and suggest that targeting the ubiquitin-proteasome system would be an effective countermeasure.

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

International Nuclear Information System (INIS)

Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

2009-01-01

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of 35 S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

Molecular mechanisms for synergistic effect of proteasome inhibitors with platinum-based therapy in solid tumors.

Science.gov (United States)

Chao, Angel; Wang, Tzu-Hao

2016-02-01

The successful development of the proteasome inhibitor bortezomib as an anticancer drug has improved survival in patients with multiple myeloma. With the emergence of the newly US Food and Drug Administration-approved proteasome inhibitor carfilzomib, ongoing trials are investigating this compound and other proteasome inhibitors either alone or in combination with other chemotherapy drugs. However, in solid tumors, the efficacy of proteasome inhibitors has not lived up to expectations. Results regarding the potential clinical efficacy of bortezomib combined with other agents in the treatment of solid tumors are eagerly awaited. Recent identification of the molecular mechanisms (involving apoptosis and autophagy) by which bortezomib and cisplatin can overcome chemotherapy resistance and sensitize tumor cells to anticancer therapy can provide insights into the development of novel therapeutic strategies for patients with solid malignancies. Copyright © 2016. Published by Elsevier B.V.

Gambogic Acid Is a Tissue-Specific Proteasome Inhibitor In Vitro and In Vivo

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Xiaofen Li

2013-01-01

Full Text Available Gambogic acid (GA is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

Analyzing proteasomal subunit expression reveals Rpt4 as a prognostic marker in stage II colorectal cancer.

LENUS (Irish Health Repository)

2012-02-01

Colorectal cancer is a leading cause of cancer-related deaths worldwide. Early diagnosis and treatment of colorectal cancer is the key to improving survival rates and as such a need exists to identify patients who may benefit from adjuvant chemotherapy. The dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in oncogenesis and cancer cell survival, and proteasome inhibitors are in clinical use for a number of malignancies including multiple myeloma. In our study, we examined the protein expression of several key components of the UPS in colorectal cancer using immunohistochemistry to determine expression levels of ubiquitinylated proteins and the proteasomal subunits, 20S core and Rpt4 in a cohort of 228 patients with colon cancer. Multivariate Cox analysis revealed that neither the intensity of either ubiquitinylated proteins or the 20S core was predictive in either Stage II or III colon cancer for disease free survival or overall survival. In contrast, in Stage II patients increased Rpt4 staining was significantly associated with disease free survival (Cox proportional hazard ratio 0.605; p = 0.0217). Our data suggest that Rpt4 is an independent prognostic variable for Stage II colorectal cancer and may aid in the decision of which patients undergo adjuvant chemotherapy.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities

Energy Technology Data Exchange (ETDEWEB)

Uozumi, Naoki; Matsumoto, Hotaru [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan)

2016-05-27

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. -- Highlights: •Click chemistry detects O-propargyl-puromycin (OP-Puro) signals in the nucleus. •OP-Puro accumulates at PML-NBs during abortive proteasome activities. •SUMO and ubiquitin are promiscuously associated with OP-Puro at PML-NBs. •The nucleus may function in immature protein homeostasis.

Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities

International Nuclear Information System (INIS)

Uozumi, Naoki; Matsumoto, Hotaru; Saitoh, Hisato

2016-01-01

The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. -- Highlights: •Click chemistry detects O-propargyl-puromycin (OP-Puro) signals in the nucleus. •OP-Puro accumulates at PML-NBs during abortive proteasome activities. •SUMO and ubiquitin are promiscuously associated with OP-Puro at PML-NBs. •The nucleus may function in immature protein homeostasis.

Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

Science.gov (United States)

Crow, Marni S; Cristea, Ileana M

2017-04-01

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells

NARCIS (Netherlands)

Ramos de Carvalho, J. Emanuel; Verwoert, Milan T.; Vogels, Ilse M. C.; Schipper-Krom, Sabine; van Noorden, Cornelis J. F.; Reits, Eric A.; Klaassen, Ingeborg; Schlingemann, Reinier O.

2018-01-01

Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome

Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

Science.gov (United States)

Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

2011-06-03

Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

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Hyewon Byun

2017-03-01

Full Text Available Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP and a C-terminal protein (Rem-CT. SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.

Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Science.gov (United States)

Cascella, Roberta; Fani, Giulia; Capitini, Claudia; Rusmini, Paola; Poletti, Angelo; Cecchi, Cristina; Chiti, Fabrizio

2017-12-01

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum.

Science.gov (United States)

Verchot, Jeanmarie

2016-11-19

The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus-host interactions will provide new targets for crop improvement.

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum

Directory of Open Access Journals (Sweden)

Jeanmarie Verchot

2016-11-01

Full Text Available The endoplasmic reticulum (ER is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Science.gov (United States)

Park, Chul Min; Lee, Kyunghoon; Jun, Sun-Hee; Song, Sang Hoon; Song, Junghan

2017-08-15

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R 2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia. Copyright © 2017 Elsevier B.V. All rights reserved.

The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

Directory of Open Access Journals (Sweden)

Victoria M Virador

Full Text Available Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.Staurosporine (STS was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A within the proline-rich center of BAG3 (PXXP and the C-terminal LEAD site (BAG3(E516A/D518A. PXXP deletion mutant and BAG3(E345A/D347A, or BAG3(E516A/D518A respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

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

Science.gov (United States)

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

2015-12-01

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

Short Chemical Ischemia Triggers Phosphorylation of eIF2α and Death of SH-SY5Y Cells but not Proteasome Stress and Heat Shock Protein Response in both SH-SY5Y and T98G Cells.

Science.gov (United States)

Klacanova, Katarina; Pilchova, Ivana; Klikova, Katarina; Racay, Peter

2016-04-01

Both translation arrest and proteasome stress associated with accumulation of ubiquitin-conjugated protein aggregates were considered as a cause of delayed neuronal death after transient global brain ischemia; however, exact mechanisms as well as possible relationships are not fully understood. The aim of this study was to compare the effect of chemical ischemia and proteasome stress on cellular stress responses and viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Chemical ischemia was induced by transient treatment of the cells with sodium azide in combination with 2-deoxyglucose. Proteasome stress was induced by treatment of the cells with bortezomib. Treatment of SH-SY5Y cells with sodium azide/2-deoxyglucose for 15 min was associated with cell death observed 24 h after treatment, while glioblastoma T98G cells were resistant to the same treatment. Treatment of both SH-SY5Y and T98G cells with bortezomib was associated with cell death, accumulation of ubiquitin-conjugated proteins, and increased expression of Hsp70. These typical cellular responses to proteasome stress, observed also after transient global brain ischemia, were not observed after chemical ischemia. Finally, chemical ischemia, but not proteasome stress, was in SH-SY5Y cells associated with increased phosphorylation of eIF2α, another typical cellular response triggered after transient global brain ischemia. Our results showed that short chemical ischemia of SH-SY5Y cells is not sufficient to induce both proteasome stress associated with accumulation of ubiquitin-conjugated proteins and stress response at the level of heat shock proteins despite induction of cell death and eIF2α phosphorylation.

Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome.

Science.gov (United States)

Lázaro, Silvia; Gamarra, David; Del Val, Margarita

2015-12-01

Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1-inhibitable manner.

Science.gov (United States)

Méndez-Hernández, Lucía E; Robledo-Rivera, Angelica Y; Macías-Silva, Marina; Calera, Mónica R; Sánchez-Olea, Roberto

2017-11-01

Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation. © 2017 Federation of European Biochemical Societies.

Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

OpenAIRE

Tawa, N E; Odessey, R; Goldberg, A L

1997-01-01

Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlyso...

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

International Nuclear Information System (INIS)

Pajonk, Frank; Ophoven, Arndt van; Weissenberger, Christian; McBride, William H

2005-01-01

By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products

NARCIS (Netherlands)

Platteel, Anouk C M; Mishto, Michele; Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Busch, Dirk H; Kloetzel, Peter M; Sijts, Alice J A M

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed

The Anti-Apoptotic Activity of BAG3 Is Restricted by Caspases and the Proteasome

OpenAIRE

Virador, Victoria M.; Davidson, Ben; Czechowicz, Josephine; Mai, Alisha; Kassis, Jareer; Kohn, Elise C.

2009-01-01

Background Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis. Methodology/Principal Findings Staurosporine (STS) was used as a tool to test ...

Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals

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Ilaria Postiglione

2015-08-01

Full Text Available Photofrin/photodynamic therapy (PDT at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53+/+ and H1299 (p53−/− cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System

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Stephen R. Armstrong

2016-12-01

Full Text Available The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

Proteasome subunit expression analysis and chemosensitivity in relapsed paediatric acute leukaemia patients receiving bortezomib-containing chemotherapy

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Denise Niewerth

2016-09-01

Full Text Available Abstract Background Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML and acute lymphocytic leukaemia (ALL. Methods We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children’s Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1 and AML (COG-AAML07P1. Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12 obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test. Results Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both β5i/β5 and β1i/β1 subunits (p = 0.004 and p < 0.001. These ratios correlated with therapy response in AML patients; β1i/β1 ratios were significantly higher (p = 0.028 between patients who did (n = 4 and did not reach complete remission (CR (n = 8, although for β5i/β5 ratios, this did not reach significance. For ALL patients, the subunit ratios were also higher for patients who showed a good early response to therapy but this relation was not statistically significant. Overall, for this study, the patients were treated with combination therapy, so response was not only attributed to proteasome inhibition. Moreover, the leukaemic blast cells were not purified for these samples. Conclusions These first ex vivo results encourage further studies into relative proteasome subunit expression to improve proteasome inhibition-containing therapy and as a potential indicator of bortezomib response in acute leukaemia.

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

Science.gov (United States)

Hakozaki, T; Laughlin, T; Zhao, S; Wang, J; Deng, D; Jewell-Motz, E; Elstun, L

2013-07-01

2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation. © 2013 The Authors BJD © 2013 British Association of Dermatologists.

REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Chai, Fan; Liang, Yan [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Bi, Jiong [Laboratory of General Surgery, First Affiliated Hospital, Sun Yet-sen University, Guangzhou 510080 (China); Chen, Li; Zhang, Fan [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Cui, Youhong [Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Jiang, Jun, E-mail: jcbd@medmail.com.cn [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

2015-01-02

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

Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

NARCIS (Netherlands)

Hougardy, BMT; Maduro, JH; van der Zee, AGJ; de Groot, DJA; van den Heuvel, FAJ; de Vries, EGE; de Jong, S

2006-01-01

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize

Interplay between Molecular Chaperones and the Ubiquitin-Proteasome System in Targeting of Misfolded Proteins for Degradation

DEFF Research Database (Denmark)

Poulsen, Esben Guldahl

interacting with purified 26S proteasomes, and the subsequent characterization of two novel proteasome interacting proteins. The third study was aimed at analyzing the chaperone-assisted pathway leading to degradation of misfolded kinetochore proteins in S. pombe. In this study chaperones, E2s, E3s and DUBs...

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination

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Laura Novellasdemunt

2017-10-01

Full Text Available The tumor suppressor gene adenomatous polyposis coli (APC is mutated in most colorectal cancers (CRCs, resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs.

Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

International Nuclear Information System (INIS)

Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J.

2006-01-01

We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1α associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome

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

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Jaime M. Ross

2015-08-01

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

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

Science.gov (United States)

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

2015-01-01

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

Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals.

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Amit Kumar Singh Gautam

Full Text Available Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a origin and identification of an intrinsic degradation signal in the substrate, b identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably

The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster.

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Pomatto, Laura C D; Wong, Sarah; Carney, Caroline; Shen, Brenda; Tower, John; Davies, Kelvin J A

2017-04-01

Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C ( Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H 2 O 2 ) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H 2 O 2 , and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H 2 O 2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.

Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.

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Zhao, Wenting; Bonem, Matthew; McWhite, Claire; Silberg, Jonathan J; Segatori, Laura

2014-04-08

The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation.

Science.gov (United States)

Yagi-Utsumi, Maho; Sikdar, Arunima; Kozai, Toshiya; Inoue, Rintaro; Sugiyama, Masaaki; Uchihashi, Takayuki; Yagi, Hirokazu; Satoh, Tadashi; Kato, Koichi

2018-01-01

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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

2015-12-01

Taurine depletion leads to impaired mitochondrial function, as characterized by reduced ATP production and elevated superoxide generation. These defects can fundamentally alter cardiomyocyte function and if left unchanged can result in cell death. To protect against these stresses, cardiomyocytes possess quality control processes, such as the ubiquitin-proteasome system (UPS) and autophagy, which can rejuvenate cells through the degradation of damaged proteins and organelles. Hence, the present study tested the hypothesis that reactive oxygen species generated by damaged mitochondria initiates UPS and autophagy in the taurine-deficient heart. Using transgenic mice lacking the taurine transporter (TauTKO) as a model of taurine deficiency, it was shown that the levels of ubiquitinated protein were elevated, an effect associated with a decrease in ATP-dependent 26S β5 proteasome activity. Treating the TauTKO mouse with the mitochondria-specific antioxidant, mitoTEMPO, largely abolished the increase in ubiquitinated protein content. The TauTKO heart was also associated with impaired autophagy, characterized by an increase in the initiator, Beclin-1, and autophagosome content, but a defect in the generation of active autophagolysosomes. Although mitoTEMPO treatment only restores the oxidative balance within the mitochondria, it appeared to completely disrupt the crosstalk between the damaged mitochondria and the quality control processes. Thus, mitochondrial oxidative stress is the main trigger initiating the quality control systems in the taurine-deficient heart. We conclude that the activation of the UPS and autophagy is another fundamental function of mitochondria.

Combination of Proteasomal Inhibitors Lactacystin and MG132 Induced Synergistic Apoptosis in Prostate Cancer Cells

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Robert B. Shirley

2005-12-01

Full Text Available The proteasome inhibitor Velcade (bortezomib/PS-341 has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego, CA and MG132 (Biomol International, Plymouth Meeting, PA may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKα, IKKβ, and IKKγ proteins and NFκB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFκB axis.

The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

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Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

2011-01-01

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

CCR 20th anniversary commentary: Preclinical study of proteasome inhibitor bortezomib in head and neck cancer.

Science.gov (United States)

Allen, Clint T; Conley, Barbara; Sunwoo, John B; Van Waes, Carter

2015-03-01

In a study published in the May 1, 2001, issue of Clinical Cancer Research, Sunwoo and colleagues provided evidence for proteasome inhibition of NF-κB and tumorigenesis, supporting early-phase clinical trials in solid malignancies of the upper aerodigestive tract. Subsequent clinical studies uncovered a dichotomy of responses in patients with hematopoietic and solid malignancies, and the mechanisms of resistance. ©2015 American Association for Cancer Research.

Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma.

Science.gov (United States)

Vogl, Dan T; Stadtmauer, Edward A; Tan, Kay-See; Heitjan, Daniel F; Davis, Lisa E; Pontiggia, Laura; Rangwala, Reshma; Piao, Shengfu; Chang, Yunyoung C; Scott, Emma C; Paul, Thomas M; Nichols, Charles W; Porter, David L; Kaplan, Janeen; Mallon, Gayle; Bradner, James E; Amaravadi, Ravi K

2014-08-01

The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy.

Predicting proteasomal cleavage sites: a comparison of available methods

DEFF Research Database (Denmark)

Saxova, P.; Buus, S.; Brunak, Søren

2003-01-01

-terminal, in particular, of CTL epitopes is cleaved precisely by the proteasome, whereas the N-terminal is produced with an extension, and later trimmed by peptidases in the cytoplasm and in the endoplasmic reticulum. Recently, three publicly available methods have been developed for prediction of the specificity...

The Proteasome Inhibitor MG-132 Protects Hypoxic SiHa Cervical Carcinoma Cells after Cyclic Hypoxia/Reoxygenation from Ionizing Radiation

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Frank Pajonk

2006-12-01

Full Text Available INTRODUCTION: Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of radiation therapy. This study was designed to determine how varying cycles of hypoxia/reoxygenation affect the response of cervical carcinoma cells irradiated under oxic and hypoxic conditions and whether this could be modulated by proteasome inhibition. MATERIALS AND METHODS: Plateau-phase SiHa cervical carcinoma cells in culture were exposed to varying numbers of 30-minute cycles of hypoxia/reoxygenation directly before irradiation under oxic or hypoxic conditions. 26S Proteasome activity was blocked by addition of MG-132. Clonogenic survival was measured by a colonyforming assay. RESULTS: Under oxic conditions, repeated cycles of hypoxia/reoxygenation decreased the clonogenic survival of SiHa cells. This effect was even more pronounced after the inhibition of 26S proteasome complex. In contrast, under hypoxic conditions, SiHa cells were radioresistant, as expected, but this was increased by proteasome inhibition. CONCLUSIONS: Proteasome inhibition radiosensitizes oxygenated tumor cells but may also protect tumor cells from ionizing radiation under certain hypoxic conditions.

The cleavage product of amyloid-β protein precursor sAβPPα modulates BAG3-dependent aggresome formation and enhances cellular proteasomal activity.

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Renziehausen, Jana; Hiebel, Christof; Nagel, Heike; Kundu, Arpita; Kins, Stefan; Kögel, Donat; Behl, Christian; Hajieva, Parvana

2015-01-01

Alzheimer's disease (AD) is the major age-associated form of dementia characterized by gradual cognitive decline. Aberrant cleavage of the amyloid-β protein precursor (AβPP) is thought to play an important role in the pathology of this disease. Two principal AβPP processing pathways exist: amyloidogenic cleavage of AβPP resulting in production of the soluble N-terminal fragment sAβPPβ, amyloid-β (Aβ), which accumulates in AD brain, and the AβPP intracellular domain (AICD) sAβPPα, p3 and AICD are generated in the non-amyloidogenic pathway. Prevalence of amyloidogenic versus non-amyloidogenic processing leads to depletion of sAβPPα and an increase in Aβ. Although sAβPPα is a well-accepted neurotrophic protein, molecular effects of this fragment remains unknown. Different studies reported impaired protein degradation pathways in AD brain, pointing to a role of disturbed proteasomal activity in the pathogenesis of this disease. Here we studied the possible role of sAβPPα in Bag3-mediated selective macroautophagy and proteasomal degradation. Employing human IMR90 cells, HEK 293 cells, and primary neurons, we demonstrate that sAβPPα prevents the proteotoxic stress-induced increase of Bag3 at the protein and at the mRNA level indicating a transcriptional regulation. Intriguingly, p62 and LC3, two other key players of autophagy, were not affected. Moreover, the formation and the accumulation of disease-related protein aggregates were significantly reduced by sAβPPα. Interestingly, there was a significant increase of proteasomal activity by sAβPPα as demonstrated by using various proteasome substrates. Our findings demonstrate that sAβPPα modulates Bag3 expression, aggresome formation, and proteasomal activity, thereby providing first evidence for a function of sAβPPα in the regulation of proteostasis.

Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies

NARCIS (Netherlands)

Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H.; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L.; Hink, Mark A.; Bates, Gillian P.; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

2014-01-01

Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that

Molecular characterization of 26S proteasome regulatory subunit in ...

African Journals Online (AJOL)

Trichophyton verrucosum is a zoophilic dermatophyte, which causes dermatophytosis infection in human as well as animals. 26S proteasome is an important protein in eukaryotic cells that is involved with degradation of unneeded or damaged proteins, when tagged with ubiquitin. In this study, we characterized the 26S ...

Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

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Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

2016-10-14

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

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Li Xing'an

2010-08-01

Full Text Available Abstract Background Cooperation of constituents of the ubiquitin proteasome system (UPS with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase.

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Nishikawa, Hiroyuki; Ooka, Seido; Sato, Ko; Arima, Kei; Okamoto, Joji; Klevit, Rachel E; Fukuda, Mamoru; Ohta, Tomohiko

2004-02-06

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver.

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Blaber, Elizabeth A; Pecaut, Michael J; Jonscher, Karen R

2017-09-27

Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-'omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver

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Elizabeth A. Blaber

2017-09-01

Full Text Available Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-‘omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

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Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

A genome-wide siRNA screen identifies proteasome addiction as a vulnerability of basal-like triple-negative breast cancer cells

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Petrocca, Fabio; Altschuler, Gabriel; Tan, Shen Mynn; Mendillo, Marc L.; Yan, Haoheng; Jerry, D. Joseph; Kung, Andrew L.; Hide, Winston; Ince, Tan A.; Lieberman, Judy

2013-01-01

Summary Basal-like triple negative breast cancers (TNBC) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes - basal-like BPLER and myoepithelial HMLER. Expression of the screen’s 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence. PMID:23948298

Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

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Aleksandra Glogowska

2012-05-01

Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

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Kremsner Peter G

2008-09-01

Full Text Available Abstract Background The emergence and spread of Plasmodium falciparum resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of P. falciparum. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon. Methods Epoxomicin, YU101, YU102, MG132, MG115, Z-L3-VS, Ada-Ahx3-L3-VS, lactacystin, bortezomib (Velcade®, gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized in vitro drug susceptibility assay against P. falciparum laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2 production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99% were calculated. Results Amongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10] and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2] as well as in field isolates (IC50: 8.5 nM. The comparator drug artesunate was even more active (IC50: 1.0 nM, whereas all strains were chloroquine-resistant (IC50: 113 nM. Conclusion The peptide α',β'-epoxyketone epoxomicin is highly active against P. falciparum regardless the grade of the parasite's chloroquine

Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

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Rasmussen, A B; Zocca, M-B; Bonefeld, C M

2004-01-01

Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent...... on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...... to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell...

Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

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Hartmann-Petersen, R; Tanaka, K; Hendil, K B

2001-01-01

and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross......-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7......, these data establish the relative orientations of ATPases with respect to the 20S proteasome....

Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

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Julia S Gelman

Full Text Available Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours, and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

Proteasome Activators, PA28α and PA28β, Govern Development of Microvascular Injury in Diabetic Nephropathy and Retinopathy

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Saeed Yadranji Aghdam

2016-01-01

Full Text Available Diabetic nephropathy (DN and diabetic retinopathy (DR are major complications of type 1 and type 2 diabetes. DN and DR are mainly caused by injury to the perivascular supporting cells, the mesangial cells within the glomerulus, and the pericytes in the retina. The genes and molecular mechanisms predisposing retinal and glomerular pericytes to diabetic injury are poorly characterized. In this study, the genetic deletion of proteasome activator genes, PA28α and PA28β genes, protected the diabetic mice in the experimental STZ-induced diabetes model against renal injury and retinal microvascular injury and prolonged their survival compared with wild type STZ diabetic mice. The improved wellbeing and reduced renal damage was associated with diminished expression of Osteopontin (OPN and Monocyte Chemoattractant Protein-1 (MCP-1 in the glomeruli of STZ-injected PA28α/PA28β double knockout (Pa28αβDKO mice and also in cultured mesangial cells and retinal pericytes isolated from Pa28αβDKO mice that were grown in high glucose. The mesangial PA28-mediated expression of OPN under high glucose conditions was suppressed by peptides capable of inhibiting the binding of PA28 to the 20S proteasome. Collectively, our findings demonstrate that diabetic hyperglycemia promotes PA28-mediated alteration of proteasome activity in vulnerable perivascular cells resulting in microvascular injury and development of DN and DR.

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination.

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Novellasdemunt, Laura; Foglizzo, Valentina; Cuadrado, Laura; Antas, Pedro; Kucharska, Anna; Encheva, Vesela; Snijders, Ambrosius P; Li, Vivian S W

2017-10-17

The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Functional interplay between mitochondrial and proteasome activity in skin aging

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Kozie, Rafa; Greussing, Ruth; Maier, Andrea B.; Declercq, Lieve; Jansen-Dürr, Pidder

According to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby contribute to the aging process. On the other hand, a pivotal role of the proteasome, as a main proteolytic

Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form

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Sugiyama, Masaaki, E-mail: sugiyama@rri.kyoto-u.ac.jp [Research Reactor Institute, Kyoto University, Osaka 590-0494 (Japan); Sahashi, Hiroki [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Kurimoto, Eiji [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Faculty of Pharmacy, Meijo University, Nagoya 468-8503 (Japan); Takata, Shin-ichi [J-PARC Center, Japan Atomic Energy Agency, Ibaraki 319-1195 (Japan); Yagi, Hirokazu; Kanai, Keita; Sakata, Eri [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Minami, Yasufumi [Department of Biotechnology, Maebashi Institute of Technology, Gunma 371-0816 (Japan); Tanaka, Keiji [Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Kato, Koichi, E-mail: kkatonmr@ims.ac.jp [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787 (Japan); Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787 (Japan)

2013-03-01

Highlights: ► Homologous α and β subunits are alternatively arranged in the PA28 heptameric ring. ► The flexible loops of the three α subunits surround the site of substrate entry. ► The loops serve as gatekeepers that selectively hinder passage of longer peptides. - Abstract: A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four β subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

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

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

2016-03-25

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

Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

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Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

2017-05-01

Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

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Barré-Sinoussi Françoise

2009-05-01

Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

26S proteasome and insulin-like growth factor-1 in serum of dogs suffering from malignant tumors.

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Gerke, Ingrid; Kaup, Franz-Josef; Neumann, Stephan

2018-04-01

Studies in humans have shown that the ubiquitin-proteasome pathway and the insulin-like growth factor axis are involved in carcinogenesis, thus, components of these systems might be useful as prognostic markers and constitute potential therapeutic targets. In veterinary medicine, only a few studies exist on this topic. Here, serum concentrations of 26S proteasome (26SP) and insulin-like growth factor-1 (IGF-1) were measured by canine enzyme-linked immunosorbent assay (ELISA) in 43 dogs suffering from malignant tumors and 21 clinically normal dogs (control group). Relationships with tumor size, survival time, body condition score (BCS), and tumor entity were assessed. The median 26SP concentration in the tumor group was non-significantly higher than in the control group. However, dogs with mammary carcinomas displayed significantly increased 26SP levels compared to the control group and dogs with tumor size less than 5 cm showed significantly increased 26SP concentrations compared to dogs with larger tumors and control dogs. 26SP concentrations were not correlated to survival time or BCS. No significant difference in IGF-1 levels was found between the tumor group and the control group; however, IGF-1 concentrations displayed a larger range of values in the tumor group. Dogs with tumors greater than 5 cm showed significantly higher IGF-1 levels than dogs with smaller tumors. The IGF-1 concentrations were positively correlated to survival time, but no correlation with BCS was found. Consequently, serum 26SP concentrations seem to be increased in some dogs suffering from malignant tumors, especially in dogs with mammary carcinoma and smaller tumors. Increased serum IGF-1 concentrations could be an indication of large tumors and a poor prognosis.

An anti-HIV-1 compound that increases steady-state expression of apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G.

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Ejima, Tomohiko; Hirota, Mayuko; Mizukami, Tamio; Otsuka, Masami; Fujita, Mikako

2011-10-01

Human apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) 3G (A3G) is an antiviral protein that blocks HIV-1 replication. However, the antiviral activity of A3G is overcome by the HIV-1 protein Vif. This inhibitory function of Vif is related to its ability to degrade A3G in the proteasome. This finding prompted us to examine the activities of 4-(dimethylamino)-2,6-bis[(N-(2-[(2-nitrophenyl)dithio]ethyl)amino)methyl]pyridine (SN-2) and SN-3. We found that 5 µM SN-2 increases the expression of A3G to a level much higher than that observed in the absence of Vif, without affecting the level of Vif expression. The proteasome inhibitor MG-132 increased the level of both A3G and Vif expression. These results demonstrate that A3G is ubiquitinated and degraded in the proteasome by a factor other than Vif, and that SN-2 selectively inhibits these processes. Furthermore, 5 µM SN-2 significantly inhibited the MAGI cell infectivity of wild-type HIV-1. These findings may contribute to the development of a novel anti-HIV-1 drug.

Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.

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Radwan, Marta; Wilkinson, David J; Hui, Wang; Destrument, Auriane P M; Charlton, Sarah H; Barter, Matt J; Gibson, Beth; Coulombe, Josée; Gray, Douglas A; Rowan, Andrew D; Young, David A

2015-08-01

To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are

Age-related and sex-specific differences in proteasome activity in individual Drosophila flies from wild type, longevity-selected and stress resistant strains

DEFF Research Database (Denmark)

Hansen, Tina Østergaard; Sarup, Pernille Merete; Loeschcke, Volker

2012-01-01

with that in C1 males. However, in longevity-selected LS1 flies the proteasome activity was significantly lower compared to C1 flies, but the sex differences were maintained to some extent. Five other stress resistant lines also had significantly reduced proteasome activity in both sexes. During ageing...... and that increased lifespan and stress resistance lead to a reduction in proteasome activity and recession of the age-related decline observed in control females....

Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

DEFF Research Database (Denmark)

Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

2015-01-01

In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...... inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair...

Genetic polymorphisms in antioxidative enzymes are associated to FEV(1) in smokers independently of asthma

DEFF Research Database (Denmark)

Malling, T H; Sigsgaard, Torben; Andersen, Charlotte Brasch

2012-01-01

), GSTT1 (gene copy number), and GSTM1 (gene copy number). We found no associations between these genotypes and the asthma phenotypes. For the 201 subjects identified as current smokers and recruited via random sampling, an association was seen between increasing number of genotypes coding for high...... of genotypes coding for low antioxidative enzyme activity. The present study does not support the hypothesis that asthma is associated with genotypes of these major antioxidative enzymes. However, we speculate that since we see an impact of these genotypes on lung function in young adult smokers, polymorphisms...... in antioxidative enzymes may contribute to the range of susceptibility of smokers have to COPD....

Yeast Sgf73/Ataxin-7 serves to anchor the deubiquitination module into both SAGA and Slik(SALSA HAT complexes

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Lee Kenneth K

2009-02-01

Full Text Available Abstract Spinocerebellar ataxia (SCA is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1–3, 6–10, 12, and 17 result from a trinucleotide repeat expansion in the DNA-coding sequence. Intriguingly, recent work has demonstrated that increased trinucleotde expansions in the SCA7 gene result in defect in the function of the SAGA histone acetyltransferase complex. The SCA7 gene encodes a subunit of the SAGA complex. This subunit is conserved in yeast as the SGF73 gene. We demonstrate that Sgf73 is required to recruit the histone deubiquitination module into both SAGA and the related SliK(SALSA complex, and to maintain levels of histone ubiquitination, which is necessary for regulation of transcription at a number of genes.

Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

Energy Technology Data Exchange (ETDEWEB)

Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail: kawamots@mail.nih.gov

2014-08-08

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

The role of the ubiquitin proteasome system in the memory process.

Science.gov (United States)

Lip, Philomena Z Y; Demasi, Marilene; Bonatto, Diego

2017-01-01

Quite intuitive is the notion that memory formation and consolidation is orchestrated by protein synthesis because of the synaptic plasticity necessary for those processes. Nevertheless, recent advances have begun accumulating evidences of a high requirement for protein degradation on the molecular mechanisms of the memory process in the mammalian brain. Because degradation determines protein half-life, degradation has been increasingly recognized as an important intracellular regulatory mechanism. The proteasome is the main player in the degradation of intracellular proteins. Proteasomal substrates are mainly degraded after a post-translational modification by a poly-ubiquitin chain. Latter process, namely poly-ubiquitination, is highly regulated at the step of the ubiquitin molecule transferring to the protein substrate mediated by a set of proteins whose genes represent almost 2% of the human genome. Understanding the role of polyubiquitin-mediated protein degradation has challenging researchers in many fields of investigation as a new source of targets for therapeutic intervention, e.g. E3 ligases that transfer ubiquitin moieties to the substrate. The goal of present work was to uncover mechanisms underlying memory processes regarding the role of the ubiquitin-proteasome system (UPS). For that purpose, preceded of a short review on UPS and memory processes a top-down systems biology approach was applied to establish central proteins involved in memory formation and consolidation highlighting their cross-talking with the UPS. According to that approach, the pattern of expression of several elements of the UPS were found overexpressed in regions of the brain involved in processing cortical inputs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

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Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying

2018-01-01

Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

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

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

2015-04-01

Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected

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K. Jeena

2012-08-01

Full Text Available White spot syndrome virus (WSSV is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2 in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

Directory of Open Access Journals (Sweden)

Anita Collavoli

2011-01-01

Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

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

NARCIS (Netherlands)

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

2014-01-01

The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's,

Lansoprazole Upregulates Polyubiquitination of the TNF Receptor-Associated Factor 6 and Facilitates Runx2-mediated Osteoblastogenesis.

Science.gov (United States)

Mishima, Kenichi; Kitoh, Hiroshi; Ohkawara, Bisei; Okuno, Tatsuya; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

2015-12-01

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models.

Science.gov (United States)

Staines, Katherine A; Prideaux, Matt; Allen, Steve; Buttle, David J; Pitsillides, Andrew A; Farquharson, Colin

2016-06-01

The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. © 2015 The Authors. Journal of

Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

OpenAIRE

El Magraoui, Fouzi; Reidick, Christina; Meyer, Hemut E.; Platta, Harald W.

2015-01-01

Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-recep...

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway.

Science.gov (United States)

Tomasello, Marianna F; Nardon, Chiara; Lanza, Valeria; Di Natale, Giuseppe; Pettenuzzo, Nicolò; Salmaso, Stefano; Milardi, Danilo; Caliceti, Paolo; Pappalardo, Giuseppe; Fregona, Dolores

2017-09-29

The gold(III)-dithiocarbamate complex AuL12 (dibromo [ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate] gold(III)), is endowed with promising in vitro/in vivo antitumor activity and toxicological profile. Here, we report our recent strategies to improve its water solubility and stability under physiological conditions along with our efforts for unravelling its tangled mechanism of action. We used three types of α-cyclodextrins (CDs), namely β-CD, Me-β-CD and HP-β-CD to prepare aqueous solutions of AuL12. The ability of these natural oligosaccharide carriers to enhance water solubility of hydrophobic compounds, allowed drug stability of AuL12 to be investigated. Moreover, pharmacokinetic experiments were first carried out for a gold(III) coordination compound, after i.v. injection of the nanoformulation AuL12/HP-β-CD to female mice. The gold content in the blood samples was detected at scheduled times by AAS (atomic absorption spectrometry) analysis, highlighting a fast biodistribution with a t β1/2 of few minutes and a slow escretion (t α1/2 of 14.3 h). The in vitro cytotoxic activity of AuL12 was compared with the AuL12/HP-β-CD mixture against a panel of three human tumor cell lines (i.e., HeLa, KB and MCF7). Concerning the mechanism of action, we previously reported the proteasome-inhibitory activity of some our gold(III)-based compounds. In this work, we moved from the proteasome target to upstream of the important ubiquitin-proteasome pathway, testing the effects of AuL12 on the polyubiquitination reactions involving the Ub-activating (E1) and -conjugating (E2) enzymes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

Science.gov (United States)

Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

2005-01-01

Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

Inhibition of nuclear factor-κB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

International Nuclear Information System (INIS)

Van Waes, Carter; Chang, Angela A.; Lebowitz, Peter F.; Druzgal, Colleen H.; Chen, Zhong; Elsayed, Yusri A.; Sunwoo, John B.; Rudy, Susan; Morris, John C.; Mitchell, James B.; Camphausen, Kevin; Gius, David; Adams, Julian; Sausville, Edward A.; Conley, Barbara A.

2005-01-01

Purpose: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-κB (NF-κB) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: The tolerability and response to bortezomib 0.6 mg/m 2 and 0.9 mg/m 2 given twice weekly concurrent with daily reirradiation to 50-70 Gy was explored. Blood proteasome inhibition and NF-κB-modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-κB phospho-p65, apoptosis, and expression of NF-κB-modulated mRNAs were compared in serial biopsies from accessible tumors. Results: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m 2 and 0.9 mg/m 2 /dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m 2 was 32%, 16%, and 7% and after 0.9 mg/m 2 was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-κB activity, apoptosis, and expression of NF-κB-modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-κB-modulated cytokines in 1 patient with a major tumor reduction. Conclusions: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m 2 and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-κB localization, apoptosis, and NF-κB-modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary radiotherapy or reirradiation may merit future investigation

Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

Science.gov (United States)

Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

2009-07-01

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

Design, synthesis and docking studies of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids.

Science.gov (United States)

Shi, Jingmiao; Lei, Meng; Wu, Wenkui; Feng, Huayun; Wang, Jia; Chen, Shanshan; Zhu, Yongqiang; Hu, Shihe; Liu, Zhaogang; Jiang, Cheng

2016-04-15

A series of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids were designed and synthesized. Their structures were elucidated by (1)H NMR, (13)C NMR, LC-MS and HRMS. These compounds were evaluated for their β5 subunit inhibitory activities of human proteasome. The results showed that dipeptidyl boronic acid inhibitors composed of αα-amino acids were as active as bortezomib. Interestingly, the activities of those derived from αβ-amino acids lost completely. Of all the inhibitors, compound 22 (IC50=4.82 nM) was the most potent for the inhibition of proteasome activity. Compound 22 was also the most active against three MM cell lines with IC50 values less than 5 nM in inhibiting cell growth assays. Molecular docking studies displayed that 22 fitted very well in the β5 subunit active pocket of proteasome. Copyright © 2016. Published by Elsevier Ltd.

Chaperoning Proteins for Destruction: Diverse Roles of Hsp70 Chaperones and their Co-Chaperones in Targeting Misfolded Proteins to the Proteasome

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Ayala Shiber

2014-07-01

Full Text Available Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases.

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

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Sheng Li

2016-06-01

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

Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients

DEFF Research Database (Denmark)

Anvar, Seyed Yahya; hoen, Peter Ac; Venema, Andrea

2011-01-01

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset progressive muscle disorder caused by a poly-alanine expansion mutation in the Poly(A) Binding Protein Nuclear 1 (PABPN1). The molecular mechanisms that regulate disease onset and progression are largely unknown. In order to identify...... molecular pathways that are consistently associated with OPMD, we performed an integrated high-throughput transcriptome study in affected muscles of OPMD animal models and patients. The ubiquitin-proteasome system (UPS) was found to be the most consistently and significantly OPMD-deregulated pathway across...

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

Science.gov (United States)

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017

Proteasome inhibition by new dual warhead containing peptido vinyl sulfonyl fluorides

NARCIS (Netherlands)

Brouwer, Arwin J; Herrero Álvarez, Natalia; Ciaffoni, Adriano; van de Langemheen, Helmus; Liskamp, Rob M J

2016-01-01

The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long

Presence of insoluble Tau following rotenone exposure ameliorates basic pathways associated with neurodegeneration

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Rodrigo S. Chaves

2016-12-01

Full Text Available Protein aggregation is an important feature of neurodegenerative disorders. In Alzheimer's disease (AD protein aggregates are composed of hyperphosphorylated Tau and amyloid beta peptide (Aβ. Despite the involvement and identification of the molecular composition of these aggregates, their role in AD pathophysiology is not fully understood. However, depositions of these insoluble aggregates are typically reported as pathogenic and toxic for cell homeostasis. New evidences suggest that the deposition of these aggregates is a protective mechanism that preserves cell from toxic insults associated with the early stages of neurodegenerative diseases. To better understand the biological role of the protein aggregation with regard its effects in cellular homeostasis, the present study investigated the role of insoluble Tau and Tau aggregates on crucial cellular parameters such as redox homeostasis, proteasome activity and autophagy in hippocampal cell cultures and hippocampus of aged Lewis rats using a rotenone-induced aggregation model. Neurons were exposed to rotenone in different concentrations and exposure times aiming to determine the interval required for Tau aggregation. Our experimental design allowed us to demonstrate that rotenone exposure induces Tau hyperphosphorylation and aggregation in a concentration and time-dependent manner. Oxidative stress triggered by rotenone exposure was observed with the absence of Tau aggregates and was reduced or absent when Tau aggregates were present. This reduction of oxidative stress along with the presence of insoluble Tau was independent of alterations in antioxidant enzymes activities or cell death. In addition, rotenone induced oxidative stress was mainly associated with decrease in proteasome activity and autophagy flux. Conversely, when insoluble Tau appeared, autophagy turns to be overactivated while proteasome activity remained low. Our studies significantly advance the understanding that Tau

Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

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Clerc, Jérôme; Florea, Bogdan I; Kraus, Marianne; Groll, Michael; Huber, Robert; Bachmann, André S; Dudler, Robert; Driessen, Christoph; Overkleeft, Herman S; Kaiser, Markus

2009-11-02

The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

Geldanamycin-induced degradation of Chk1 is mediated by proteasome

International Nuclear Information System (INIS)

Nomura, M.; Nomura, N.; Yamashita, J.

2005-01-01

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

Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome

DEFF Research Database (Denmark)

Kesmir, Can; van Noort, V.; de Boer, R.J.

2003-01-01

not yet been quantified how different the specificity of two forms of the proteasome are. The main question, which still lacks direct evidence, is whether the immunoproteasome generates more MHC ligands. Here we use bioinformatics tools to quantify these differences and show that the immunoproteasome...

Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

Science.gov (United States)

Sutovsky, Peter; Manandhar, Gaurishankar; Laurincik, Jozef; Letko, Juraj; Caamaño, Jose Nestor; Day, Billy N; Lai, Liangxue; Prather, Randall S; Sharpe-Timms, Kathy L; Zimmer, Randall; Sutovsky, Miriam

2005-03-01

Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells.

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Ravi K Anchoori

Full Text Available 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 α-β 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.

Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway

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Raúl Sánchez-Lanzas

2017-12-01

Full Text Available Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7 by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag or C-terminus (V5 of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag or C-terminus (V5 were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging. While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro.

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Kai Wang

Full Text Available Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI+] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

Science.gov (United States)

Wang, Kai; Redeker, Virginie; Madiona, Karine; Melki, Ronald; Kabani, Mehdi

2015-01-01

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI +] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI +] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems. PMID:26115123

Interactive effects of CO₂ and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

Energy Technology Data Exchange (ETDEWEB)

Götze, Sandra [Alfred Wegener Institute, Helmholtz Centre for Polar, Marine Research, Functional Ecology, 27570 Bremerhaven (Germany); Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States); Matoo, Omera B. [Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States); Beniash, Elia [Department of Oral Biology, University of Pittsburgh, Pittsburgh, PA (United States); Saborowski, Reinhard [Alfred Wegener Institute, Helmholtz Centre for Polar, Marine Research, Functional Ecology, 27570 Bremerhaven (Germany); Sokolova, Inna M., E-mail: isokolov@uncc.edu [Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States)

2014-04-01

Highlights: • Elevated P_CO₂ enhanced accumulation of Cu and Cd in the gills of mollusks. • The proteasome activities were affected by metals but robust to elevated P_CO₂. • Exposure to Cd and Cu had opposite effects on the proteasome activity. • Combined exposure to Cu and elevated P_CO₂ negatively affected energy status. - Abstract: Increased anthropogenic emission of CO₂ changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO₂ and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves—Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4–5 weeks to clean seawater (control) and to either 50 μg L⁻¹ Cu or 50 μg L⁻¹ Cd at one of three partial pressures of CO₂ P_CO₂ ~395, ~800 and ~1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated P_CO₂ enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated P_CO₂, but P_CO₂ modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome

Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

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J. M. H. Geddes

2016-01-01

Full Text Available The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis.

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

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

2000-01-01

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

Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages.

Science.gov (United States)

Ohta, Yuko; McKinney, E Churchill; Criscitiello, Michael F; Flajnik, Martin F

2002-01-15

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.

Gene expression for peroxisome-associated enzymes in hepatocellular carcinomas induced by ciprofibrate, a hypolipidemic compound

International Nuclear Information System (INIS)

Rao, M.S.; Nemali, M.R.; Reddy, J.K.

1986-01-01

Administration of hypolipidemic compounds leads to marked proliferation of peroxisomes and peroxisome-associated enzymes (PAE) in the livers of rodents and non-rodent species. The increase peroxisome-associated enzymes such as fatty acid β-oxidation system and catalase is shown to be due to an increase in the levels of mRNA. In this experiment they have examined hepatocellular carcinomas (HCC), induced in male F-344 rats by ciprofibrate (0.025%, w/w for 60 weeks), for gene expression of PAE. Total RNA was purified from HCC as well as from control and ciprofibrate (0.025% for 2 weeks) fed rat livers. Northern blot analysis was performed using [32/sub p/]cDNA probes for albumin, fatty acetyl-CoA oxidase, enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and catalase. mRNA levels in HCC for albumin, fatty acid β-oxidation enzymes and catalase were comparable with those levels observed in the livers of rats given ciprofibrate for 2 weeks. In control livers the mRNAs for β-oxidation enzymes were low. Albumin mRNA levels in all the 3 groups were comparable. Additional studies are necessary to determine whether the increased level of mRNAs for the β-oxidation enzymes in HCC is due to the effect of ciprofibrate or to the gene amplification

Potential role of proteasome on c-jun related signaling in hypercholesterolemia induced atherosclerosis

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Erdi Sozen

2014-01-01

Full Text Available Atherosclerosis and its complications are major causes of death all over the world. One of the major risks of atherosclerosis is hypercholesterolemia. During atherosclerosis, oxidized low density lipoprotein (oxLDL regulates CD36-mediated activation of c-jun amino terminal kinase-1 (JNK1 and modulates matrix metalloproteinase (MMP induction which stimulates inflammation with an invasion of monocytes. Additionally, inhibition of proteasome leads to an accumulation of c-jun and phosphorylated c-jun and activation of activator protein-1 (AP-1 related increase of MMP expression. We have previously reported a significant increase in cluster of differentiation 36 (CD36 mRNA levels in hypercholesterolemic rabbits and shown that vitamin E treatment prevented the cholesterol induced increase in CD36 mRNA expression. In the present study, our aim is to identify the signaling molecules/transcription factors involved in the progression of atherosclerosis following CD36 activation in an in vivo model of hypercholesterolemic (induced by 2% cholesterol containing diet rabbits. In this direction, proteasomal activities by fluorometry and c-jun, phospo c-jun, JNK1, MMP-9 expressions by quantitative RT-PCR and immunoblotting were tested in aortic tissues. The effects of vitamin E on these changes were also investigated in this model. As a result, c-jun was phosphorylated following decreased proteasomal degradation in hypercholesterolemic group. MMP-9 expression was also increased in cholesterol group rabbits contributing to the development of atherosclerosis. In addition, vitamin E showed its effect by decreasing MMP-9 levels and phosphorylation of c-jun.

Interactive effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

International Nuclear Information System (INIS)

Götze, Sandra; Matoo, Omera B.; Beniash, Elia; Saborowski, Reinhard; Sokolova, Inna M.

2014-01-01

Highlights: • Elevated P CO 2 enhanced accumulation of Cu and Cd in the gills of mollusks. • The proteasome activities were affected by metals but robust to elevated P CO 2 . • Exposure to Cd and Cu had opposite effects on the proteasome activity. • Combined exposure to Cu and elevated P CO 2 negatively affected energy status. - Abstract: Increased anthropogenic emission of CO 2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO 2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves—Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4–5 weeks to clean seawater (control) and to either 50 μg L −1 Cu or 50 μg L −1 Cd at one of three partial pressures of CO 2 (P CO 2 ∼395, ∼800 and ∼1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated P CO 2 enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated P CO 2 , but P CO 2 modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO 2 levels. In contrast, trypsin- and caspase-like activities of

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

Science.gov (United States)

Lu, Ying; Wu, Jiayi; Dong, Yuanchen; Chen, Shuobing; Sun, Shuangwu; Ma, Yong-Bei; Ouyang, Qi; Finley, Daniel; Kirschner, Marc W; Mao, Youdong

2017-07-20

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5 Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

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Qureshi Asaf A

2012-07-01

Full Text Available Abstract Background Altered immune function during ageing results in increased production of nitric oxide (NO and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin in lipopolysaccharide (LPS-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. Results The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P 40%; P 60%; P 40%; P P  Conclusions The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli

Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

Science.gov (United States)

Lavorgna, Alfonso; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

Amyloid-β secretion, generation, and lysosomal sequestration in response to proteasome inhibition

DEFF Research Database (Denmark)

Agholme, Lotta; Hallbeck, Martin; Benedikz, Eirikur

2012-01-01

, as the autophagosome has been suggested as a site of amyloid-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPP(Swe) transfected SH-SY5Y neuroblastoma cells. We show...

Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways.

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Jonathan Wills

Full Text Available SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.

50Hz Extremely Low Frequency Electromagnetic Fields Enhance Protein Carbonyl Groups Content in Cancer Cells: Effects on Proteasomal Systems

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A. M. Eleuteri

2009-01-01

Full Text Available Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24–72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.

The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

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Dijk, Marianne van; Murphy, Eoin [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); Morrell, Ruth [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Knapper, Steven [Department of Haematology, School of Medicine, Cardiff University, Heath Park, CF14 4XN Cardiff (United Kingdom); O' Dwyer, Michael [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Samali, Afshin; Szegezdi, Eva, E-mail: eva.szegezdi@nuigalway.ie [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland)

2011-03-15

Acute myeloid leukemia (AML) is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification), which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

Associated liver enzymes with hyperlipidemic profile in type 2 diabetes patients.

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Al-Jameil, Noura; Khan, Farah A; Arjumand, Sadia; Khan, Mohammad F; Tabassum, Hajera

2014-01-01

Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and is associated with dyslipidemia and disturbed liver function. Aim of the present work is to assess the liver enzymes and to find its association with hyperlipidemic profile in T2DM. Total of 157 subjects were studied and divided into two groups; diabetes (n=81) and non-diabetes (n=76). Various biochemical parameters like fasting glucose, post prandial glucose, HbA1c, total cholesterol (TC), triglycerides (Tg), high density lipoprotein cholesterol (HDL-C), alanine amino transferase (ALT), aspartate amino transferase (AST) and gamma-glutamyl transferase (GGT) were analyzed by ROCHE module Cobas 6000 (C501 & C601) analyzer, kits were procured by ROCHE diagnostics. Low density lipoprotein cholesterol (LDL-C) was estimated by Freidwald's formula. Statistical analysis was performed by applying student t test and Pearson's correlation coefficient, at 0.0001 and 0.05 level of significance, respectively. All the glycemic control parameters, lipid profile parameters except HDL-C and liver enzymes were found increased in diabetes group and significantly differ from non-diabetes group (p>0.0001). ALT showed significant positive correlation with fasting glucose, post prandial glucose, HbA1c, TC, Tg, LDL-C and GGT at p>0.05. AST showed very weak relation with all parameters while GGT was positively associated with fasting glucose, post prandial glucose, HbA1c, TC, Tg, LDL-C and ALT at p>0.05. In conclusion, T2DM incline to elevate liver enzymes, especially ALT and GGT were of significance. Routine screening of ALT and GGT in T2DM patients may assists early detection of liver abnormalities and to arrest the progress of disease.

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

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Maria Emilia Figueiredo-Pereira

2015-01-01

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

Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma.

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Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans

2016-06-07

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

Skeletal muscle myotubes of the severely obese exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux

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Bollinger, Lance M.; Powell, Jonathan J. S.; Houmard, Joseph A.; Witczak, Carol A.; Brault, Jeffrey J.

2015-01-01

Objective Whole-body protein metabolism is dysregulated with obesity. Our goal was to determine if activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Methods We utilized primary Human Skeletal Muscle cell (HSkM) cultures since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean (BMI ≤ 26.0 kg/m2) and 8 severely obese (BMI ≥ 39.0) women were examined basally and when stimulated to atrophy (serum and amino acid starvation). Results HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in the lean and an increase in HSkM from obese subjects. HSkMC from the obese also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from lean and obese individuals under basal and starved conditions. Conclusions Our data indicate that muscle cells of the lean and severely obese have innate differences in management of protein degradation, which may explain their metabolic differences. PMID:26010327

Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals

Czech Academy of Sciences Publication Activity Database

Karabínová, Pavla; Kubelka, Michal; Šušor, Andrej

2011-01-01

Roč. 346, č. 1 (2011), s. 1-9 ISSN 0302-766X R&D Projects: GA ČR GAP502/10/0944; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50450515 Keywords : Oocyte * Proteasome * Meiosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.114, year: 2011

Health behavior associated with liver enzymes among obese Korean adolescents, 2009-2014.

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Lee, Eun-Young; Choi, Hwa Young; Cho, Hyunsoon; Kim, Bo Hyun; Ki, Moran

2018-01-01

Obesity is major risk factor for liver health. This study aimed to clarify whether specific health behaviors were associated with liver function in obese adolescents in Korea. Based on national school health examination data from 2009 to 2014, 25,142 obese or overweight students were examined for aspartate transaminase and alanine transaminase levels, and health behaviors. Multiple logistic regression was used to calculate the odds ratio for liver enzyme elevation. Subjects who thought of themselves as "very fat" had a 1.6 times higher odds ratio for liver enzyme elevations than those who thought of themselves as "normal." Those who consumed fast food 3 to 5 times weekly had 1.3 times higher odds ratio (OR = 1.27, 95% confidence interval = 1.05-1.54) for the elevation of ALT than those who did not consume fast food. Those who took sugar-sweetened beverage 3 to 5 times weekly had 1.2 times higher odds ratio (OR = 1.24, 95% confidence interval = 1.07-1.42) for the elevation of ALT than those who did not take it. Those who played computer game more than 2 hours a day showed 1.1 times higher odds ratio (OR = 1.10, 95% confidence interval = 1.01-1.21) for the elevation of ALT than those who did not. Specific food item and its frequency of consumption were identified for the positive and negative association with the elevation of liver enzymes. Self-image of body shape, sleeping time and need of help for alcohol or smoking problems also showed substantial association with the elevation.

Selective increase of in vivo firing frequencies in DA SN neurons after proteasome inhibition in the ventral midbrain.

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Subramaniam, Mahalakshmi; Kern, Beatrice; Vogel, Simone; Klose, Verena; Schneider, Gaby; Roeper, Jochen

2014-09-01

The impairment of protein degradation via the ubiquitin-proteasome system (UPS) is present in sporadic Parkinson's disease (PD), and might play a key role in selective degeneration of vulnerable dopamine (DA) neurons in the substantia nigra pars compacta (SN). Further evidence for a causal role of dysfunctional UPS in familial PD comes from mutations in parkin, which results in a loss of function of an E3-ubiquitin-ligase. In a mouse model, genetic inactivation of an essential component of the 26S proteasome lead to widespread neuronal degeneration including DA midbrain neurons and the formation of alpha-synuclein-positive inclusion bodies, another hallmark of PD. Studies using pharmacological UPS inhibition in vivo had more mixed results, varying from extensive degeneration to no loss of DA SN neurons. However, it is currently unknown whether UPS impairment will affect the neurophysiological functions of DA midbrain neurons. To answer this question, we infused a selective proteasome inhibitor into the ventral midbrain in vivo and recorded single DA midbrain neurons 2 weeks after the proteasome challenge. We found a selective increase in the mean in vivo firing frequencies of identified DA SN neurons in anesthetized mice, while those in the ventral tegmental area (VTA) were unaffected. Our results demonstrate that a single-hit UPS inhibition is sufficient to induce a stable and selective hyperexcitability phenotype in surviving DA SN neurons in vivo. This might imply that UPS dysfunction sensitizes DA SN neurons by enhancing 'stressful pacemaking'. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

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Lyons, Lisa C.; Gardner, Jacob S.; Gandour, Catherine E.; Krishnan, Harini C.

2017-01-01

We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in "Aplysia" using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through…

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

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Dogra, Nilambra; Mukhopadhyay, Tapas

2012-01-01

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 IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, 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. PMID:22745125

High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma

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Arshid, Samina; Tahir, Muhammad; Fontes, Belchor

2017-01-01

and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using...... functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome...... degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal...

Potential roles for ubiquitin and the proteasome during ribosome biogenesis

Czech Academy of Sciences Publication Activity Database

Stavreva, D. A.; Kawasaki, M.; Dundr, M.; Koberna, Karel; Müller, W. G.; Tsujimura-Takahashi, T.; Komatsu, W.; Hayano, T.; Isobe, T.; Raška, Ivan; Misteli, T.; Takahashi, N.; McNally, J. G.

2006-01-01

Roč. 26, č. 13 (2006), s. 5131-5145 ISSN 0270-7306 R&D Projects: GA MŠk(CZ) LC535; GA ČR(CZ) GA304/05/0374; GA ČR(CZ) GA304/04/0692 Grant - others:NIH(US) Intramural Research Program; Ministry of Education(JP) Pioneer Research grant Institutional research plan: CEZ:AV0Z50110509 Keywords : the role of the ubikvitin * proteasome system in ribosome biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.773, year: 2006

Proteasome LMP2/β1i subunit as biomarker for human uterine leiomyosarcoma

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Takuma Hayashi

2014-02-01

Full Text Available Uterine leiomyosarcoma (Ut-LMS develops more frequently in the myometrium of the uterine body than in the uterine cervix. Although the development of gynecological tumors is often correlated with the secretion of female hormones that of Ut-LMS does not, and its risk factor(s remain unknown. Importantly, a diagnostic biomarker that can distinguish malignant tumor Ut-LMS from benign tumor leiomyoma (LMA, has yet to be established. Therefore, the risk factor(s associated with Ut-LMS need to be examined in order to establish a diagnosis and clinical treatment method. Mice with a homozygous deficiency for the proteasome b-ring subunit, low-molecular mass polypeptide (LMP2/b1i spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. In recent studies, we showed that LMP2/b1i expression was absent in human Ut-LMS, but present in other human uterine mesenchymal tumors including uterine LMA. Moreover, LMP2/b1i is also known to negatively regulate human Ut-LMS tumorigenesis. Additional experiments furthermore revealed the differential expression of cyclin E and calponin h1 in human uterine mesenchymal tumors. Therefore, LMP2/b1i is a potential diagnostic biomarker when combined with the candidate molecules, cyclin E and calponin h1 for human Ut-LMS, and may be a targeted molecule for a new therapeutic approach.---------------------------------------------Cite this article as: Hayashi T, Horiuchi A Aburatani H, Ishiko O, Yaegashi N, Kanai Y, Zharhary D, Tonegawa S, Konishi I. Proteasome LMP2/ß1i subunit as biomarker for human uterine leiomyosarcoma. Int J Cancer Ther Oncol 2014; 2(1:02018.DOI: http://dx.doi.org/10.14319/ijcto.0201.8

Monoamine oxidase A gene polymorphisms and enzyme activity associated with risk of gout in Taiwan aborigines.

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Tu, Hung-Pin; Ko, Albert Min-Shan; Wang, Shu-Jung; Lee, Chien-Hung; Lea, Rod A; Chiang, Shang-Lun; Chiang, Hung-Che; Wang, Tsu-Nai; Huang, Meng-Chuan; Ou, Tsan-Teng; Lin, Gau-Tyan; Ko, Ying-Chin

2010-02-01

Taiwanese aborigines have a high prevalence of hyperuricemia and gout. Uric acid levels and urate excretion have correlated with dopamine-induced glomerular filtration response. MAOs represent one of the major renal dopamine metabolic pathways. We aimed to identify the monoamine oxidase A (MAOA, Xp11.3) gene variants and MAO-A enzyme activity associated with gout risk. This study was to investigate the association between gout and the MAOA single-nucleotide polymorphisms (SNPs) rs5953210, rs2283725, and rs1137070 as well as between gout and the COMT SNPs rs4680 Val158Met for 374 gout cases and 604 controls. MAO-A activity was also measured. All three MAOA SNPs were significantly associated with gout. A synonymous MAOA SNP, rs1137070 Asp470Asp, located in exon 14, was associated with the risk of having gout (P = 4.0 x 10(-5), adjusted odds ratio 1.46, 95% confidence intervals [CI]: 1.11-1.91). We also showed that, when compared to individuals with the MAOA GAT haplotype, carriers of the AGC haplotype had a 1.67-fold (95% CI: 1.28-2.17) higher risk of gout. Moreover, we found that MAOA enzyme activity correlated positively with hyperuricemia and gout (P for trend = 2.00 x 10(-3) vs. normal control). We also found that MAOA enzyme activity by rs1137070 allele was associated with hyperuricemia and gout (P for trend = 1.53 x 10(-6) vs. wild-type allele). Thus, our results show that some MAOA alleles, which have a higher enzyme activity, predispose to the development of gout.

Health behavior associated with liver enzymes among obese Korean adolescents, 2009-2014.

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Eun-Young Lee

Full Text Available Obesity is major risk factor for liver health. This study aimed to clarify whether specific health behaviors were associated with liver function in obese adolescents in Korea.Based on national school health examination data from 2009 to 2014, 25,142 obese or overweight students were examined for aspartate transaminase and alanine transaminase levels, and health behaviors. Multiple logistic regression was used to calculate the odds ratio for liver enzyme elevation.Subjects who thought of themselves as "very fat" had a 1.6 times higher odds ratio for liver enzyme elevations than those who thought of themselves as "normal." Those who consumed fast food 3 to 5 times weekly had 1.3 times higher odds ratio (OR = 1.27, 95% confidence interval = 1.05-1.54 for the elevation of ALT than those who did not consume fast food. Those who took sugar-sweetened beverage 3 to 5 times weekly had 1.2 times higher odds ratio (OR = 1.24, 95% confidence interval = 1.07-1.42 for the elevation of ALT than those who did not take it. Those who played computer game more than 2 hours a day showed 1.1 times higher odds ratio (OR = 1.10, 95% confidence interval = 1.01-1.21 for the elevation of ALT than those who did not.Specific food item and its frequency of consumption were identified for the positive and negative association with the elevation of liver enzymes. Self-image of body shape, sleeping time and need of help for alcohol or smoking problems also showed substantial association with the elevation.

Screen for ISG15-crossreactive deubiquitinases.

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André Catic

2007-07-01

Full Text Available The family of ubiquitin-like molecules (UbLs comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs might exist, capable of recognizing both ubiquitin and ISG15.We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1, USP13 (IsoT3, and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43. USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo

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Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.

2012-01-01

Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah−/−Ku70−/− double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy. PMID:22486314

Association of canalicular membrane enzymes with bile acid micelles and lipid aggregates in human and rat bile.

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Accatino, L; Pizarro, M; Solís, N; Koenig, C S

1995-01-18

This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the celular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.

Regulation of mIκBNS stability through PEST-mediated degradation by proteasome

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Park, Koog Chan; Jeong, Jiyeong; Kim, Keun Il, E-mail: kikim@sookmyung.ac.kr

2014-01-24

Highlights: • mIκBNS is degraded rapidly by proteasome without ubiquitylation. • N-terminal PEST sequence is responsible for the unstable nature of mIκBNS. • PEST sequence is not critical for nuclear localization of mIκBNS. • There is single bona fide NLS at the C-terminus of mIκBNS. - Abstract: Negative regulatory proteins in a cytokine signaling play a critical role in restricting unwanted excess activation of the signaling pathway. At the same time, negative regulatory proteins need to be removed rapidly from cells to respond properly to the next incoming signal. A nuclear IκB protein called IκBNS is known to inhibit a subset of NF-κB target genes upon its expression by NF-κB activation. Here, we show a mechanism to control the stability of mIκBNS which might be important for cells to prepare the next round signaling. We found that mIκBNS is a short-lived protein of which the stability is controlled by proteasome, independent of ubiquitylation process. We identified that the N-terminal PEST sequence in mIκBNS was critical for the regulation of stability.

Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

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Tingting Chen

2017-11-01

Full Text Available Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson’s disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.

Immunoproteasome in the blood plasma of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors.

Science.gov (United States)

Matuszczak, E; Sankiewicz, A; Debek, W; Gorodkiewicz, E; Milewski, R; Hermanowicz, A

2018-01-01

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). Twenty-seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P appendicitis. © 2017 British Society for Immunology.

Resveratrol protects leukemic cells against cytotoxicity induced by proteasome inhibitors via induction of FOXO1 and p27Kip1

International Nuclear Information System (INIS)

Niu, Xiao-Fang; Liu, Bao-Qin; Du, Zhen-Xian; Gao, Yan-Yan; Li, Chao; Li, Ning; Guan, Yifu; Wang, Hua-Qin

2011-01-01

It was reported recently that resveratrol could sensitize a number of cancer cells to the antitumoral effects of some conventional chemotherapy drugs. The current study was designed to investigate whether resveratrol could sensitize leukemic cells to proteasome inhibitors. Leukemic cells were treated with MG132 alone or in combination with resveratrol. Cell viability was investigated using MTT assay, and induction of apoptosis and cell cycle distribution was measured using flow cytometry. Western blot and real-time RT-PCR were used to investigate the expression of FOXO1 and p27 Kip1 . CHIP was performed to investigate the binding of FOXO1 to the p27 Kip1 promoter. Resveratrol strongly reduced cytotoxic activities of proteasome inhibitors against leukemic cells. MG132 in combination with resveratrol caused cell cycle blockade at G1/S transition via p27 Kip1 accumulation. Knockdown of p27 Kip1 using siRNA dramatically attenuated the protective effects of resveratrol on cytotoxic actions of proteasome inhibitors against leukemic cells. Resveratrol induced FOXO1 expression at the transcriptional level, while MG132 increased nuclear distribution of FOXO1. MG132 in combination with resveratrol caused synergistic induction of p27 Kip1 through increased recruitment of FOXO1 on the p27 Kip1 promoter. Resveratrol may have the potential to negate the cytotoxic effects of proteasome inhibitors via regulation of FOXO1 transcriptional activity and accumulation of p27 Kip1

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

International Nuclear Information System (INIS)

Pinak, Miroslav

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

2001-07-01

The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

Enzyme recycling in lignocellulosic biorefineries

DEFF Research Database (Denmark)

Jørgensen, Henning; Pinelo, Manuel

2017-01-01

platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

Science.gov (United States)

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

Thioredoxin 1 in Prostate Tissue Is Associated with Gleason Score, Erythrocyte Antioxidant Enzyme Activity, and Dietary Antioxidants

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Terrence M. Vance

2015-01-01

Full Text Available Background. Prostate cancer is the most common noncutaneous cancer and second leading cause of cancer-related mortality in men in the US. Growing evidence suggests that oxidative stress is involved in prostate cancer. Methods. In this study, thioredoxin 1 (Trx 1, an enzyme and subcellular indicator of redox status, was measured in prostate biopsy tissue from 55 men from the North Carolina-Louisiana Prostate Cancer Project. A pathologist blindly scored levels of Trx 1. The association between Trx 1 and the Gleason score, erythrocyte antioxidant enzyme activity, and dietary antioxidant intake was determined using Fisher’s exact test. Results. Trx 1 levels in benign prostate tissue in men with incident prostate cancer were positively associated with the Gleason score (P=0.01 and inversely associated with dietary antioxidant intake (P=0.03. In prostate cancer tissue, Trx 1 levels were associated with erythrocyte glutathione peroxidase activity (P=0.01. No association was found for other erythrocyte enzymes. Greater Gleason score of malignant tissue corresponds to a greater difference in Trx 1 levels between malignant and benign tissue (P=0.04. Conclusion. These results suggest that the redox status of prostate tissue is associated with prostate cancer grade and both endogenous and exogenous antioxidants.

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

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Antje eVoigt

2013-03-01

Full Text Available 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.

Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation*

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Choi, Seung-Won; Song, Jun-Kyu; Yim, Ye-Seal; Yun, Ho-Geun; Chun, Kyung-Hee

2015-01-01

Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G1 transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G1 population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy. PMID:25691573

H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

Science.gov (United States)

Belle, Jad I; Nijnik, Anastasia

2014-05-01

Posttranslational modifications of histone H2A through the attachment of ubiquitin or poly-ubiquitin conjugates are common in mammalian genomes and play an important role in the regulation of chromatin structure, gene expression, and DNA repair. Histone H2A deubiquitinases (H2A-DUBs) are a group of structurally diverse enzymes that catalyze the removal ubiquitin from histone H2A. In this review we provide a concise summary of the mechanisms that mediate histone H2A ubiquitination in mammalian cells, and review our current knowledge of mammalian H2A-DUBs, their biochemical activities, and recent developments in our understanding of their functions in mammalian physiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

Science.gov (United States)

Oresic, Kristina; Mueller, Britta; Tortorella, Domenico

2009-06-01

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

International Nuclear Information System (INIS)

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

2009-01-01

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

Abnormal serum pancreatic enzymes, but not pancreatitis, are associated with an increased risk of malignancy in patients with intraductal papillary mucinous neoplasms.

Science.gov (United States)

Roch, Alexandra M; Parikh, Janak A; Al-Haddad, Mohammad A; DeWitt, John M; Ceppa, Eugene P; House, Michael G; Nakeeb, Attila; Schmidt, C Max

2014-10-01

Pancreatitis is associated with intraductal papillary mucinous neoplasm (IPMN). This association is in part due to inflammation from pancreatic ductal obstruction. Although the correlation between pancreatitis and the malignant potential of IPMN is unclear, the 2012 International Consensus Guidelines (ICG) consider pancreatitis a "worrisome feature." We hypothesized that serum pancreatic enzymes, markers of inflammation, are a better predictor of malignancy than pancreatitis in patients with IPMN. Between 1992 and 2012, 364 patients underwent resection for IPMN at a single university hospital. In the past decade, serum amylase and lipase were collected prospectively as an inflammatory marker in 203 patients with IPMN at initial surveillance and "cyst clinic" visits. The latest serum pancreatic enzyme values within 3 months preoperatively were studied. Pancreatitis was defined according to the 2012 revision of the Atlanta Consensus. Of the 203 eligible patients, there were 76 with pancreatitis. Pancreatitis was not associated with an increased rate of malignancy (P = .51) or invasiveness (P = .08). Serum pancreatic enzymes categorically outside of normal range (high or low) were also not associated with malignancy or invasiveness. In contrast, as a continuous variable, the higher the serum pancreatic enzymes were, the greater the rate of invasive IPMN. Of the 127 remaining patients without pancreatitis, serum pancreatic enzymes outside of normal range (low and high) were each associated with a greater rate of malignancy (P enzyme levels above normal range (high) were associated with a greater rate of invasiveness (P = .02). In patients with IPMN without a history of pancreatitis, serum pancreatic enzymes outside of the normal range are associated with a greater risk of malignancy. In patients with a history of pancreatitis, there is a positive correlation between the levels of serum pancreatic enzymes and the presence of invasive IPMN. These data suggest

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu.

Science.gov (United States)

Vielberg, Marie-Theres; Bauer, Verena C; Groll, Michael

2018-03-02

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O γ , Asp17O δ1 and Lys32N ε , it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH 2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified. Copyright © 2018 Elsevier Ltd. All rights reserved.

Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

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Bingrong Tang

Full Text Available 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-lactacystinβ-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.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

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Britta Stadelmann

2014-12-01

Full Text Available A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ, a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Profiling the orphan enzymes

Science.gov (United States)

2014-01-01

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

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Sayem Miah

Full Text Available Breast tumor kinase (BRK, also known as protein tyrosine kinase 6 (PTK6, is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Science.gov (United States)

Miah, Sayem; Goel, Raghuveera Kumar; Dai, Chenlu; Kalra, Natasha; Beaton-Brown, Erika; Bagu, Edward T; Bonham, Keith; Lukong, Kiven E

2014-01-01

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells

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Wei Huang

2017-08-01

Full Text Available Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

OpenAIRE

Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

2018-01-01

Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants.

Science.gov (United States)

Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

2018-01-01

Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat.

Science.gov (United States)

Jadeja, Shahnawaz D; Mansuri, Mohmmad Shoab; Singh, Mala; Dwivedi, Mitesh; Laddha, Naresh C; Begum, Rasheedunnisa

2017-01-01

Autoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo. To explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat. PSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis. The frequency of 'TT' genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively). PSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

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Sylvain Cardinaud

2011-05-01

Full Text Available Cytotoxic CD8+ T cells (CTLs play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF. We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D and a majority encoding an asparagine (Q9VF/5N. We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

Science.gov (United States)

Guilbert, Solenn M; Lambert, Herman; Rodrigue, Marc-Antoine; Fuchs, Margit; Landry, Jacques; Lavoie, Josée N

2018-02-05

BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

The association between angiotensin-converting enzyme gene polymorphism and coronary calcification - The Rotterdam Coronary Calcification Study

NARCIS (Netherlands)

Oei, HHS; Sayed-Tabatabaei, FA; Hofman, A; Oudkerk, M; van Duijn, CM; Witteman, JCM

Background: An insertion/deletion (I/D) polymorphism in the gene encoding angiotensin-converting enzyme (ACE) has been associated with serum ACE levels. The association between the ACE I/D polymorphism and coronary heart disease is unclear. Electron-beam-computed tomography (EBT) is a technique to

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

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David S Reis

2013-10-01

Full Text Available 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 polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-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 highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

SUMOylation of Blimp-1 promotes its proteasomal degradation.

Science.gov (United States)

Shimshon, Livnat; Michaeli, Avital; Hadar, Rivka; Nutt, Stephen L; David, Yael; Navon, Ami; Waisman, Ari; Tirosh, Boaz

2011-08-04

B lymphocyte induced maturation protein-1 (Blimp-1) is a transcription repressor of the Krueppel-like family. Blimp-1 plays important roles in developmental processes, such as of germ cells and hair follicle stem cells. In B lymphocytes Blimp-1 orchestrates the terminal differentiation into plasma cells. We discovered that Blimp-1 undergoes SUMOylation by SUMO-1. This SUMOylation is modulated by the SUMO protease SENP1. While Blimp-1 is relatively stable in 293T cells, a fusion with SUMO1 rendered it to rapid proteasomal degradation. Increase in SENP1 activity stabilized Blimp-1, while a decrease promoted its degradation. Our data indicate that SUMOylation of Blimp-1 regulates its intracellular stability. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

Science.gov (United States)

Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

2015-01-01

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

Chemical analysis of Greek pollen - Antioxidant, antimicrobial and proteasome activation properties

Directory of Open Access Journals (Sweden)

Gonos Efstathios

2011-06-01

Full Text Available Abstract Background Pollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated. Results Three different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique. Conclusions The Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile.

Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

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Denamur, Sophie; Boland, Lidvine [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Beyaert, Maxime [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Verstraeten, Sandrine L. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Fillet, Marianne [University of Liege, CIRM, Department of Pharmacy, Laboratory for the Analysis of Medicines, Quartier Hopital, Avenue Hippocrate, 15, B36, Tower 4, 4000 Liège 1 (Belgium); Tulkens, Paul M. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Bontemps, Françoise [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Mingeot-Leclercq, Marie-Paule [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium)

2016-10-15

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

International Nuclear Information System (INIS)

Denamur, Sophie; Boland, Lidvine; Beyaert, Maxime; Verstraeten, Sandrine L.; Fillet, Marianne; Tulkens, Paul M.; Bontemps, Françoise; Mingeot-Leclercq, Marie-Paule

2016-01-01

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

The proteasome of the differently-diverged eukaryote Giardia lamblia and its role in remodeling of the microtubule-based cytoskeleton.

Science.gov (United States)

Ray, Atrayee; Sarkar, Srimonti

2017-08-01

Giardia lamblia is the causative agent of the diarrheal disease giardiasis, against which only a limited number of drugs are currently available. Increasing reports of resistance to these drugs makes it necessary to identify new cellular targets for designing the next generation of anti-giardial drugs. Towards this goal, therapeutic agents that target the parasitic cellular machinery involved in the functioning of the unique microtubule-based cytoskeleton of the Giardia trophozoites are likely to be effective as microtubule function is not only important for the survival of trophozoites within the host, but also their extensive remodeling is necessary during the transition from trophozoites to cysts. Thus, drugs that affect microtubule remodeling have the potential to not only kill the disease-causing trophozoites, but also inhibit transmission of cysts in the community. Recent studies in other model organisms have indicated that the proteasome plays an integral role in the formation and remodeling of the microtubule-based cytoskeleton. This review draws attention to the various processes by which the giardial proteasome may impact the functioning of its microtubule cytoskeleton and highlights the possible differences of the parasitic proteasome and some of other cellular machinery involved in microtubule remodeling, compared to that of the higher eukaryotic host.

Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

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Yang M

2014-08-01

Full Text Available Ming Yang,1 Huizhong Su,1 Tomoyoshi Soga,2 Kamil R Kranc,3 Patrick J Pollard1 1Cancer Biology and Metabolism Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 2Institute for Advanced Biosciences, Keio University, Mizukami, Tsuruoka, Yamagata, Japan; 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Abstract: The hypoxia-inducible factor (HIF prolyl hydroxylase domain enzymes (PHDs regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2 availability enables its association with the von Hippel-Lindau (VHL tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. Keywords: prolyl hydroxylase domain (PHD

Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

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Bianca Baudisch

2018-02-01

Full Text Available Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

KAUST Repository

Magnacca, A.; Persiconi, I.; Nurzia, E.; Caristi, S.; Meloni, F.; Barnaba, V.; Paladini, F.; Raimondo, D.; Fiorillo, M. T.; Sorrentino, R.

2012-01-01

-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B

Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

Science.gov (United States)

Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

2016-08-17

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

Energy Technology Data Exchange (ETDEWEB)

Strom, Bjorn O. [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.no [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway)

2012-01-27

Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

Model-Informed Drug Development for Ixazomib, an Oral Proteasome Inhibitor.

Science.gov (United States)

Gupta, Neeraj; Hanley, Michael J; Diderichsen, Paul M; Yang, Huyuan; Ke, Alice; Teng, Zhaoyang; Labotka, Richard; Berg, Deborah; Patel, Chirag; Liu, Guohui; van de Velde, Helgi; Venkatakrishnan, Karthik

2018-02-15

Model-informed drug development (MIDD) was central to the development of the oral proteasome inhibitor ixazomib, facilitating internal decisions (switch from body surface area (BSA)-based to fixed dosing, inclusive phase III trials, portfolio prioritization of ixazomib-based combinations, phase III dose for maintenance treatment), regulatory review (model-informed QT analysis, benefit-risk of 4 mg dose), and product labeling (absolute bioavailability and intrinsic/extrinsic factors). This review discusses the impact of MIDD in enabling patient-centric therapeutic optimization during the development of ixazomib. © 2017 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3β inhibition-independent pathways.

Science.gov (United States)

Doeppner, Thorsten R; Kaltwasser, Britta; Sanchez-Mendoza, Eduardo H; Caglayan, Ahmet B; Bähr, Mathias; Hermann, Dirk M

2017-03-01

Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3β inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0-9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen-glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3β inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis.

Meta-analysis of genome-wide association studies on the intolerance of angiotensin-converting enzyme inhibitors

NARCIS (Netherlands)

Mahmoudpour, Seyed H.; Veluchamy, Abirami; Siddiqui, Moneeza K.; Asselbergs, Folkert W.; Souverein, Patrick C.; De Keyser, Catherine E.; Hofman, Albert; Lang, Chim C.; Doney, Alexander S.F.; Stricker, Bruno H.; De Boer, Anthonius; Maitland-Van Der Zee, Anke H.; Palmer, Colin N.A.

2017-01-01

Objectives To identify single nucleotide polymorphisms (SNPs) associated with switching from an angiotensin-converting enzyme (ACE)-inhibitor to an angiotensin receptor blocker. Methods Two cohorts of patients starting ACE-inhibitors were identified within the Rotterdam Study in the Netherlands and

CHD8, A Novel Beta-Catenin Associated Chromatin Remodeling Enzyme, Regulates Androgen Receptor Mediated Gene Transcription

National Research Council Canada - National Science Library

Bochar, Daniel A

2008-01-01

.... To better understand the function of beta-catenin in AR mediated transcription, we have identified a novel chromatin remodeling enzyme, CHD8, that can associate with beta-catenin and functions in AR...

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

Energy Technology Data Exchange (ETDEWEB)

Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

2009-08-28

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

Mouse models of two missense mutations in actin-binding domain 1 of dystrophin associated with Duchenne or Becker muscular dystrophy.

Science.gov (United States)

McCourt, Jackie L; Talsness, Dana M; Lindsay, Angus; Arpke, Robert W; Chatterton, Paul D; Nelson, D'anna M; Chamberlain, Christopher M; Olthoff, John T; Belanto, Joseph J; McCourt, Preston M; Kyba, Michael; Lowe, Dawn A; Ervasti, James M

2018-02-01

Missense mutations in the dystrophin protein can cause Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) through an undefined pathomechanism. In vitro studies suggest that missense mutations in the N-terminal actin-binding domain (ABD1) cause protein instability, and cultured myoblast studies reveal decreased expression levels that can be restored to wild-type with proteasome inhibitors. To further elucidate the pathophysiology of missense dystrophin in vivo, we generated two transgenic mdx mouse lines expressing L54R or L172H mutant dystrophin, which correspond to missense mutations identified in human patients with DMD or BMD, respectively. Our biochemical, histologic and physiologic analysis of the L54R and L172H mice show decreased levels of dystrophin which are proportional to the phenotypic severity. Proteasome inhibitors were ineffective in both the L54R and L172H mice, yet mice homozygous for the L172H transgene were able to express even higher levels of dystrophin which caused further improvements in muscle histology and physiology. Given that missense dystrophin is likely being degraded by the proteasome but whole body proteasome inhibition was not possible, we screened for ubiquitin-conjugating enzymes involved in targeting dystrophin to the proteasome. A myoblast cell line expressing L54R mutant dystrophin was screened with an siRNA library targeting E1, E2 and E3 ligases which identified Amn1, FBXO33, Zfand5 and Trim75. Our study establishes new mouse models of dystrophinopathy and identifies candidate E3 ligases that may specifically regulate dystrophin protein turnover in vivo. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling

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Bangjun Zhou

2018-05-01

Full Text Available In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2 with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

Science.gov (United States)

Andrade, Ana Camila; Fróes, Adriana; Lopes, Fabyano Álvares Cardoso; Thompson, Fabiano L; Krüger, Ricardo Henrique; Dinsdale, Elizabeth; Bruce, Thiago

2017-07-01

Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

Science.gov (United States)

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Tay–Sachs disease mutations in HEXA target the α chain of hexosaminidase A to endoplasmic reticulum–associated degradation

Science.gov (United States)

Dersh, Devin; Iwamoto, Yuichiro; Argon, Yair

2016-01-01

Loss of function of the enzyme β-hexosaminidase A (HexA) causes the lysosomal storage disorder Tay–Sachs disease (TSD). It has been proposed that mutations in the α chain of HexA can impair folding, enzyme assembly, and/or trafficking, yet there is surprisingly little known about the mechanisms of these potential routes of pathogenesis. We therefore investigated the biosynthesis and trafficking of TSD-associated HexA α mutants, seeking to identify relevant cellular quality control mechanisms. The α mutants E482K and G269S are defective in enzymatic activity, unprocessed by lysosomal proteases, and exhibit altered folding pathways compared with wild-type α. E482K is more severely misfolded than G269S, as observed by its aggregation and inability to associate with the HexA β chain. Importantly, both mutants are retrotranslocated from the endoplasmic reticulum (ER) to the cytosol and are degraded by the proteasome, indicating that they are cleared via ER-associated degradation (ERAD). Leveraging these discoveries, we observed that manipulating the cellular folding environment or ERAD pathways can alter the kinetics of mutant α degradation. Additionally, growth of patient fibroblasts at a permissive temperature or with chemical chaperones increases cellular Hex activity by improving mutant α folding. Therefore modulation of the ER quality control systems may be a potential therapeutic route for improving some forms of TSD. PMID:27682588

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8(+) T Cells during Bacterial Infection

NARCIS (Netherlands)

Platteel, Anouk C M|info:eu-repo/dai/nl/375805613; Liepe, Juliane; Textoris-Taube, Kathrin; Keller, Christin; Henklein, Petra; Schalkwijk, Hanna H; Cardoso, Rebeca; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M|info:eu-repo/dai/nl/115553843

2017-01-01

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to

The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System.

Science.gov (United States)

Fuchs, Adrian C D; Alva, Vikram; Maldoner, Lorena; Albrecht, Reinhard; Hartmann, Marcus D; Martin, Jörg

2017-06-06

Proteasomes are self-compartmentalizing proteases that function at the core of the cellular protein degradation machinery in eukaryotes, archaea, and some bacteria. Although their evolutionary history is under debate, it is thought to be linked to that of the bacterial protease HslV and the hypothetical bacterial protease Anbu (ancestral beta subunit). Here, together with an extensive bioinformatic analysis, we present the first biophysical characterization of Anbu. Anbu forms a dodecameric complex with a unique architecture that was only accessible through the combination of X-ray crystallography and small-angle X-ray scattering. While forming continuous helices in crystals and electron microscopy preparations, refinement of sections from the crystal structure against the scattering data revealed a helical open-ring structure in solution, contrasting the ring-shaped structures of proteasome and HslV. Based on this primordial architecture and exhaustive sequence comparisons, we propose that Anbu represents an ancestral precursor at the origin of self-compartmentalization. Copyright © 2017 Elsevier Ltd. All rights reserved.

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

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Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

The Role of Epibionts of Bacteria of the Genus Pseudoalteromonas and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges.

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Kravchuk, O I; Lavrov, A I; Finoshin, A D; Gornostaev, N G; Georgiev, A A; Abaturova, S B; Mikhailov, V S; Lyupina, Yu V

2018-03-01

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

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Sophie A Comyn

2016-07-01

Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

Structural Basis for the Species-Selective Binding of N,C-Capped Dipeptides to the Mycobacterium tuberculosis Proteasome

Energy Technology Data Exchange (ETDEWEB)

Hsu, Hao-Chi [Van Andel Research Institute, Grand Rapids, Michigan 49503, United States; Singh, Pradeep K.; Fan, Hao; Wang, Rong [NMR; Sukenick, George [NMR; Nathan, Carl; Lin, Gang; Li, Huilin [Van Andel Research Institute, Grand Rapids, Michigan 49503, United States

2016-12-27

The Mycobacterium tuberculosis (Mtb) 20S proteasome is vital for the pathogen to survive under nitrosative stress in vitro and to persist in mice. To qualify for drug development, inhibitors targeting Mtb 20S must spare both the human constitutive proteasome (c-20S) and immunoproteasome (i-20S). We recently reported members of a family of noncovalently binding dipeptide proteasome inhibitors that are highly potent and selective for Mtb 20S over human c-20S and i-20S. To understand the structural basis of their potency and selectivity, we have studied the structure–activity relationship of six derivatives and solved their cocrystal structures with Mtb 20S. The dipeptide inhibitors form an antiparallel β-strand with the active site β-strands. Selectivity is conferred by several features of Mtb 20S relative to its mouse counterparts, including a larger S1 pocket, additional hydrogen bonds in the S3 pocket, and hydrophobic interactions in the S4 pocket. Serine-20 and glutamine-22 of Mtb 20S interact with the dipeptides and confer Mtb-specific inhibition over c-20S and i-20S. The Mtb 20S and mammalian i-20S have a serine-27 that interacts strongly with the dipeptides, potentially explaining the higher inhibitory activity of the dipeptides toward i-20S over c-20S. This detailed structural knowledge will aid in optimizing the dipeptides as anti-tuberculosis drugs.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes

Czech Academy of Sciences Publication Activity Database

Nagyová, Eva; Scsuková, S.; Němcová, Lucie; Mlynarčíková, A.; Yi, Y.J.; Sutovky, M.; Sutovsky, P.

2012-01-01

Roč. 42, č. 1 (2012), s. 50-62 ISSN 0739-7240 R&D Projects: GA ČR GAP502/11/0593 Institutional research plan: CEZ:AV0Z50450515 Keywords : Oocyte-cumulus complex * TNFAIP6 * HAS2 * Progesterone * Ubiquitin * Proteasome Subject RIV: ED - Physiology Impact factor: 2.377, year: 2012

Characterizing restriction enzyme-associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom-designed RNA probes

DEFF Research Database (Denmark)

Sanchez Barreiro, Fatima; Garrett Vieira, Filipe Jorge; Martin, Michael David

2017-01-01

Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols......, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits...

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

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Lushchak V.I.

1998-01-01

Full Text Available The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

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Anouk C.M. Platteel

2017-08-01

Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway.

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Scott A Keith

2016-02-01

Full Text Available The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant

Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

Science.gov (United States)

de Andrade, Luiz Henrique Soares; de Moraes, Wilson Max Almeida Monteiro; Matsuo Junior, Eduardo Hiroshi; de Orleans Carvalho de Moura, Elizabeth; Antunes, Hanna Karen Moreira; Montemor, Jairo; Antonio, Ednei Luiz; Bocalini, Danilo Sales; Serra, Andrey Jorge; Tucci, Paulo José Ferreira; Brum, Patricia Chakur; Medeiros, Alessandra

2015-04-01

The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS's activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 5×/week in treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-Akt(ser473), GSK3β, and phospho-GSK3β(ser9) were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3β and phospho-GSK3β, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3β, and did not alter the expression of total Akt, total GSK3β, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS's activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR

High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

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Johanna Abrigo

2016-01-01

Full Text Available Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes.

Association between Angiotensin-Converting Enzyme Inhibitors and Troponin in Acute Coronary Syndrome

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Luiz Minuzzo

2014-12-01

Full Text Available Background: Cardiovascular disease is the leading cause of mortality in the western world and its treatment should be optimized to decrease severe adverse events. Objective: To determine the effect of previous use of angiotensin-converting enzyme inhibitors on cardiac troponin I measurement in patients with acute coronary syndrome without ST-segment elevation and evaluate clinical outcomes at 180 days. Methods: Prospective, observational study, carried out in a tertiary center, in patients with acute coronary syndrome without ST-segment elevation. Clinical, electrocardiographic and laboratory variables were analyzed, with emphasis on previous use of angiotensin-converting enzyme inhibitors and cardiac troponin I. The Pearson chi-square tests (Pereira or Fisher's exact test (Armitage were used, as well as the non-parametric Mann-Whitney's test. Variables with significance levels of 0.5 ng / mL were high blood glucose at admission (p = 0.0034 and ST-segment depression ≥ 0.5 mm in one or more leads (p = 0.0016. The use of angiotensin-converting inhibitors prior to hospitalization was associated with troponin ≤ 0.5 ng / mL (p = 0.0482. The C-statistics for this model was 0.77. Conclusion: This study showed a correlation between prior use of angiotensin-converting enzyme inhibitors and reduction in the myocardial necrosis marker troponin I in patients admitted for acute coronary syndrome without ST-segment elevation. However, there are no data available yet to state that this reduction could lead to fewer severe clinical events such as death and re-infarction at 180 days.

Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5 expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

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Jitesh D Kawedia

Full Text Available The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5, we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70% decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.

Hypoxia and Hypoxia Mimetics Decrease Aquaporin 5 (AQP5) Expression through Both Hypoxia Inducible Factor-1α and Proteasome-Mediated Pathways

Science.gov (United States)

Kawedia, Jitesh D.; Yang, Fan; Sartor, Maureen A.; Gozal, David; Czyzyk-Krzeska, Maria; Menon, Anil G.

2013-01-01

The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels. PMID:23469202

Cancer-associated myositis associated with oesophageal adenocarcinoma arising in Barrett's oesophagus without serum myogenic enzymes elevation: an example suggesting the importance of MRI.

Science.gov (United States)

Sasaki, Yosuke; Shimizu, Hiroshige; Nemoto, Tetsuo; Urita, Yoshihisa

2016-04-21

The strong association between myositis and malignancy has been well recognised. Cancer-associated myositis (CAM) is thought to be a cross-reaction to regenerating muscle tissue similar to tumour antigen. We report a case of CAM due to oesophageal adenocarcinoma arising in Barrett's oesophagus without elevation of myogenic enzymes, diagnosed by MRI and repeated endoscopy. Elderly onset, prominent symptoms, lack of interstitial pneumonia, poorer response to immunosuppressive therapies, and the combination of negative conventional myositis-related antibodies and positive anti-p155/140 antibody may help to distinguish CAM from idiopathic inflammatory myopathy. As the prognosis of patients with CAM depends on the malignancy, aggressive diagnosis of CAM and the causative malignancy is required. Our experience underscores the importance of avoiding the over-reliance on serum myogenic enzymes for excluding CAM and recognising MRI as a useful diagnostic tool of myositis. 2016 BMJ Publishing Group Ltd.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

Science.gov (United States)

Bardag-Gorce, Fawzia; Oliva, Joan; Li, Jun; French, Barbara A; French, Samuel W

2010-06-01

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome. Copyright 2010 Elsevier Inc. All rights reserved.

Shotgun Proteomics of Aspergillus niger Microsomes upon d-Xylose Induction▿ †

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de Oliveira, José Miguel P. Ferreira; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2010-01-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which—many of them hypothetical proteins—were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles. PMID:20453123

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

Science.gov (United States)

Ferreira de Oliveira, José Miguel P; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2010-07-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

Isolation and enzyme bioprospection of endophytic bacteria associated with plants of Brazilian mangrove ecosystem.

Science.gov (United States)

Castro, Renata A; Quecine, Maria Carolina; Lacava, Paulo T; Batista, Bruna D; Luvizotto, Danice M; Marcon, Joelma; Ferreira, Anderson; Melo, Itamar S; Azevedo, João L

2014-01-01

The mangrove ecosystem is a coastal tropical biome located in the transition zone between land and sea that is characterized by periodic flooding, which confers unique and specific environmental conditions on this biome. In these ecosystems, the vegetation is dominated by a particular group of plant species that provide a unique environment harboring diverse groups of microorganisms, including the endophytic microorganisms that are the focus of this study. Because of their intimate association with plants, endophytic microorganisms could be explored for biotechnologically significant products, such as enzymes, proteins, antibiotics and others. Here, we isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. After identifying the isolates, the bacterial communities were evaluated for enzyme production. Protease activity was observed in 75% of the isolates, while endoglucanase activity occurred in 62% of the isolates. Bacillus showed the highest activity rates for amylase and esterase and endoglucanase. To our knowledge, this is the first reported diversity analysis performed on endophytic bacteria obtained from the branches of mangrove trees and the first overview of the specific enzymes produced by different bacterial genera. This work contributes to our knowledge of the microorganisms and enzymes present in mangrove ecosystems.

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

Science.gov (United States)

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

2013-01-01

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.

Angiotensin converting enzyme insertion/deletion polymorphism: association with ethnic origin.

Science.gov (United States)

Barley, J; Blackwood, A; Carter, N D; Crews, D E; Cruickshank, J K; Jeffery, S; Ogunlesi, A O; Sagnella, G A

1994-08-01

To determine the distribution of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in several ethnic groups: Caucasian Europeans, Black Nigerians, Samoan Polynesians and Yanomami Indians. The ratio of the frequencies of the II, ID and DD genotypes were 1:2:1 in the Europeans, but there was a tendency towards a higher frequency of the D allele in the Nigerians. In contrast, the Samoans and the Yanomami Indians displayed a much higher frequency of the I allele than of the D allele. The relationship between ACE genotype and disease in these latter groups is still not known, but the present results clearly suggest that ethnic origin should be carefully considered in the increasing number of studies on the association between I/D ACE genotype and disease aetiology.

Sperm surface protein AQ1 spermadhesin and ubiquitin-proteasome pathway in porcine anti-polyspermy defense

Czech Academy of Sciences Publication Activity Database

Jonáková, Věra; Postlerová, Pavla; Young-Joo, Y.; Sutovsky, P.; Pěknicová, Jana

2012-01-01

Roč. 67, Issue Supplement s1 (2012), s. 8-9 ISSN 1046-7408. [13th International Symposium for Immunology of reproduction "From the roots to the tops of Reproductive Immunology". 22.06.2012-24.06.2012, Varna] R&D Projects: GA ČR(CZ) GA523/09/1793; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Keywords : spermadhesin * ubiquitin * proteasome Subject RIV: CE - Biochemistry

Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

Science.gov (United States)

Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

2010-10-01

Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

Science.gov (United States)

Steely, Andrea M; Willoughby, Jamin A; Sundar, Shyam N; Aivaliotis, Vasiliki I; Firestone, Gary L

2017-10-01

Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria.

Science.gov (United States)

Martin, Marjolaine; Vandermies, Marie; Joyeux, Coline; Martin, Renée; Barbeyron, Tristan; Michel, Gurvan; Vandenbol, Micheline

2016-01-01

Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two "plurigenomic" (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the "great screen anomaly". Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon "plurigenomic" library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

Science.gov (United States)

Schnerch, Dominik; Schüler, Julia; Follo, Marie; Felthaus, Julia; Wider, Dagmar; Klingner, Kathrin; Greil, Christine; Duyster, Justus; Engelhardt, Monika; Wäsch, Ralph

2017-03-28

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

Activation of interfacial enzymes at membrane surfaces

DEFF Research Database (Denmark)

Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi

2006-01-01

A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

The Ubiquitin-Proteasome System and Microvascular Complications of Diabetes

Directory of Open Access Journals (Sweden)

Saeed Yadranji Aghdam

2013-01-01

Full Text Available The ubiquitin-proteasome system (UPS is the mainstay of protein quality control which regulates cell cycle, differentiation and various signal transduction pathways in eukaryotic cells. The timely and selective degradation of surplus and/or aberrant proteins by the UPS is essential for normal cellular physiology. Any disturbance, delay or exaggeration in the process of selection, sequestration, labeling for degradation and degradation of target proteins by the UPS will compromise cellular and tissue homeostasis. High blood glucose or hyperglycemia caused by diabetes disrupts normal vascular function in several target organs including the retina and kidney resulting in the development of diabetic retinopathy (DR and diabetic nephropathy (DN. We and others have shown that hyperglycemia and oxidative stress modulate UPS activity in the retina and kidney. The majority of studies have focused on the kidney and provided insights into the contribution of dysregulated UPS to microvascular damage in DN. The eye is a unique organ in which a semi-fluid medium, the vitreous humor, separates the neural retina and its anastomosed blood vessels from the semi-solid lens tissue. The complexity of the cellular and molecular components of the eye may require a normal functioning and well tuned UPS for healthy vision. Altered UPS activity may contribute to the development of retinal microvascular complications of diabetes. A better understanding of the molecular nature of the ocular UPS function under normal and diabetic conditions is essential for development of novel strategies targeting its activity. This review will discuss the association of retinal vascular cell UPS activity with microvascular damage in DR with emphasis on alterations of the PA28 subunits of the UPS.

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies.

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Ciechanover, Aaron; Kwon, Yong Tae

2015-03-13

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

Melatonin-Induced Temporal Up-Regulation of Gene Expression Related to Ubiquitin/Proteasome System (UPS in the Human Malaria Parasite Plasmodium falciparum

Directory of Open Access Journals (Sweden)

Fernanda C. Koyama

2014-12-01

Full Text Available There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

Proteasome and NF-kappaB inhibiting phaeophytins from the green alga Cladophora fascicularis.

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Huang, Xinping; Li, Min; Xu, Bo; Zhu, Xiaobin; Deng, Zhiwei; Lin, Wenhan

2007-03-21

Chemical examination of the green alga Cladophora fascicularis resulted in the isolation and characterization of a new porphyrin derivative, porphyrinolactone (1), along with five known phaeophytins 2-6 and fourteen sterols and cycloartanes. The structure of 1 was determined on the basis of spectroscopic analyses and by comparison of its NMR data with those of known phaeophytins. Compounds 1-6 displayed moderate inhibition of tumor necrosis factor alpha (TNF-alpha) induced nuclear factor-kappaB (NF-kappaB) activation, while 2 and 4 displayed potential inhibitory activity toward proteasome chymotripsin-like activation. The primary structure-activity relationship was also discussed.

Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction

International Nuclear Information System (INIS)

He, Li-Cai; Xu, Han-Zhang; Gu, Zhi-Min; Liu, Chuan-Xu; Chen, Guo-Qiang; Wang, Yue-Fei; Wen, Dong-Hua; Wu, Ying-Li

2011-01-01

Research highlights: → Chemotherapeutic drugs or UV treatment reduces Ikaros prior to caspase-3 activation. → Etoposide treatment does not alter the mRNA but shortens the half-life of Ikaros. → MG132 or epoxomicin but not calpeptin inhibits etoposide-induced Ikaros degradation. → Overexpression of Ikaros accelerates etoposide-induced apoptosis in NB4 cells. -- Abstract: Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3 h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

Science.gov (United States)

Negahdar, Maria; Aukrust, Ingvild; Molnes, Janne; Solheim, Marie H; Johansson, Bente B; Sagen, Jørn V; Dahl-Jørgensen, Knut; Kulkarni, Rohit N; Søvik, Oddmund; Flatmark, Torgeir; Njølstad, Pål R; Bjørkhaug, Lise

2014-01-25

GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

DGAT enzymes and triacylglycerol biosynthesis

Science.gov (United States)

Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

2008-01-01

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

Science.gov (United States)

Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

2016-10-15

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Downregulated kynurenine 3-monooxygenase gene expression and enzyme activity in schizophrenia and genetic association with schizophrenia endophenotypes.

Science.gov (United States)

Wonodi, Ikwunga; Stine, O Colin; Sathyasaikumar, Korrapati V; Roberts, Rosalinda C; Mitchell, Braxton D; Hong, L Elliot; Kajii, Yasushi; Thaker, Gunvant K; Schwarcz, Robert

2011-07-01

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, is an antagonist at N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors and modulates glutamate, dopamine, and acetylcholine signaling. Cortical kynurenic acid concentrations are elevated in the brain and cerebrospinal fluid of schizophrenia patients. The proximal cause may be an impairment of kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme at the branching point of the kynurenine pathway. To examine KMO messenger RNA expression and KMO enzyme activity in postmortem tissue from the frontal eye field (FEF; Brodmann area 6) obtained from schizophrenia individuals compared with healthy control individuals and to explore the relationship between KMO single-nucleotide polymorphisms and schizophrenia oculomotor endophenotypes. Case-control postmortem and clinical study. Maryland Brain Collection, outpatient clinics. Postmortem specimens from schizophrenia patients (n = 32) and control donors (n = 32) and a clinical sample of schizophrenia patients (n = 248) and healthy controls (n = 228). Comparison of quantitative KMO messenger RNA expression and KMO enzyme activity in postmortem FEF tissue between schizophrenia patients and controls and association of KMO single-nucleotide polymorphisms with messenger RNA expression in postmortem FEF and schizophrenia and oculomotor endophenotypes (ie, smooth pursuit eye movements and oculomotor delayed response). In postmortem tissue, we found a significant and correlated reduction in KMO gene expression and KMO enzyme activity in the FEF in schizophrenia patients. In the clinical sample, KMO rs2275163 was not associated with a diagnosis of schizophrenia but showed modest effects on predictive pursuit and visuospatial working memory endophenotypes. Our results provide converging lines of evidence implicating reduced KMO activity in the etiopathophysiology of schizophrenia and related neurocognitive deficits.

CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

Science.gov (United States)

Cheong, Jit Kong; Gunaratnam, Lakshman; Hsu, Stephen I-Hong

2008-04-25

Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

Phage lytic enzymes: a history.

Science.gov (United States)

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

Mechanism of Action of Bortezomib and the New Proteasome Inhibitors on Myeloma Cells and the Bone Microenvironment: Impact on Myeloma-Induced Alterations of Bone Remodeling

Directory of Open Access Journals (Sweden)

Fabrizio Accardi

2015-01-01

Full Text Available Multiple myeloma (MM is characterized by a high capacity to induce alterations in the bone remodeling process. The increase in osteoclastogenesis and the suppression of osteoblast formation are both involved in the pathophysiology of the bone lesions in MM. The proteasome inhibitor (PI bortezomib is the first drug designed and approved for the treatment of MM patients by targeting the proteasome. However, recently novel PIs have been developed to overcome bortezomib resistance. Interestingly, several preclinical data indicate that the proteasome complex is involved in both osteoclast and osteoblast formation. It is also evident that bortezomib either inhibits osteoclast differentiation induced by the receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL or stimulates the osteoblast differentiation. Similarly, the new PIs including carfilzomib and ixazomib can inhibit bone resorption and stimulate the osteoblast differentiation. In a clinical setting, PIs restore the abnormal bone remodeling by normalizing the levels of bone turnover markers. In addition, a bone anabolic effect was described in responding MM patients treated with PIs, as demonstrated by the increase in the osteoblast number. This review summarizes the preclinical and clinical evidence on the effects of bortezomib and other new PIs on myeloma bone disease.

In parkinsonian substantia nigra, alpha-synuclein is modified by acrolein, a lipid-peroxidation product, and accumulates in the dopamine neurons with inhibition of proteasome activity.

Science.gov (United States)

Shamoto-Nagai, M; Maruyama, W; Hashizume, Y; Yoshida, M; Osawa, T; Riederer, P; Naoi, M

2007-01-01

alpha-Synuclein (alphaSYN) plays a central role in the neural degeneration of Parkinson's disease (PD) through its conformational change. In PD, alphaSYN, released from the membrane, accumulates in the cytoplasm and forms Lewy body. However, the mechanism behind the translocation and conformational change of alphaSYN leading to the cell death has not been well elucidated. This paper reports that in the dopamine neurons of the substantia nigra containing neuromelanin from PD patients, alphaSYN was modified with acrolein (ACR), an aldehyde product of lipid peroxidation. Histopathological observation confirmed the co-localization of protein immunoreactive to anti-alphaSYN and ACR antibody. By Western blot analyses of samples precipitated with either anti-alphaSYN or anti-ACR antibody, increase in ACR-modified alphaSYN was confirmed in PD brain. Modification of recombinant alphaSYN by ACR enhanced its oligomerization, and at higher ACR concentrations alphaSYN was fragmented and polymerized forming a smear pattern in SDS-PAGE. ACR reduced 20S proteasome activity through the direct modification of the proteasome proteins and the production of polymerized ACR-modified proteins, which inhibited proteasome activity in vitro. These results suggest that ACR may initiate vicious cycle of modification and aggregation of proteins, including alphaSYN, and impaired proteolysis system, to cause neuronal death in PD.

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

Science.gov (United States)

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Epigenetic regulation of lncRNA connects ubiquitin-proteasome system with infection-inflammation in preterm births and preterm premature rupture of membranes.

Science.gov (United States)

Luo, Xiucui; Pan, Jing; Wang, Leilei; Wang, Peirong; Zhang, Meijiao; Liu, Meilin; Dong, Ziqing; Meng, Qian; Tao, Xuguang; Zhao, Xinliang; Zhong, Julia; Ju, Weina; Gu, Yang; Jenkins, Edmund C; Brown, W Ted; Shi, Qingxi; Zhong, Nanbert

2015-02-15

Preterm premature rupture of membranes (PPROM) is responsible for one third of all preterm births (PTBs). We have recently demonstrated that long noncoding RNAs (lncRNAs) are differentially expressed in human placentas derived from PPROM, PTB, premature rupture of the membranes (PROM), and full-term birth (FTB), and determined the major biological pathways involved in PPROM. Here, we further investigated the relationship of lncRNAs, which are differentially expressed in spontaneous PTB (sPTB) and PPROM placentas and are found to overlap a coding locus, with the differential expression of transcribed mRNAs at the same locus. Ten lncRNAs (five up-regulated and five down-regulated) and the lncRNA-associated 10 mRNAs (six up- and four down-regulated), which were identified by microarray in comparing PPROM vs. sPTB, were then validated by real-time quantitative PCR. A total of 62 (38 up- and 24 down-regulated) and 1,923 (790 up- and 1,133 down-regulated) lncRNAs were identified from placentas of premature labor (sPTB + PPROM), as compared to those from full-term labor (FTB + PROM) and from premature rupture of membranes (PPROM + PROM), as compared to those from non-rupture of membranes (sPTB + FTB), respectively. We found that a correlation existed between differentially expressed lncRNAs and their associated mRNAs, which could be grouped into four categories based on the gene strand (sense or antisense) of lncRNA and its paired transcript. These findings suggest that lncRNA regulates mRNA transcription through differential mechanisms. Differential expression of the transcripts PPP2R5C, STAM, TACC2, EML4, PAM, PDE4B, STAM, PPP2R5C, PDE4B, and EGFR indicated a co-expression among these mRNAs, which are involved in the ubiquitine-proteasome system (UPS), in addition to signaling transduction and beta adrenergic signaling, suggesting that imbalanced regulation of UPS may present an additional mechanism underlying the premature rupture of membrane in PPROM

Association of Angiotensin-Converting Enzyme Genotype, Insertion/Deletion Polymorphism and Saphenous Vein Graft Atherosclerosis in Iranian Patients

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Neda Zeinali

2015-10-01

Full Text Available ABSTRACT OBJECTIVE: The aim of this study was to evaluate possible interactions among Angiotensin-I converting enzyme genotype, insertion/deletion polymorphism and atherosclerosis of vein grafts in Iranian patients, and characterize their clinical and demographic profile. METHODS: In this cross-sectional study, patients who underwent coronary artery bypass graft surgery more than five years ago, were included for angiographic analysis. Atherosclerosis was determined by quantitative angiography and adjusted Gensini score. The gene angiotensin converting enzyme I/D polymorphism was detected by polymerase chain reaction. RESULTS: A total of 102 patients participated in this study. Eighty-four patients were male. The frequency distribution of DD, ID and II polymorphism were 23.6%, 62.7% and 13.7% respectively. There were no differences among genotypic groups in age, sex, number of risk factors, number of vein grafts and months since bypass surgery. According to adjusted Gensini score [0.18±0.12 (II vs. 0.11±0.09 (ID and 0.1±0.09 (DD P=0.021] the II genotype was associated with severity of vein graft atherosclerosis. CONCLUSION: Although there are conflicting results about gene angiotensin converting enzyme I/D polymorphism and the degree of venous bypass graft degeneration, this study suggests an association between ACE genotype II and atherosclerosis of saphenous vein grafts, however, large samples considering clinical, demographic and ethnic profile are necessary to confirm these results.

Association of antioxidative enzymes with the synergistic effect of selenium and UV irradiation in enhancing plant growth

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T. XUE

2008-12-01

Full Text Available Selenium (Se is able to defend human and animal cells against UV(B stress. Higher plants are generally considered not to require Se but to have a low tolerance to it. However, recently it has been demonstrated that Se is able to protect also plants against UV-induced oxidative stress and even to promote the growth of plants subjected to high-energy light. In the present study the effects of Se on antioxidative enzymes possibly associated with this synergistic effect were investigated. Ryegrass and lettuce were grown in soil supplemented with Se at 0, 0.1 or 1.0 mg kg-1 under normal light or subjected to UV episodes. Lipid peroxidation and the changes of antioxidative enzymes were measured at two growing stages. The positive synergistic effect of the lower Se dosage and UV was found to be at least partly associated with the antioxidative role of Se through increased glutathione peroxidase (GSH-Px and catalase (CAT activity, whereas ascorbate peroxidase (APX responded negatively to both factors. The contribution of the other enzymes studied seemed to be plant-specific: glutathione S-transferase (GST increased in both ryegrass assays and superoxide dismutase (SOD in the first lettuce assay. At the higher addition level Se acted as a pro-oxidant and diminished fresh weight yields. UV irradiation alleviated the toxicity coincidently with increase of CAT in ryegrass and SOD in lettuce.;

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

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Yao Dai

Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

Genetic variants of methyl metabolizing enzymes and epigenetic regulators: Associations with promoter CpG island hypermethylation in colorectal cancer

NARCIS (Netherlands)

Vogel, S. de; Wouters, K.A.D.; Gottschalk, R.W.H.; Schooten, F.J. van; Goeij, A.F.P.M. de; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den; Weijenberg, M.P.; Engeland, M. van

2009-01-01