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

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...

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

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 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.

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).

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.

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

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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 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.

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.

Discovery of multiple interacting partners of gankyrin, a proteasomal chaperone and an oncoprotein--evidence for a common hot spot site at the interface and its functional relevance.

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Nanaware, Padma P; Ramteke, Manoj P; Somavarapu, Arun K; Venkatraman, Prasanna

2014-07-01

Gankyrin, a non-ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin-proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line- MDA-MB-435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA-MB-231 cells in which the endogenous CLIC1 is silenced, trans-expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein-protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. © 2013 Wiley Periodicals, Inc.

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

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.

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

Features of proteasome functioning in malignant tumors

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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.

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.

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

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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.

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

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...

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.

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.

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.

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.

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.

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

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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

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.

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.

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

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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

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

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

2016-06-01

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

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

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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.

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.

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.

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.

Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development.

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Amin Al-Shami

Full Text Available Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/- mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/- mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/- mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.

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 ...

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...

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

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

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.

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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.

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.

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

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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.

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.

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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.

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

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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.

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

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta

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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

2014-01-01

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

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

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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

2016-01-01

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

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.

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.

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

DEFF Research Database (Denmark)

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....

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.

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

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

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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.

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

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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.

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.

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].

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

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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...

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.

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.

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.

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.

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.

Antitumorigenic effect of proteasome inhibitors on insulinoma cells

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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...

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

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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.

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

Proteasome inhibitor carfilzomib interacts synergistically with histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells.

Science.gov (United States)

Gao, Minjie; Gao, Lu; Tao, Yi; Hou, Jun; Yang, Guang; Wu, Xiaosong; Xu, Hongwei; Tompkins, Van S; Han, Ying; Wu, Huiqun; Zhan, Fenghuang; Shi, Jumei

2014-06-01

In the present study, we investigated the interactions between proteasome inhibitor carfilzomib (CFZ) and histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells. Coexposure of cells to minimally lethal concentrations of CFZ with very low concentration of vorinostat resulted in synergistic antiproliferative effects and enhanced apoptosis in Jurkat T-leukemia cells, accompanied with the sharply increased reactive oxygen species (ROS), the striking decrease in the mitochondrial membrane potential (MMP), the increased release of cytochrome c, the enhanced activation of caspase-9 and -3, and the cleavage of PARP. The combined treatment of Jurkat cells pre-treated with ROS scavengers N-acetylcysteine (NAC) significantly blocked the loss of mitochondrial membrane potential, suggesting that ROS generation was a former event of the loss of mitochondrial membrane potential. Furthermore, NAC also resulted in a marked reduction in apoptotic cells, indicating a critical role for increased ROS generation by combined treatment. In addition, combined treatment arrested the cell cycle in G2-M phase. These results imply that CFZ interacted synergistically with vorinostat in Jurkat T-leukemia cells, which raised the possibility that the combination of carfilzomib with vorinostat may represent a novel strategy in treating T-cell Leukemia. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

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

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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

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

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.

PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana

DEFF Research Database (Denmark)

Cho, Seok Keun; Bae, Hansol; Ryu, Moonyoung

2015-01-01

and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only......Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22...

Studies on terrein as a new class of proteasome inhibitors

International Nuclear Information System (INIS)

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)

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

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

Science.gov (United States)

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.

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.

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

Science.gov (United States)

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.

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.

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

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

Association of proteasomal activity with metastasis in luminal breast cancer

Science.gov (United States)

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.

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

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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.

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...

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

Science.gov (United States)

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.

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.

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...

The use of activity based protein profiling to study proteasome biology

NARCIS (Netherlands)

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).

Interactive affective sharing versus non-interactive affective sharing in work groups : Comparative effects of group affect on work group performance and dynamics

NARCIS (Netherlands)

Klep, Annefloor; Wisse, Barbara; Van Der Flier, Henk

This study explores whether the dynamic path to group affect, which is characterized by interactive affective sharing processes, yields different effects on task performance and group dynamics than the static path to group affect, which arises from non-interactive affective sharing. The results of

Interactive affective sharing versus non-interactive affective sharing in work groups: Comparative effects of group affect on work group performance and dynamics

NARCIS (Netherlands)

Klep, A.H.M.; Wisse, B.M.; van der Flier, H.

2011-01-01

This study explores whether the dynamic path to group affect, which is characterized by interactive affective sharing processes, yields different effects on task performance and group dynamics than the static path to group affect, which arises from non-interactive affective sharing. The results of

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

Science.gov (United States)

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

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

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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

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

Structural Basis for the Species-Selective Binding of N,C-Capped Dipeptides to the Mycobacterium tuberculosis Proteasome

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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.

Nonverbal synchrony and affect in dyadic interactions

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Wolfgang eTschacher

2014-11-01

Full Text Available In an experiment on dyadic social interaction, we invited participants to verbal interactions in cooperative, competitive, and 'fun task' conditions. We focused on the link between interactants' affectivity and their nonverbal synchrony, and explored which further variables contributed to affectivity: interactants' personality traits, sex, and the prescribed interaction tasks. Nonverbal synchrony was quantified by the coordination of interactants' body movement, using an automated video-analysis algorithm (Motion Energy Analysis, MEA. Traits were assessed with standard questionnaires of personality, attachment, interactional style, psychopathology and interpersonal reactivity. We included 168 previously unacquainted individuals who were randomly allocated to same-sex dyads (84 females, 84 males, mean age 27.3 years. Dyads discussed four topics of general interest drawn from an urn of eight topics, and finally engaged in a fun interaction. Each interaction lasted five minutes. In between interactions, participants repeatedly assessed their affect. Using hierarchical linear modeling, we found moderate to strong effect sizes for synchrony to occur, especially in competitive and fun task conditions. Positive affect was associated positively with synchrony, negative affect was associated negatively. As for causal direction, data supported the interpretation that synchrony entailed affect rather than vice versa. The link between nonverbal synchrony and affect was strongest in female dyads. The findings extend previous reports of synchrony and mimicry associated with emotion in relationships and suggest a possible mechanism of the synchrony-affect correlation.

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

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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.

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.

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

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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...

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.

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.

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

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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

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

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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

Prediction of proteasome cleavage motifs by neural networks

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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...

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

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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

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

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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

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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.

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.

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

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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...

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.

Affective Computing and Intelligent Interaction

CERN Document Server

2012-01-01

2012 International Conference on Affective Computing and Intelligent Interaction (ICACII 2012) was the most comprehensive conference focused on the various aspects of advances in Affective Computing and Intelligent Interaction. The conference provided a rare opportunity to bring together worldwide academic researchers and practitioners for exchanging the latest developments and applications in this field such as Intelligent Computing, Affective Computing, Machine Learning, Business Intelligence and HCI.   This volume is a collection of 119 papers selected from 410 submissions from universities and industries all over the world, based on their quality and relevancy to the conference. All of the papers have been peer-reviewed by selected experts.  

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.

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.

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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.

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.

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.

Yeast Interacting Proteins Database: YML064C, YLR377C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available d or autophagy-mediated degradation depending on growth conditions; interacts with Vid30p Rows with this pre...er proteasome-mediated or autophagy-mediated degradation depending on growth conditions; interacts with Vid3

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.

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

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.

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.

Mood Swings: An Affective Interactive Art System

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Bialoskorski, Leticia S. S.; Westerink, Joyce H. D. M.; van den Broek, Egon L.

The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on the integration of a framework for affective movements and a color model. This enables Mood Swings to recognize affective movement characteristics as expressed by a person and display a color that matches the expressed emotion. With that, a unique interactive system is introduced, which can be considered as art, a game, or a combination of both.

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...

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.

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.

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.

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.

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

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu.

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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.

Non-covalent interaction between polyubiquitin and GTP cyclohydrolase 1 dictates its degradation.

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Yu Zhao

Full Text Available GTP cyclohydrolase 1 (GTPCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4. GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131 in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.

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

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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.

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.

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

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

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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.

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

Energy Technology Data Exchange (ETDEWEB)

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

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.

Come, See and Experience Affective Interactive Art

NARCIS (Netherlands)

Nijholt, Antinus; Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; Reidsma, Dennis; van den Broek, Egon; Hondorp, G.H.W.

2009-01-01

The progress in the field of affective computing enables the realization of affective consumer products, affective games, and affective art. This paper describes the affective interactive art system Mood Swings, which interprets and visualizes affect expressed by a person. Mood Swings is founded on

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...

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

Directory of Open Access Journals (Sweden)

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.

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.

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

Directory of Open Access Journals (Sweden)

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

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

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

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

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

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Affective Interface Adaptations in the Musickiosk Interactive Entertainment Application

Science.gov (United States)

Malatesta, L.; Raouzaiou, A.; Pearce, L.; Karpouzis, K.

The current work presents the affective interface adaptations in the Musickiosk application. Adaptive interaction poses several open questions since there is no unique way of mapping affective factors of user behaviour to the output of the system. Musickiosk uses a non-contact interface and implicit interaction through emotional affect rather than explicit interaction where a gesture, sound or other input directly maps to an output behaviour - as in traditional entertainment applications. PAD model is used for characterizing the different affective states and emotions.

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.

Affective Computing used in an imaging interaction paradigm

DEFF Research Database (Denmark)

Schultz, Nette

2003-01-01

This paper combines affective computing with an imaging interaction paradigm. An imaging interaction paradigm means that human and computer communicates primarily by images. Images evoke emotions in humans, so the computer must be able to behave emotionally intelligent. An affective image selection...

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

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...

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

Science.gov (United States)

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

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.

Melatonin-Induced Temporal Up-Regulation of Gene Expression Related to Ubiquitin/Proteasome System (UPS in the Human Malaria Parasite Plasmodium falciparum

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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.

The Affective Regulation of Social Interaction

Science.gov (United States)

Clore, Gerald L.; Pappas, Jesse

2007-01-01

The recent publication of David Heise's "Expressive Order" (2007) provides an occasion for discussing some of the key ideas in Affect Control Theory. The theory proposes that a few dimensions of affective meaning provide a common basis for interrelating personal identities and social actions. It holds that during interpersonal interactions, social…

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

Science.gov (United States)

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.

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.

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.

Yeast Interacting Proteins Database: YLR377C, YLR377C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available sis pathway, required for glucose metabolism; undergoes either proteasome-mediated or autophagy-mediated degradation depending...utophagy-mediated degradation depending on growth conditions; interacts with Vid30p Rows with this prey as p...d or autophagy-mediated degradation depending on growth conditions; interacts wit...me-mediated or autophagy-mediated degradation depending on growth conditions; int

Urban Interaction and Affective Experience

DEFF Research Database (Denmark)

Fritsch, Jonas; Brynskov, Martin

2008-01-01

As interactive digital technologies become a still more integrated and complex part of the everyday physical, social and cultural spaces we inhabit, research into these spaces’ dynamics and struc-tures needs to formulate adequate methods of analysis and dis-course. In this position paper we argue...... in favor of three points in that direction: First we argue that interaction – and the definition of interaction – is central to unfold the potential of digital urban media, from big, shared screens and media facades to small pri-vate, networked mobile and embedded platforms. Then we argue that an affective...... approach holds potential to address important aspects of the design of such blended digital spaces, extending beyond traditional interaction design. And finally we argue for the importance of construction, i.e. actual interventions of consider-able scale....

Best of Affective Computing and Intelligent Interaction 2013 in Multimodal Interactions

NARCIS (Netherlands)

Soleymani, Mohammad; Soleymani, M.; Pun, T.; Pun, Thierry; Nijholt, Antinus

The fifth biannual Humaine Association Conference on Affective Computing and Intelligent Interaction (ACII 2013) was held in Geneva, Switzerland. This conference featured the recent advancement in affective computing and relevant applications in education, entertainment and health. A number of

Affective loop experiences: designing for interactional embodiment.

Science.gov (United States)

Höök, Kristina

2009-12-12

Involving our corporeal bodies in interaction can create strong affective experiences. Systems that both can be influenced by and influence users corporeally exhibit a use quality we name an affective loop experience. In an affective loop experience, (i) emotions are seen as processes, constructed in the interaction, starting from everyday bodily, cognitive or social experiences; (ii) the system responds in ways that pull the user into the interaction, touching upon end users' physical experiences; and (iii) throughout the interaction the user is an active, meaning-making individual choosing how to express themselves-the interpretation responsibility does not lie with the system. We have built several systems that attempt to create affective loop experiences with more or less successful results. For example, eMoto lets users send text messages between mobile phones, but in addition to text, the messages also have colourful and animated shapes in the background chosen through emotion-gestures with a sensor-enabled stylus pen. Affective Diary is a digital diary with which users can scribble their notes, but it also allows for bodily memorabilia to be recorded from body sensors mapping to users' movement and arousal and placed along a timeline. Users can see patterns in their bodily reactions and relate them to various events going on in their lives. The experiences of building and deploying these systems gave us insights into design requirements for addressing affective loop experiences, such as how to design for turn-taking between user and system, how to create for 'open' surfaces in the design that can carry users' own meaning-making processes, how to combine modalities to create for a 'unity' of expression, and the importance of mirroring user experience in familiar ways that touch upon their everyday social and corporeal experiences. But a more important lesson gained from deploying the systems is how emotion processes are co-constructed and experienced

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.

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

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.

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

Science.gov (United States)

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.

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.

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...

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

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...

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

Science.gov (United States)

Patrick, B A; Jaiswal, A K

2012-10-04

Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

Mood swings: design and evaluation of affective interactive art

NARCIS (Netherlands)

Bialoskorski, Leticia S.S.; Westerink, Joyce H.D.M.; van den Broek, Egon

2009-01-01

The field of affective computing is concerned with developing emphatic products, such as affective consumer products, affective games, and affective art. This paper describes Mood Swings, an affective interactive art system, which interprets and visualizes affect expressed by a person. Mood Swings

Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

Science.gov (United States)

Broering, R; Trippler, M; Werner, M; Real, C I; Megger, D A; Bracht, T; Schweinsberg, V; Sitek, B; Eisenacher, M; Meyer, H E; Baba, H A; Weber, F; Hoffmann, A-C; Gerken, G; Schlaak, J F

2016-05-01

The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV. © 2016 John Wiley & Sons Ltd.

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 ...

Thiacloprid affects trophic interaction between gammarids and mayflies

International Nuclear Information System (INIS)

Englert, D.; Bundschuh, M.; Schulz, R.

2012-01-01

Neonicotinoid insecticides like thiacloprid enter agricultural surface waters, where they may affect predator–prey-interactions, which are of central importance for ecosystems as well as the functions these systems provide. The effects of field relevant thiacloprid concentrations on the leaf consumption of Gammarus fossarum (Amphipoda) were assessed over 96 h (n = 13–17) in conjunction with its predation on Baetis rhodani (Ephemeroptera) nymphs. The predation by Gammarus increased significantly at 0.50–1.00 μg/L. Simultaneously, its leaf consumption decreased with increasing thiacloprid concentration. As a consequence of the increased predation at 1.00 μg/L, gammarids' dry weight rose significantly by 15% compared to the control. At 4.00 μg/L, the reduced leaf consumption was not compensated by an increase in predation causing a significantly reduced dry weight of Gammarus (∼20%). These results may finally suggest that thiacloprid adversely affects trophic interactions, potentially translating into alterations in ecosystem functions, like leaf litter breakdown and aquatic-terrestrial subsidies. - Highlights: ► Field relevant thiacloprid concentrations affected gammarid and mayfly interaction. ► Gammarus leaf consumption and predation success is adversely affected. ► Gammarus growth increased due to higher predation at 1.0 μg thiacloprid/L. ► The study's results are discussed in the context of ecosystem functions. - Field relevant thiacloprid concentrations affect species interactions, which may translate to alterations in ecosystem functions.

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.

Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast

DEFF Research Database (Denmark)

Stone, Miranda; Hartmann-Petersen, Rasmus; Seeger, Michael

2004-01-01

. Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity. Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes. The human orthologues of these enzymes are known as Uch37 and Usp14......, respectively. We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome. In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6...... becomes essential when activity of these subunits is compromised by conditional mutations. Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may...

Association between social interaction and affect in nursing home residents with dementia.

Science.gov (United States)

Jao, Ying-Ling; Loken, Eric; MacAndrew, Margaret; Van Haitsma, Kimberly; Kolanowski, Ann

2018-06-01

Social interactions that lead to positive affect are fundamental to human well-being. However, individuals with dementia are challenged to achieve positive social interaction. It is unclear how social interactions influence affect in people with dementia. This study examined the association between social interactions and affect in nursing home residents with dementia. This repeated measures study used baseline data from a clinical trial in which 126 residents from 12 nursing homes were enrolled. Participants were video recorded twice daily on five days. Ratings of social interaction and affect were taken from the videotapes using the Interacting with People subscale of the Passivity in Dementia and the Philadelphia Geriatric Center Apparent Affect Rating Scale. Linear mixed models were used for analysis. Social interaction was significantly related to higher interest and pleasure at within- and between-person levels. Social interaction significantly predicted anxiety and sadness at the between-person level only. Residents with higher cognitive function also displayed greater pleasure. Greater interest and anxiety was evident during the afternoon hours. This study supports the impact of social interactions on positive and negative affect. Findings can guide intervention development, aimed at promoting positive social interactions and improving affect for people with dementia.

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

Science.gov (United States)

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.

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.

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

Role of the Ubiquitin Proteasome System in Regulating Skin Pigmentation

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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.

Facial Affect Reciprocity in Dyadic Interactions

Science.gov (United States)

2012-09-01

regulators of social interaction. In the developmental literature, this concept has been investigated under the rubric of social referencing...The communication of affects in monkeys: Cooperative reward conditioning. Journal of Genetic Psychology, 108, 121– 134. Miller, R. E., Banks, J

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

Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

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Florian Full

2017-10-01

Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

Directory of Open Access Journals (Sweden)

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.

Affect in Human-Robot Interaction

Science.gov (United States)

2014-01-01

Werry, I., Rae, J., Dickerson, P., Stribling, P., & Ogden, B. (2002). Robotic Playmates: Analysing Interactive Competencies of Children with Autism ...WE-4RII. IEEE International Conference on Intelligent Robots and Systems, Edmonton, Canada. 35. Moravec, H. (1988). Mind Children : The Future of...and if so when and where? • What approaches, theories , representations, and experimental methods inform affective HRI research? Report Documentation

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

Energy Technology Data Exchange (ETDEWEB)

Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Ghazawi, Feras [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); MacPherson, Paul, E-mail: pmacpherson@toh.on.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Division of Infectious Diseases, The Ottawa Hospital General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada)

2016-11-15

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

International Nuclear Information System (INIS)

Sugden, Scott; Ghazawi, Feras; MacPherson, Paul

2016-01-01

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

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

DEFF Research Database (Denmark)

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...

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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.

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.

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

Science.gov (United States)

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

Functional interplay between mitochondrial and proteasome activity in skin aging

NARCIS (Netherlands)

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

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.

Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I.

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Gori-Savellini, Gianni; Valentini, Melissa; Cusi, Maria Grazia

2013-06-01

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71-79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.

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

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

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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.

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

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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

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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...

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.

Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

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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.

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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.

Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341.

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Fang, Yong; Hu, Yi; Wu, Peng; Wang, Beibei; Tian, Yuan; Xia, Xi; Zhang, Qinghua; Chen, Tong; Jiang, Xuefeng; Ma, Quanfu; Xu, Gang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Meng, Li

2011-05-01

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

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.

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

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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

Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

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Boucher Charles AB

2010-07-01

Full Text Available Abstract Background The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics. Results Our analyses show that human proteins interacting with HIV form a densely connected and central sub-network within the total human protein interaction network. The evaluation of this sub-network for connectivity and centrality resulted in a set of proteins essential for the HIV life-cycle. Remarkably, we were able to associate proteins involved in RNA polymerase II transcription with hubs and proteasome formation with bottlenecks. Inferred network motifs show significant over-representation of positive and negative feedback patterns between virus and host. Strikingly, such patterns have never been reported in combined virus-host systems. Conclusions HIV infection results in a reprioritization of cellular processes reflected by an increase in the relative importance of transcriptional machinery and proteasome formation. We conclude that during the evolution of HIV, some patterns of interaction have been selected for resulting in a system where virus proteins preferably interact with central human proteins for direct control and with proteasomal proteins for indirect control over the cellular processes. Finally, the patterns described by network motifs illustrate how virus and host interact with one another.

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

International Nuclear Information System (INIS)

Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

2009-01-01

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface α5β1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1α) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1α mediated transcriptional activity as well as HIF-1α mediated angiogenic sprouting of ECs. HIF-1α plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1α activities.

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

Energy Technology Data Exchange (ETDEWEB)

Tandle, Anita T. [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Calvani, Maura; Uranchimeg, Badarch [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Zahavi, David [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Melillo, Giovanni [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Libutti, Steven K., E-mail: slibutti@montefiore.org [Department of Surgery, Montefiore-Einstein Center for Cancer Care, Albert Einstein College of Medicine, Greene Medical Arts Pavilion, 4th Floor 3400, Bainbridge Avenue, Bronx, New York 10467 (United States)

2009-07-01

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

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

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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,

Intrinsically Disordered Segments Affect Protein Half-Life in the Cell and during Evolution

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Robin van der Lee

2014-09-01

Full Text Available Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast, mouse, and human proteins with terminal or internal intrinsically disordered segments have significantly shorter half-lives than proteins without these features. The lengths of the disordered segments that affect protein half-life are compatible with the structure of the proteasome. Divergence in terminal and internal disordered segments in yeast proteins originating from gene duplication leads to significantly altered half-life. Many paralogs that are affected by such changes participate in signaling, where altered protein half-life will directly impact cellular processes and function. Thus, natural variation in the length and position of disordered segments may affect protein half-life and could serve as an underappreciated source of genetic variation with important phenotypic consequences.

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

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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

Affective Dynamics in Triadic Peer Interactions in Early Childhood

NARCIS (Netherlands)

Lavictoire, L.A.; Snyder, J.; Stoolmiller, M.; Hollenstein, T.P.

2012-01-01

In interpersonal interaction research, moving beyond dyadic to triadic dynamics can be analytically daunting. We explored the affective states expressed during triadic peer interactions to understand how patterns were associated with childhood psychopathology and sociometric status. High-risk

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.

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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.

BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

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Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

2017-08-26

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

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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.

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

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.

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.

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.

Affective processes in human-automation interactions.

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Merritt, Stephanie M

2011-08-01

This study contributes to the literature on automation reliance by illuminating the influences of user moods and emotions on reliance on automated systems. Past work has focused predominantly on cognitive and attitudinal variables, such as perceived machine reliability and trust. However, recent work on human decision making suggests that affective variables (i.e., moods and emotions) are also important. Drawing from the affect infusion model, significant effects of affect are hypothesized. Furthermore, a new affectively laden attitude termed liking is introduced. Participants watched video clips selected to induce positive or negative moods, then interacted with a fictitious automated system on an X-ray screening task At five time points, important variables were assessed including trust, liking, perceived machine accuracy, user self-perceived accuracy, and reliance.These variables, along with propensity to trust machines and state affect, were integrated in a structural equation model. Happiness significantly increased trust and liking for the system throughout the task. Liking was the only variable that significantly predicted reliance early in the task. Trust predicted reliance later in the task, whereas perceived machine accuracy and user self-perceived accuracy had no significant direct effects on reliance at any time. Affective influences on automation reliance are demonstrated, suggesting that this decision-making process may be less rational and more emotional than previously acknowledged. Liking for a new system may be key to appropriate reliance, particularly early in the task. Positive affect can be easily induced and may be a lever for increasing liking.

Ordinary Social Interaction and the Main Effect Between Perceived Support and Affect.

Science.gov (United States)

Lakey, Brian; Vander Molen, Randy J; Fles, Elizabeth; Andrews, Justin

2016-10-01

Relational regulation theory hypothesizes that (a) the main effect between perceived support and mental health primarily reflects ordinary social interaction rather than conversations about stress and how to cope with it, and (b) the extent to which a provider regulates a recipient's mental health primarily reflects the recipient's personal taste (i.e., is relational), rather than the provider's objective supportiveness. In three round-robin studies, participants rated each other on supportiveness and the quality of ordinary social interaction, as well as their own affect when interacting with each other. Samples included marines about to deploy to Afghanistan (N = 100; 150 dyads), students sharing apartments (N = 64; 96 dyads), and strangers (N = 48; 72 dyads). Perceived support and ordinary social interaction were primarily relational, and most of perceived support's main effect on positive affect was redundant with ordinary social interaction. The main effect between perceived support and affect emerged among strangers after brief text conversations, and these links were partially verified by independent observers. Findings for negative affect were less consistent with theory. Ordinary social interaction appears to be able to explain much of the main effect between perceived support and positive affect. © 2015 Wiley Periodicals, Inc.

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...

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.

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.

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

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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.

Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport

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Zaarur, Nava; Meriin, Anatoli B.; Bejarano, Eloy; Xu, Xiaobin; Gabai, Vladimir L.; Cuervo, Ana Maria

2014-01-01

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species. PMID:24469403

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.

Plastome-Genome Interactions Affect Plastid Transmission in Oenothera

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Chiu, W. L.; Sears, B. B.

1993-01-01

Plastids of Oenothera, the evening primrose, can be transmitted to the progeny from both parents. In a constant nuclear background, the frequency of biparental plastid transmission is determined by the types of plastid genomes (plastomes) involved in the crosses. In this study, the impact of nuclear genomes on plastid inheritance was analyzed. In general, the transmission efficiency of each plastome correlated strongly with its compatibility with the nuclear genome of the progeny, suggesting that plastome-genome interactions can influence plastid transmission by affecting the efficiency of plastid multiplication after fertilization. Lower frequencies of plastid transmission from the paternal side were observed when the pollen had poor vigor due to an incompatible plastome-genome combination, indicating that plastome-genome interactions may also affect the input of plastids at fertilization. Parental traits that affect the process of fertilization can also have an impact on plastid transmission. Crosses using maternal parents with long styles or pollen with relatively low growth capacity resulted in reduced frequencies of paternal plastid transmission. These observations suggest that degeneration of pollen plastids may occur as the time interval between pollination and fertilization is lengthened. PMID:8462856

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

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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.

Pesticide interactions with soils affected by olive oil mill wastewater

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Keren, Yonatan; Bukhanovsky, Nadezhda; Borisover, Mikhail

2013-04-01

Soil pesticide sorption is well known to affect the fate of pesticides, their bioavailability and the potential to contaminate air and water. Soil - pesticide interactions may be strongly influenced by soil organic matter (SOM) and organic matter (OM)-rich soil amendments. One special OM source in soils is related to olive oil production residues that may include both solid and liquid wastes. In the Mediterranean area, the olive oil production is considered as an important field in the agricultural sector. Due to the significant rise in olive oil production, the amount of wastes is growing respectively. Olive oil mill waste water (OMWW) is the liquid byproduct in the so-called "three phase" technological process. Features of OMWW include the high content of fatty aliphatic components and polyphenols and their often-considered toxicity. One way of OMWW disposal is the land spreading, e.g., in olive orchards. The land application of OMWW (either controlled or not) is supposed to affect the multiple soil properties, including hydrophobicity and the potential of soils to interact with pesticides. Therefore, there is both basic and applied interest in elucidating the interactions between organic compounds and soils affected by OMWW. However, little is known about the impact of OMWW - soil interactions on sorption of organic compounds, and specifically, on sorption of agrochemicals. This paper reports an experimental study of sorption interactions of a series of organic compounds including widely used herbicides such as diuron and simazine, in a range of soils that were affected by OMWW (i) historically or (ii) in the controlled land disposal experiments. It is demonstrated that there is a distinct increase in apparent sorption of organic chemicals in soils affected by OMWW. In selected systems, this increase may be explained by increase in SOM content. However, the SOM quality places a role: the rise in organic compound - soil interactions may both exceed the SOM

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

Directory of Open Access Journals (Sweden)

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.

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

Directory of Open Access Journals (Sweden)

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

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...

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

Energy Technology Data Exchange (ETDEWEB)

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.

Affective interactions using virtual reality: the link between presence and emotions.

Science.gov (United States)

Riva, Giuseppe; Mantovani, Fabrizia; Capideville, Claret Samantha; Preziosa, Alessandra; Morganti, Francesca; Villani, Daniela; Gaggioli, Andrea; Botella, Cristina; Alcañiz, Mariano

2007-02-01

Many studies showed the ability of movies and imagery techniques to elicit emotions. Nevertheless, it is less clear how to manipulate the content of interactive media to induce specific emotional responses. In particular, this is true for the emerging medium virtual reality (VR), whose main feature is the ability to induce a feeling of "presence" in the computer-generated world experienced by the user. The main goal of this study was to analyze the possible use of VR as an affective medium. Within this general goal, the study also analyzed the relationship between presence and emotions. The results confirmed the efficacy of VR as affective medium: the interaction with "anxious" and "relaxing" virtual environments produced anxiety and relaxation. The data also showed a circular interaction between presence and emotions: on one side, the feeling of presence was greater in the "emotional" environments; on the other side, the emotional state was influenced by the level of presence. The significance of these results for the assessment of affective interaction is discussed.

50Hz Extremely Low Frequency Electromagnetic Fields Enhance Protein Carbonyl Groups Content in Cancer Cells: Effects on Proteasomal Systems

Directory of Open Access Journals (Sweden)

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

Energy Technology Data Exchange (ETDEWEB)

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.

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.

Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma.

Science.gov (United States)

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

Science.gov (United States)

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

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.

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

What Makes Sports Fans Interactive? Identifying Factors Affecting Chat Interactions in Online Sports Viewing.

Science.gov (United States)

Ko, Minsam; Yeo, Jaeryong; Lee, Juyeong; Lee, Uichin; Jang, Young Jae

2016-01-01

Sports fans are able to watch games from many locations using TV services while interacting with other fans online. In this paper, we identify the factors that affect sports viewers' online interactions. Using a large-scale dataset of more than 25 million chat messages from a popular social TV site for baseball, we extract various game-related factors, and investigate the relationships between these factors and fans' interactions using a series of multiple regression analyses. As a result, we identify several factors that are significantly related to viewer interactions. In addition, we determine that the influence of these factors varies according to the user group; i.e., active vs. less active users, and loyal vs. non-loyal users.

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.

Factors affecting quality of social interaction park in Jakarta

Science.gov (United States)

Mangunsong, N. I.

2018-01-01

The existence of social interactions park in Jakarta is an oasis in the middle of a concrete jungle. Parks is a response to the need for open space as a place of recreation and community interaction. Often the social interaction parks built by the government does not function as expected, but other functions such as a place to sell, trash, unsafe so be rarely visited by visitors. The purpose of this study was to analyze the factors that affect the quality of social interaction parks in Jakarta by conducting descriptive analysis and correlation analysis of the variables assessment. The results of the analysis can give an idea of social interactions park based on community needs and propose the development of social interactioncity park. The object of study are 25 social interaction parks in 5 municipalities of Jakarta. The method used is descriptive analysis method, correlation analysis using SPSS 19 and using crosstab, chi-square tests. The variables are 5 aspects of Design, Plants composition: Selection type of plant (D); the beauty and harmony (Ind); Maintenance and fertility (P); Cleanliness and Environmental Health (BS); Specificity (Drainage, Multi Function garden, Means, Concern/Mutual cooperation, in dense settlements) (K). The results of analysis show that beauty is the most significant correlation with the value of the park followed by specificity, cleanliness and maintenance. Design was not the most significant variable affecting the quality of the park. The results of this study can be used by the Department of Parks and Cemeteries as input in managing park existing or to be developed and to improve the quality of social interaction park in Jakarta.

Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

Science.gov (United States)

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…

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

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

What Makes Sports Fans Interactive? Identifying Factors Affecting Chat Interactions in Online Sports Viewing.

Directory of Open Access Journals (Sweden)

Minsam Ko

Full Text Available Sports fans are able to watch games from many locations using TV services while interacting with other fans online. In this paper, we identify the factors that affect sports viewers' online interactions. Using a large-scale dataset of more than 25 million chat messages from a popular social TV site for baseball, we extract various game-related factors, and investigate the relationships between these factors and fans' interactions using a series of multiple regression analyses. As a result, we identify several factors that are significantly related to viewer interactions. In addition, we determine that the influence of these factors varies according to the user group; i.e., active vs. less active users, and loyal vs. non-loyal users.

What Makes Sports Fans Interactive? Identifying Factors Affecting Chat Interactions in Online Sports Viewing

Science.gov (United States)

Yeo, Jaeryong; Lee, Juyeong

2016-01-01

Sports fans are able to watch games from many locations using TV services while interacting with other fans online. In this paper, we identify the factors that affect sports viewers’ online interactions. Using a large-scale dataset of more than 25 million chat messages from a popular social TV site for baseball, we extract various game-related factors, and investigate the relationships between these factors and fans’ interactions using a series of multiple regression analyses. As a result, we identify several factors that are significantly related to viewer interactions. In addition, we determine that the influence of these factors varies according to the user group; i.e., active vs. less active users, and loyal vs. non-loyal users. PMID:26849568

With love, from me to you: Embedding social interactions in affective neuroscience.

Science.gov (United States)

Gilam, Gadi; Hendler, Talma

2016-09-01

Human emotional experiences naturally occur while interacting in a spontaneous, dynamic and response contingent fashion with other humans. This resonates with both theoretical considerations as well as neuroimaging findings that illustrate the nexus between the "social" and "emotional" brain suggesting a domain-general organization of the brain. Nevertheless, most knowledge in affective neuroscience stems from studying the brain in isolation from its natural social environment. Whether social interactions are constitutive or not to the understanding of other people's intentions, incorporating such interactions is clearly required for ecological validity. Moreover, since interpersonal interactions may influence emotional experiences and expressions, interactive paradigms may advance the theoretical understanding of what emotions are and what about them is social, and will correspondingly characterize their underlying neural substrates. We highlight the recent conceptual and experimental advances of bringing realistic social interactions into the neuroimaging lab; review emotion-induction paradigms and consider their congruency with features of social interactions; and emphasize the importance of embedding such spontaneous and dynamic interactive paradigms in the field of affective neuroscience. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Regulation of mIκBNS stability through PEST-mediated degradation by proteasome

Energy Technology Data Exchange (ETDEWEB)

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.

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

Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

Directory of Open Access Journals (Sweden)

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.

The effect of pathological narcissism on interpersonal and affective processes in social interactions.

Science.gov (United States)

Wright, Aidan G C; Stepp, Stephanie D; Scott, Lori N; Hallquist, Michael N; Beeney, Joseph E; Lazarus, Sophie A; Pilkonis, Paul A

2017-10-01

Narcissism has significant interpersonal costs, yet little research has examined behavioral and affective patterns characteristic of narcissism in naturalistic settings. Here we studied the effect of narcissistic features on the dynamic processes of interpersonal behavior and affect in daily life. We used interpersonal theory to generate transactional models of social interaction (i.e., linkages among perceptions of others' behavior, affect, and one's own behavior) predicted to be characteristic of narcissism. Psychiatric outpatients (N = 102) completed clinical interviews and a 21-day ecological momentary assessment protocol using smartphones. After social interactions (N = 5,781), participants reported on perceptions of their interaction partner's behavior (scored along the dimensions of dominant-submissive and affiliative-quarrelsome), their own affect, and their own behavior. Multilevel structural equation modeling was used to examine dynamic links among behavior and affect across interactions, and the role of narcissism in moderating these links. Results showed that perceptions of others' dominance did not predict dominant behavior, but did predict quarrelsome behavior, and this link was potentiated by narcissism. Furthermore, the link between others' dominance and one's own quarrelsome behavior was mediated by negative affect. Moderated mediation was also found: Narcissism amplified the link between ratings of others' dominance and one's own quarrelsomeness and negative affect. Narcissism did not moderate the link between other dominance and own dominance, nor the link between other affiliation and own affiliation. These results suggest that narcissism is associated with specific interpersonal and affective processes, such that sensitivity to others' dominance triggers antagonistic behavior in daily life. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

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

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.

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

Directory of Open Access Journals (Sweden)

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.

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.

Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation*

Science.gov (United States)

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

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

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

Directory of Open Access Journals (Sweden)

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.

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.

Science.gov (United States)

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.

Integral UBL domain proteins: a family of proteasome interacting proteins

DEFF Research Database (Denmark)

Hartmann-Petersen, Rasmus; Gordon, Colin

2004-01-01

The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact...

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

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.

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.

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.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

Energy Technology Data Exchange (ETDEWEB)

Maeda, Tomoji, E-mail: t-maeda@nichiyaku.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Nakajima, Toshihiro [Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku, Shinjyuku, Tokyo, Tokyo, 160-8402 (Japan); Komano, Hiroto, E-mail: hkomano@iwate-med.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan)

2016-05-13

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

International Nuclear Information System (INIS)

Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

2016-01-01

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

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.

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.

Plant functional diversity affects climate-vegetation interaction

Science.gov (United States)

Groner, Vivienne P.; Raddatz, Thomas; Reick, Christian H.; Claussen, Martin

2018-04-01

We present how variations in plant functional diversity affect climate-vegetation interaction towards the end of the African Humid Period (AHP) in coupled land-atmosphere simulations using the Max Planck Institute Earth system model (MPI-ESM). In experiments with AHP boundary conditions, the extent of the green Sahara varies considerably with changes in plant functional diversity. Differences in vegetation cover extent and plant functional type (PFT) composition translate into significantly different land surface parameters, water cycling, and surface energy budgets. These changes have not only regional consequences but considerably alter large-scale atmospheric circulation patterns and the position of the tropical rain belt. Towards the end of the AHP, simulations with the standard PFT set in MPI-ESM depict a gradual decrease of precipitation and vegetation cover over time, while simulations with modified PFT composition show either a sharp decline of both variables or an even slower retreat. Thus, not the quantitative but the qualitative PFT composition determines climate-vegetation interaction and the climate-vegetation system response to external forcing. The sensitivity of simulated system states to changes in PFT composition raises the question how realistically Earth system models can actually represent climate-vegetation interaction, considering the poor representation of plant diversity in the current generation of land surface models.

SOCIAL INTERACTIONS, STRESSFUL EVENTS AND NEGATIVE AFFECT AT WORK - A MICROANALYTIC APPROACH

NARCIS (Netherlands)

PEETERS, MCW; BUUNK, BP; SCHAUFELI, WB

1995-01-01

In the present study a daily event-recording method, the DIRO (Daily Interaction Record in Organizations), was employed for assessing social interactions, stressful events and negative affect at work. Forty-one secretaries filled out the records during the course of a week. This made it possible to

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.

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

OpenAIRE

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...

Hydrological Conditions Affect the Interspecific Interaction between Two Emergent Wetland Species

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Jian Zhou

2018-01-01

Full Text Available Hydrological conditions determine the distribution of plant species in wetlands, where conditions such as water depth and hydrological fluctuations are expected to affect the interspecific interactions among emergent wetland species. To test such effects, we conducted a greenhouse experiment with three treatment categories, interspecific interaction (mixed culture or monoculture, water depth (10 or 30 cm depth, and hydrological fluctuation (static or fluctuating water level, and two common emergent wetland plant species, Scirpus planiculumis Fr. (Cyperaceae and Phragmites australis var. baiyangdiansis (Gramineae. An increase in the water depth significantly restrained the growth of both S. planiculumis and P. australis, while hydrological fluctuations did not obviously alter the growth of either species. In addition, both water depth and hydrological fluctuations significantly affected the interspecific interaction between these two wetland species. P. australis benefited from interspecific interaction under increasing water depth and hydrological fluctuations, and the RII values were clearly positive for plants grown at a water depth that fluctuated around 30 cm. The results may have some implications for understanding how S. planiculumis and P. australis, as well as wetland communities, respond to the natural variation or human modification of hydrological conditions.

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.

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.

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

International Nuclear Information System (INIS)

Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu; Kim, Ki-Jeong; Park, Chang-Jin; Kim, Young-Jin; Park, Ohkmae K.; Paek, Kyung-Hee

2006-01-01

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

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.

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.

How social factors and behavioural strategies affect feeding and social interaction patterns in pigs.

Science.gov (United States)

Boumans, Iris J M M; de Boer, Imke J M; Hofstede, Gert Jan; Bokkers, Eddie A M

2018-04-26

Animals living in groups compete for food resources and face food conflicts. These conflicts are affected by social factors (e.g. competition level) and behavioural strategies (e.g. avoidance). This study aimed to deepen our understanding of the complex interactions between social factors and behavioural strategies affecting feeding and social interaction patterns in animals. We focused on group-housed growing pigs, Sus scrofa, which typically face conflicts around the feeder, and of which patterns in various competitive environments (i.e. pig:feeder ratio) have been documented soundly. An agent-based model was developed to explore how interactions among social factors and behavioural strategies can affect various feeding and social interaction patterns differently under competitive situations. Model results show that pig and diet characteristics interact with group size and affect daily feeding patterns (e.g. feed intake and feeding time) and conflicts around the feeder. The level of competition can cause a turning point in feeding and social interaction patterns. Beyond a certain point of competition, meal-based (e.g. meal frequency) and social interaction patterns (e.g. displacements) are determined mainly by behavioural strategies. The average daily feeding time can be used to predict the group size at which this turning point occurs. Under the model's assumptions, social facilitation was relatively unimportant in the causation of behavioural patterns in pigs. To validate our model, simulated patterns were compared with empirical patterns in conventionally housed pigs. Similarities between empirical and model patterns support the model results. Our model can be used as a tool in further research for studying the effects of social factors and group dynamics on individual variation in feeding and social interaction patterns in pigs, as well as in other animal species. Copyright © 2018 Elsevier Inc. All rights reserved.

Leader charisma and affective team climate: the moderating role of the leader's influence and interaction.

Science.gov (United States)

Hernández Baeza, Ana; Araya Lao, Cristina; García Meneses, Juliana; González Romá, Vicente

2009-11-01

In this study, we evaluate the role of leader charisma in fostering positive affective team climate and preventing negative affective climate. The analysis of a longitudinal database of 137 bank branches by means of hierarchical moderated regression shows that leader charisma has a stronger effect on team optimism than on team tension. In addition, the leader's influence and the frequency of leader-team interaction moderate the relationship between charisma and affective climate. However, whereas the leader's influence enhances the relationship between leader charisma and positive affective climate, the frequency of interaction has counterproductive effects.

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.

Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding

International Nuclear Information System (INIS)

Guinez, Celine; Mir, Anne-Marie; Leroy, Yves; Cacan, Rene; Michalski, Jean-Claude; Lefebvre, Tony

2007-01-01

Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG 2 cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property

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.

Immersion in a virtual world interactive drama and affective sciences

CERN Document Server

Mayr, Simon

2014-01-01

Interactive drama is more than just a new breed of entertainment software. As different research projects have shown, these systems can also be used for pedagogical and therapeutic purposes. The goal of these systems is to teach sophisticated problem solving skills by allowing the user to interact with compelling stories that have didactic purpose.One of the main attractions of narratives, independent of the medium in which they are presented, is that they elicit emotional response in their audiences. They have an affective impact and only engineers and authors who understand how the emotion s

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.

Dopamine D4 Receptor Polymorphism and Sex Interact to Predict Children's Affective Knowledge

Directory of Open Access Journals (Sweden)

Sharon eBen-Israel

2015-06-01

Full Text Available Affective knowledge, the ability to understand others’ emotional states, is considered to be a fundamental part in efficient social interaction. Affective knowledge can be seen as related to cognitive empathy, and in the framework of Theory of Mind (ToM as affective ToM. Previous studies found that cognitive empathy and ToM are heritable, yet little is known regarding the specific genes involved in individual variability in affective knowledge. Investigating the genetic basis of affective knowledge is important for understanding brain mechanisms underlying socio-cognitive abilities. The 7-repeat (7R allele within the third exon of the Dopamine D4 receptor gene (DRD4-III has been a focus of interest, due to accumulated knowledge regarding its relevance to individual differences in prosocial behavior. A recent study suggests that an interaction between the DRD4-III polymorphism and sex is associated with cognitive empathy among adults. We aimed to examine the same association in two childhood age groups. Children (N = 280, age 3.5 years, N = 283, age 5 years participated as part of the Longitudinal Israel Study of Twins (LIST. Affective knowledge was assessed through children’s responses to an illustrated story describing different emotional situations, told in a laboratory setting. The findings suggest a significant interaction between sex and the DRD4-III polymorphism, replicated in both age groups. Boy carriers of the 7R allele had higher affective knowledge scores than girls, whereas in the absence of the 7R there was no significant sex effect on affective knowledge. The results support the importance of DRD4-III polymorphism and sex differences to social development. Possible explanations for differences from adult findings are discussed, as are pathways for future studies.

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.

When sad groups expect to meet again : Interactive affective sharing and future interaction expectation as determinants of work groups' analytical and creative task performance

NARCIS (Netherlands)

Klep, Annefloor H. M.; Wisse, Barbara; van der Flier, Henk

2013-01-01

The present study examines the moderating role of future interaction expectation in the relationship between affective sharing and work groups' task performance. We argue that group affect, a group defining characteristic, becomes more salient to its members when it is interactively shared, and that

STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells.

Science.gov (United States)

Shin, Minkyung; Yi, Eun Hee; Kim, Byung-Hak; Shin, Jae-Cheon; Park, Jung Youl; Cho, Chung-Hyun; Park, Jong-Wan; Choi, Kang-Yell; Ye, Sang-Kyu

2016-11-30

The β-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, β-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of β-catenin. The level of β-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to β-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and β-catenin in HEK293T cells. To our knowledge, this is the first study to report that β-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated β-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active β-catenin via degradation, which stabilized SIAH-1 and increased its interaction with β-catenin. These results suggest that activated STAT3 regulates active β-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of β-catenin in HEK293T cells.

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

International Nuclear Information System (INIS)

Kusano, Shuichi; Yoshimitsu, Makoto; Hachiman, Miho; Ikeda, Masanori

2015-01-01

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

Energy Technology Data Exchange (ETDEWEB)

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Yoshimitsu, Makoto; Hachiman, Miho [Division of Hematology and Immunology, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ikeda, Masanori [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

2015-12-15

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.

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

Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

Directory of Open Access Journals (Sweden)

Brandi L Gancarz

Full Text Available Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol levels were significantly increased in strains depleted for a heat shock protein (HSF1 or proteasome components (PRE1 and RPT6, suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System.

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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.

Affective Interaction with a Virtual Character through an fNIRS Brain-Computer Interface

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Gabor Aranyi

2016-07-01

Full Text Available Affective Brain-Computer Interfaces (BCI harness Neuroscience knowledge to develop affective interaction from first principles. In this paper, we explore affective engagement with a virtual agent through Neurofeedback (NF. We report an experiment where subjects engage with a virtual agent by expressing positive attitudes towards her under a NF paradigm. We use for affective input the asymmetric activity in the dorsolateral prefrontal cortex (DL-PFC, which has been previously found to be related to the high-level affective-motivational dimension of approach/avoidance. The magnitude of left-asymmetric DL-PFC activity, measured using fNIRS and treated as a proxy for approach, is mapped onto a control mechanism for the virtual agent’s facial expressions, in which Action Units are activated through a neural network. We carried out an experiment with 18 subjects, which demonstrated that subjects are able to successfully engage with the virtual agent by controlling their mental disposition through NF, and that they perceived the agent’s responses as realistic and consistent with their projected mental disposition. This interaction paradigm is particularly relevant in the case of affective BCI as it facilitates the volitional activation of specific areas normally not under conscious control. Overall, our contribution reconciles a model of affect derived from brain metabolic data with an ecologically valid, yet computationally controllable, virtual affective communication environment.

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

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

Affect recognition and the quality of mother-infant interaction: understanding parenting difficulties in mothers with schizophrenia.

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Healy, Sarah J; Lewin, Jona; Butler, Stephen; Vaillancourt, Kyla; Seth-Smith, Fiona

2016-02-01

This study investigated the quality of mother-infant interaction and maternal ability to recognise adult affect in three study groups consisting of mothers with a diagnosis of schizophrenia, mothers with depression and healthy controls. Sixty-four mothers were recruited from a Mother and Baby Unit and local children's centres. A 5-min mother-infant interaction was coded on a number of caregiving variables. Affect recognition and discrimination abilities were tested via a series of computerised tasks. Group differences were found both in measures of affect recognition and in the mother-infant interaction. Mothers with schizophrenia showed consistent impairments across most of the parenting measures and all measures of affect recognition and discrimination. Mothers with depression fell between the mothers with schizophrenia and healthy controls on most measures. However, depressed women's parenting was not significantly poorer than controls on any of the measures, and only showed trends for differences with mothers with schizophrenia on a few measures. Regression analyses found impairments in affect recognition and a diagnosis of schizophrenia to predict the occurrence of odd or unusual speech in the mother-infant interaction. Results add to the growing body of knowledge on the mother-infant interaction in mothers with schizophrenia and mothers with depression compared to healthy controls, suggesting a need for parenting interventions aimed at mothers with these conditions. While affect recognition impairments were not found to fully explain differences in parenting among women with schizophrenia, further research is needed to understand the psychopathology of parenting disturbances within this clinical group.

Spatial heterogeneity of plant-soil feedback affects root interactions and interspecific competition.

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Hendriks, Marloes; Ravenek, Janneke M; Smit-Tiekstra, Annemiek E; van der Paauw, Jan Willem; de Caluwe, Hannie; van der Putten, Wim H; de Kroon, Hans; Mommer, Liesje

2015-08-01

Plant-soil feedback is receiving increasing interest as a factor influencing plant competition and species coexistence in grasslands. However, we do not know how spatial distribution of plant-soil feedback affects plant below-ground interactions. We investigated the way in which spatial heterogeneity of soil biota affects competitive interactions in grassland plant species. We performed a pairwise competition experiment combined with heterogeneous distribution of soil biota using four grassland plant species and their soil biota. Patches were applied as quadrants of 'own' and 'foreign' soils from all plant species in all pairwise combinations. To evaluate interspecific root responses, species-specific root biomass was quantified using real-time PCR. All plant species suffered negative soil feedback, but strength was species-specific, reflected by a decrease in root growth in own compared with foreign soil. Reduction in root growth in own patches by the superior plant competitor provided opportunities for inferior competitors to increase root biomass in these patches. These patterns did not cascade into above-ground effects during our experiment. We show that root distributions can be determined by spatial heterogeneity of soil biota, affecting plant below-ground competitive interactions. Thus, spatial heterogeneity of soil biota may contribute to plant species coexistence in species-rich grasslands. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Emotion in languaging: Language and emotion as affective, adaptive and flexible behavior in social interaction

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Thomas Wiben Jensen

2014-07-01

Full Text Available This article argues for a view on languaging as inherently affective. Informed by recent ecological tendencies within cognitive science and distributed language studies a distinction between first order languaging (language as whole-body sense making and second order language (language as system like constraints is put forward. Contrary to common assumptions within linguistics and communication studies separating language-as-a-system from language use (resulting in separations between language vs. body-language and verbal vs. non-verbal communication etc. the first/second order distinction sees language as emanating from behavior making it possible to view emotion and affect as integral parts languaging behavior. Likewise, emotion and affect are studied, not as inner mental states, but as processes of organism-environment interactions. Based on video recordings of interaction between 1 children with special needs, and 2 couple in therapy and the therapist patterns of reciprocal influences between interactants are examined. Through analyzes of affective stance and patterns of inter-affectivity it is exemplified how language and emotion should not be seen as separate phenomena combined in language use, but rather as completely intertwined phenomena in languaging behavior constrained by second order patterns.

Phase 1 clinical trial of the novel proteasome inhibitor marizomib with the histone deacetylase inhibitor vorinostat in patients with melanoma, pancreatic and lung cancer based on in vitro assessments of the combination.

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Millward, Michael; Price, Timothy; Townsend, Amanda; Sweeney, Christopher; Spencer, Andrew; Sukumaran, Shawgi; Longenecker, Angie; Lee, Lonnie; Lay, Ana; Sharma, Girish; Gemmill, Robert M; Drabkin, Harry A; Lloyd, G Kenneth; Neuteboom, Saskia T C; McConkey, David J; Palladino, Michael A; Spear, Matthew A

2012-12-01

Combining proteasome and histone deacetylase (HDAC) inhibition has been seen to provide synergistic anti-tumor activity, with complementary effects on a number of signaling pathways. The novel bi-cyclic structure of marizomib with its unique proteasome inhibition, toxicology and efficacy profiles, suggested utility in combining it with an HDAC inhibitor such as vorinostat. Thus, in this study in vitro studies assessed the potential utility of combining marizomib and vorinostat, followed by a clinical trial with the objectives of assessing the recommended phase 2 dose (RP2D), pharmacokinetics (PK), pharmacodynamics (PD), safety and preliminary anti-tumor activity of the combination in patients. Combinations of marizomib and vorinostat were assessed in vitro. Subsequently, in a Phase 1 clinical trial patients with melanoma, pancreatic carcinoma or Non-small Cell Lung Cancer (NSCLC) were given escalating doses of weekly marizomib in combination with vorinostat 300 mg daily for 16 days in 28 day cycles. In addition to standard safety studies, proteasome inhibition and pharmacokinetics were assayed. Marked synergy of marizomib and vorinostat was seen in tumor cell lines derived from patients with NSCLC, melanoma and pancreatic carcinoma. In the clinical trial, 22 patients were enrolled. Increased toxicity was not seen with the combination. Co-administration did not appear to affect the PK or PD of either drug in comparison to historical data. Although no responses were demonstrated using RECIST criteria, 61% of evaluable patients demonstrated stable disease with 39% having decreases in tumor measurements. Treatment of multiple tumor cell lines with marizomib and vorinostat resulted in a highly synergistic antitumor activity. The combination of full dose marizomib with vorinostat is tolerable in patients with safety findings consistent with either drug alone.

Acidification and warming affect both a calcifying predator and prey, but not their interaction

DEFF Research Database (Denmark)

Landes, Anja; Zimmer, Martin

2012-01-01

Both ocean warming and acidification have been demonstrated to affect the growth, performance and reproductive success of calcifying invertebrates. However, relatively little is known regarding how such environmental change may affect interspecific interactions. We separately treated green crabs...... to environmental change. Acidification negatively affected the closer-muscle length of the crusher chela and correspondingly the claw-strength increment in C. maenas. The effects of warming and/or acidification on L. littorea were less consistent but indicated weaker shells in response to acidification...... Carcinus maenas and periwinkles Littorina littorea under conditions that mimicked either ambient conditions (control) or warming and acidification, both separately and in combination, for 5 mo. After 5 mo, the predators, prey and predator-prey interactions were screened for changes in response...

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

Automatic Control of Contextual Interaction Integrated with Affection and Architectural Attentional Control

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Yanrong Jiang

2013-03-01

Full Text Available It is still a challenge for robots to interact with complex environments in a smooth and natural manner. The robot should be aware of its surroundings and inner status to make decisions accordingly and appropriately. Contexts benefit the interaction a lot, such as avoiding frequent interruptions (e.g., the explicit inputting requests and thus are essential for interaction. Other challenges, such as shifting attentional focus to a more important stimulus, etc., are also crucial in interaction control. This paper presents a hybrid automatic control approach for interaction, as well as its integration, with these multiple important factors, aiming at performing natural, human-like interactions in robots. In particular, a novel approach of architectural attentional control, based on affection is presented, which attempts to shift the attentional focus in a natural manner. Context-aware computing is combined with interaction to endow the robot with proactive abilities. The long-term interaction control approaches are described. Emotion and personality are introduced into the interaction and their influence mechanism on interaction is explored. We implemented the proposal in an interactive head robot (IHR and the experimental results indicate the effectiveness.

Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

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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

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.

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

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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

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.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

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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.

Mathematical Modeling of E6-p53 interactions in Cervical Cancer

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Khattak, Faryal; Haseeb, Muhammad; Fazal, Sahar; Bhatti, A I; Ullah, Mukhtar

2017-04-01

Background: Cervical cancer is the third most common cancer in women throughout the world. The human papillomavirus (HPV) E6 viral protein plays an essential role in proteasomal degradation of the cancer suppressant protein p53. As a result, p53 negative regulation and apoptosis relevant activities are abrogated, facilitating development of cervical cancer. Methods: A mathematical model of E6-p53 interactions was developed using mathematical laws. In-silico simulations were carried out on CellDesigner and as a test case the small molecule drug RITA was considered for its ability to rescue the functions of tumor suppressor p53 by inhibiting E6 mediated proteasomal degradation. Results: Using a computational model we scrutinized how p53 responds to RITA, and chemical reactions of this small molecule drug were incorporated to perceive the full effects. The evolved strategy allowed the p53 response and rescue of its tumor suppressor function to be delineated, RITA being found to block p53 interactions with E6 associated proteins. Conclusion: We could develop a model of E6-p53 interactions with incorporation of actions of the small molecule drug RITA. Suppression of E6 associated proteins by RITA induces accumulation of tumor suppressant p53. Using CellDesigner to encode the model ensured that it can be easily modified and extended as more data become available. This strategy should play an effective role in the development of therapies against cancer. Creative Commons Attribution License

Interaction between Task Oriented and Affective Information Processing in Cognitive Robotics

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Haazebroek, Pascal; van Dantzig, Saskia; Hommel, Bernhard

There is an increasing interest in endowing robots with emotions. Robot control however is still often very task oriented. We present a cognitive architecture that allows the combination of and interaction between task representations and affective information processing. Our model is validated by comparing simulation results with empirical data from experimental psychology.

Unsupportive social interactions and affective states: examining associations of two oxytocin-related polymorphisms.

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McInnis, Opal A; McQuaid, Robyn J; Matheson, Kimberly; Anisman, Hymie

2017-01-01

Two single-nucleotide polymorphisms (SNPs) on oxytocin-related genes, specifically the oxytocin receptor (OXTR) rs53576 and the CD38 rs3796863 variants, have been associated with alterations in prosocial behaviors. A cross-sectional study was conducted among undergraduate students (N = 476) to examine associations between the OXTR and CD38 polymorphisms and unsupportive social interactions and mood states. Results revealed no association between perceived levels of unsupportive social interactions and the OXTR polymorphism. However, A carriers of the CD38 polymorphism, a variant previously associated with elevated oxytocin, reported greater perceived peer unsupportive interactions compared to CC carriers. As expected, perceived unsupportive interactions from peers was associated with greater negative affect, which was moderated by the CD38 polymorphism. Specifically, this relation was stronger among CC carriers of the CD38 polymorphism (a variant thought to be linked to lower oxytocin). When examining whether the OXTR polymorphism moderated the relation between unsupportive social interactions from peers and negative affect there was a trend toward significance, however, this did not withstand multiple testing corrections. These findings are consistent with the perspective that a variant on an oxytocin polymorphism that may be tied to lower oxytocin is related to poor mood outcomes in association with negative social interactions. At the same time, having a genetic constitution presumed to be associated with higher oxytocin was related to increased perceptions of unsupportive social interactions. These seemingly paradoxical findings could be related to previous reports in which variants associated with prosocial behaviors were also tied to relatively more effective coping styles to deal with challenges.

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.

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

Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

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McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

2016-03-29

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

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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.

Ecological Momentary Assessment of social functioning in schizophrenia: impact of performance appraisals and affect on social interactions.

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Granholm, Eric; Ben-Zeev, Dror; Fulford, Daniel; Swendsen, Joel

2013-04-01

Research concerning the complex interplay between factors that contribute to poor social functioning in schizophrenia has been hampered by limitations of traditional measures, most notably the ecological validity and accuracy of retrospective self-report and interview measures. Computerized Ecological Momentary Assessment (EMAc) permits the real-time assessment of relationships between daily life experiences, thoughts, feelings, and behaviors. In the current study, EMAc was used to record daily social interactions, subjective performance appraisals of these interactions (e.g., "I succeeded/failed"; "I was liked/rejected"), and affect in 145 individuals with schizophrenia or schizoaffective disorder. Participants completed electronic questionnaires on a personal digital assistant (PDA) four times per day for one week. Time-lagged multilevel modeling of the data revealed that more positive interaction appraisals at any point in a day were associated with greater positive affect which, in turn, was a strong predictor of more social interactions over subsequent hours. Social functioning, therefore, was linked to positive performance beliefs about social interactions that were associated with greater positive affect. The findings suggest a useful treatment target for cognitive behavioral therapy and other psychosocial interventions that can be used to challenge defeatist beliefs and increase positive affect to enhance social functioning in schizophrenia. Published by Elsevier B.V.

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

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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.

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.

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.

Warming, CO2, and nitrogen deposition interactively affect a plant-pollinator mutualism.

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Hoover, Shelley E R; Ladley, Jenny J; Shchepetkina, Anastasia A; Tisch, Maggie; Gieseg, Steven P; Tylianakis, Jason M

2012-03-01

Environmental changes threaten plant-pollinator mutualisms and their critical ecosystem service. Drivers such as land use, invasions and climate change can affect pollinator diversity or species encounter rates. However, nitrogen deposition, climate warming and CO(2) enrichment could interact to disrupt this crucial mutualism by altering plant chemistry in ways that alter floral attractiveness or even nutritional rewards for pollinators. Using a pumpkin model system, we show that these drivers non-additively affect flower morphology, phenology, flower sex ratios and nectar chemistry (sugar and amino acids), thereby altering the attractiveness of nectar to bumble bee pollinators and reducing worker longevity. Alarmingly, bees were attracted to, and consumed more, nectar from a treatment that reduced their survival by 22%. Thus, three of the five major drivers of global environmental change have previously unknown interactive effects on plant-pollinator mutualisms that could not be predicted from studies of individual drivers in isolation. © 2012 Blackwell Publishing Ltd/CNRS.

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.

Situational motivation and perceived intensity: their interaction in predicting changes in positive affect from physical activity.

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Guérin, Eva; Fortier, Michelle S

2012-01-01

There is evidence that affective experiences surrounding physical activity can contribute to the proper self-regulation of an active lifestyle. Motivation toward physical activity, as portrayed by self-determination theory, has been linked to positive affect, as has the intensity of physical activity, especially of a preferred nature. The purpose of this experimental study was to examine the interaction between situational motivation and intensity [i.e., ratings of perceived exertion (RPE)] in predicting changes in positive affect following an acute bout of preferred physical activity, namely, running. Fourty-one female runners engaged in a 30-minute self-paced treadmill run in a laboratory context. Situational motivation for running, pre- and post-running positive affect, and RPE were assessed via validated self-report questionnaires. Hierarchical regression analyses revealed a significant interaction effect between RPE and introjection (P positive affect was considerable, with higher RPE ratings being associated with greater increases in positive affect. The implications of the findings in light of SDT principles as well as the potential contingencies between the regulations and RPE in predicting positive affect among women are discussed.

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

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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.

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

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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.

Aggresome formation is regulated by RanBPM through an interaction with HDAC6

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Louisa M. Salemi

2014-05-01

Full Text Available In conditions of proteasomal impairment, the build-up of damaged or misfolded proteins activates a cellular response leading to the recruitment of damaged proteins into perinuclear aggregates called aggresomes. Aggresome formation involves the retrograde transport of cargo proteins along the microtubule network and is dependent on the histone deacetylase HDAC6. Here we show that ionizing radiation (IR promotes Ran-Binding Protein M (RanBPM relocalization into discrete perinuclear foci where it co-localizes with aggresome components ubiquitin, dynein and HDAC6, suggesting that the RanBPM perinuclear clusters correspond to aggresomes. RanBPM was also recruited to aggresomes following treatment with the proteasome inhibitor MG132 and the DNA-damaging agent etoposide. Strikingly, aggresome formation by HDAC6 was markedly impaired in RanBPM shRNA cells, but was restored by re-expression of RanBPM. RanBPM was found to interact with HDAC6 and to inhibit its deacetylase activity. This interaction was abrogated by a RanBPM deletion of its LisH/CTLH domain, which also prevented aggresome formation, suggesting that RanBPM promotes aggresome formation through an association with HDAC6. Our results suggest that RanBPM regulates HDAC6 activity and is a central regulator of aggresome formation.

Ionizing radiation affects generation of MART-1-specific cytotoxic T cell responses by dendritic cells

International Nuclear Information System (INIS)

Liao, Y.P.; Wang, C.-C.; McBride, W.H.

2003-01-01

Full text: The human MART-1/Melan-A (MART-1) melanoma tumor antigen is known to be recognized by cytotoxic T lymphocytes (CTLs) and several groups are using this target for clinical immunotherapy. Most approaches use dendritic cells (DCs) that are potent antigen presentation cells for initiating CTL responses. In order for CTL recognition to occur, DCs must display 9-residue antigenic peptides on MHC class I molecules. These peptides are generated by proteasome degradation and then transported through the endoplasmic reticulum to the cell surface where they stabilize MHC class I expression. Our previous data showed that irradiation inhibits proteasome function and, therefore, we hypothesized that irradiation may inhibit antigen processing and CTL activation, as has been shown for proteasome inhibitors. To study the importance of irradiation effects on DCs, we studied the generation MART-1-specific CTL responses. Preliminary data showed that irradiation of murine bone marrow derived DCs did not affect expression of MHC class I, II, CD80, or CD86, as assessed by flow cytometric analyses 24-hour after irradiation. The effect of irradiation on MART-1 antigen processing by DCs was evaluated using DC transduced with adenovirus MART-1 (AdVMART1). C57BL/6 mice were immunized with AdVMART1 transduced DCs, with and without prior irradiation. IFN-γ production was measured by ELISPOT assays after 10-14 days of immunization. Prior radiation treatment resulted in a significant decrease in MART-1-specific T cell responses. The ability of irradiated and non-irradiated AdVMART1/DC vaccines to protect mice against growth of murine B16 tumors, which endogenously express murine MART-1, was also examined. AdVMART1/DC vaccination protected C57BL/6 mice against challenge with viable B16 melanoma cells while DCs irradiated (10 Gy) prior to AdVMART1 transduction abrogated protection. These results suggest that proteasome inhibition in DCs by irradiation may be a possible pathway in

Interactive effects of music tempi and intensities on grip strength and subjective affect.

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Karageorghis, C I; Cheek, P; Simpson, S D; Bigliassi, M

2018-03-01

Pretask music is widely used by athletes albeit there is scant empirical evidence to support its use. The present study extended a line of work into pretask music by examining the interactive effects of music tempo and intensity (volume) on the performance of a simple motor skill and subjective affect. A 2 × 2 within-subjects factorial design was employed with an additional no-music control, the scores from which were used as a covariate. A sample of 52 male athletes (M age  = 26.1 ± 4.8 years) was exposed to five conditions: fast/loud (126 bpm/80 dBA), fast/quiet (126 bpm/70 dBA), slow/loud (87 bpm/80 dBA), slow/quiet (87 bpm/70 dBA) music, and a no-music control. Dependent variables were grip strength, measured with a handgrip dynamometer, and subjective affect, assessed by use of the Affect Grid. The tempo and intensity components of music had interactive effects for grip strength but only main effects for subjective affect. Fast-tempo music played at a high intensity yielded the highest grip strength, while fast-tempo music played at a low-intensity resulted in much lower grip strength (M diff.  = -1.11 Force kg). For affective valence, there were main effects of tempo and intensity, with fast and loud music yielding the highest scores. For affective arousal, there was no difference between tempi although there was between intensities, with the high-intensity condition yielding higher scores. The present findings indicate the utility of fast/loud pretask music in enhancing affective valence and arousal in preparation for a simple or gross motor task. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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.

Seasonal Influences on Ground-Surface Water Interactions in an Arsenic-Affected Aquifer in Cambodia

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Richards, L. A.; Magnone, D.; Van Dongen, B.; Bryant, C.; Boyce, A.; Ballentine, C. J.; Polya, D. A.

2015-12-01

Millions of people in South and Southeast Asia consume drinking water daily which contains dangerous levels of arsenic exceeding health-based recommendations [1]. A key control on arsenic mobilization in aquifers in these areas has been controversially identified as the interaction of 'labile' organic matter contained in surface waters with groundwaters and sediments at depth [2-4], which may trigger the release of arsenic from the solid- to aqueous-phase via reductive dissolution of iron-(hyr)oxide minerals [5]. In a field site in Kandal Province, Cambodia, which is an arsenic-affected area typical to others in the region, there are strong seasonal patterns in groundwater flow direction, which are closely related to monsoonal rains [6] and may contribute to arsenic release in this aquifer. The aim of this study is to explore the implications of the high susceptibility of this aquifer system to seasonal changes on potential ground-surface water interactions. The main objectives are to (i) identify key zones where there are likely ground-surface water interactions, (ii) assess the seasonal impact of such interactions and (iii) quantify the influence of interactions using geochemical parameters (such as As, Fe, NO3, NH4, 14C, 3T/3He, δ18O, δ2H). Identifying the zones, magnitude and seasonal influence of ground-surface water interactions elucidates new information regarding potential locations/pathways of arsenic mobilization and/or transport in affected aquifers and may be important for water management strategies in affected areas. This research is supported by NERC (NE/J023833/1) to DP, BvD and CJB and a NERC PhD studentship (NE/L501591/1) to DM. References: [1] World Health Organization, 2008. [2] Charlet & Polya (2006), Elements, 2, 91-96. [3] Harvey et al. (2002), Science, 298, 1602-1606. [4] Lawson et al. (2013), Env. Sci. Technol. 47, 7085 - 7094. [5] Islam et al. (2004), Nature, 430, 68-71. [6] Benner et al. (2008) Appl. Geochem. 23(11), 3072 - 3087.

BRCA1 affects lipid synthesis through its interaction with acetyl-CoA carboxylase.

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Moreau, Karen; Dizin, Eva; Ray, Hind; Luquain, Céline; Lefai, Etienne; Foufelle, Fabienne; Billaud, Marc; Lenoir, Gilbert M; Venezia, Nicole Dalla

2006-02-10

Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase alpha (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1.ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.

JS-K, a nitric oxide pro-drug, regulates growth and apoptosis through the ubiquitin-proteasome pathway in prostate cancer cells.

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Tan, Guobin; Qiu, Mingning; Chen, Lieqian; Zhang, Sai; Ke, Longzhi; Liu, Jianjun

2017-05-26

In view of the fact that JS-K might regulate ubiquitin E3 ligase and that ubiquitin E3 ligase plays an important role in the mechanism of CRPC formation, the goal was to investigate the probable mechanism by which JS-K regulates prostate cancer cells. Proliferation inhibition by JS-K on prostate cancer cells was examined usingCCK-8 assays. Caspase 3/7 activity assays and flow cytometry were performed to examine whether JS-K induced apoptosis in prostate cancer cells. Western blotting and co-immunoprecipitation analyses investigated JS-K's effects on the associated apoptosis mechanism. Real time-PCR and Western blotting were performed to assess JS-K's effect on transcription of specific AR target genes. Western blotting was also performed to detect Siah2 and AR protein concentrations and co-immunoprecipitation to detect interactions of Siah2 and AR, NCoR1 and AR, and p300 and AR. JS-K inhibited proliferation and induced apoptosis in prostate cancer cells. JS-K increased p53 and Mdm2 concentrations and regulated the caspase cascade reaction-associated protein concentrations. JS-K inhibited transcription of AR target genes and down-regulated PSA protein concentrations. JS-K inhibited Siah2 interactions and also inhibited the ubiquitination of AR. With further investigation, JS-K was found to stabilize AR and NCoR1 interactions and diminish AR and p300 interactions. The present results suggested that JS-K might have been able to inhibit proliferation and induce apoptosis via regulation of the ubiquitin-proteasome degradation pathway, which represented a promising platform for the development of new compounds for PCa treatments.

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

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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

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

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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.

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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.

Chronic and Daily Stressors Along With Negative Affect Interact to Predict Daily Tiredness.

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Hartsell, Elizabeth N; Neupert, Shevaun D

2017-11-01

The present study examines the within-person relationship of daily stressors and tiredness and whether this depends on daily negative affect and individual differences in chronic stress. One hundred sixteen older adult participants were recruited via Amazon's Mechanical Turk for a 9-day daily diary study. Daily tiredness, daily stressors, and negative affect were measured each day, and chronic stress was measured at baseline. Daily stressors, daily negative affect, and chronic stress interacted to predict daily tiredness. People with high chronic stress who experienced an increase in daily negative affect were the most reactive to daily stressors in terms of experiencing an increase in daily tiredness. We also found that people with low levels of chronic stress were the most reactive to daily stressors when they experienced low levels of daily negative affect. Our results highlight the need for individualized and contextualized approaches to combating daily tiredness in older adults.

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.

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Loewen, Carin A; Ganetzky, Barry

2018-04-01

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23 , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23 A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23 , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease. Copyright © 2018 by the Genetics Society of America.

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.

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

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

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.

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

Sorption interactions of organic compounds with soils affected by agricultural olive mill wastewater.

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Keren, Yonatan; Borisover, Mikhail; Bukhanovsky, Nadezhda

2015-11-01

The organic compound-soil interactions may be strongly influenced by changes in soil organic matter (OM) which affects the environmental fate of multiple organic pollutants. The soil OM changes may be caused by land disposal of various OM-containing wastes. One unique type of OM-rich waste is olive mill-related wastewater (OMW) characterized by high levels of OM, the presence of fatty aliphatics and polyphenolic aromatics. The systematic data on effects of the land-applied OMW on organic compound-soil interactions is lacking. Therefore, aqueous sorption of simazine and diuron, two herbicides, was examined in batch experiments onto three soils, including untreated and OMW-affected samples. Typically, the organic compound-soil interactions increased following the prior land application of OMW. This increase is associated with the changes in sorption mechanisms and cannot be attributed solely to the increase in soil organic carbon content. A novel observation is that the OMW application changes the soil-sorbent matrix in such a way that the solute uptake may become cooperative or the existing ability of a soil sorbent to cooperatively sorb organic molecules from water may become characterized by a larger affinity. The remarkable finding of this study was that in some cases a cooperative uptake of organic molecules by soils makes itself evident in distinct sigmoidal sorption isotherms rarely observed in soil sorption of non-ionized organic compounds; the cooperative herbicide-soil interactions may be characterized by the Hill model coefficients. However, no single trend was found for the effect of applied OMW on the mechanisms of organic compound-soil interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Situational Motivation and Perceived Intensity: Their Interaction in Predicting Changes in Positive Affect from Physical Activity

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Eva Guérin

2012-01-01

Full Text Available There is evidence that affective experiences surrounding physical activity can contribute to the proper self-regulation of an active lifestyle. Motivation toward physical activity, as portrayed by self-determination theory, has been linked to positive affect, as has the intensity of physical activity, especially of a preferred nature. The purpose of this experimental study was to examine the interaction between situational motivation and intensity [i.e., ratings of perceived exertion (RPE] in predicting changes in positive affect following an acute bout of preferred physical activity, namely, running. Fourty-one female runners engaged in a 30-minute self-paced treadmill run in a laboratory context. Situational motivation for running, pre- and post-running positive affect, and RPE were assessed via validated self-report questionnaires. Hierarchical regression analyses revealed a significant interaction effect between RPE and introjection (P<.05 but not between RPE and identified regulation or intrinsic motivation. At low levels of introjection, the influence of RPE on the change in positive affect was considerable, with higher RPE ratings being associated with greater increases in positive affect. The implications of the findings in light of SDT principles as well as the potential contingencies between the regulations and RPE in predicting positive affect among women are discussed.

The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System

OpenAIRE

Fuchs, Adrian C.D.; Alva, Vikram; Maldoner, Lorena; Albrecht, Reinhard; Hartmann, Marcus D.; Martin, Jörg

2017-01-01

Summary 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 dodec...

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

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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.

Do core interpersonal and affective traits of PCL-R psychopathy interact with antisocial behavior and disinhibition to predict violence?

Science.gov (United States)

Kennealy, Patrick J; Skeem, Jennifer L; Walters, Glenn D; Camp, Jacqueline

2010-09-01

The utility of psychopathy measures in predicting violence is largely explained by their assessment of social deviance (e.g., antisocial behavior; disinhibition). A key question is whether social deviance interacts with the core interpersonal-affective traits of psychopathy to predict violence. Do core psychopathic traits multiply the (already high) risk of violence among disinhibited individuals with a dense history of misbehavior? This meta-analysis of 32 effect sizes (N = 10,555) tested whether an interaction between the Psychopathy Checklist-Revised (PCL-R; R. D. Hare, 2003) Interpersonal-Affective and Social Deviance scales predicted violence beyond the simple additive effects of each scale. Results indicate that Social Deviance is more uniquely predictive of violence (d = .40) than Interpersonal-Affective traits (d = .11), and these two scales do not interact (d = .00) to increase power in predicting violence. In fact, Social Deviance alone would predict better than the Interpersonal-Affective scale and any interaction in 81% and 96% of studies, respectively. These findings have fundamental practical implications for risk assessment and theoretical implications for some conceptualizations of psychopathy.

Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver.

Science.gov (United States)

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.

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

Foreword 3rd International Conference on Affective Computing and Intelligent Interaction - ACII 2009

NARCIS (Netherlands)

Cohn, Jeffrey; Cohn, Jeffrey; Nijholt, Antinus; Pantic, Maja

2009-01-01

It is a pleasure and an honor to have organized the Third International Conference on Affective Computing and Intelligent Interaction (ACII). The conference will be held from 10th – 12th September 2009 in Amsterdam, The Netherlands. The conference series is the premier forum for presenting research

Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella

Science.gov (United States)

Domin, Hanna; Zurita-Gutiérrez, Yazmín H.; Scotti, Marco; Buttlar, Jann; Hentschel Humeida, Ute; Fraune, Sebastian

2018-01-01

The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community. PMID:29740401

Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella

Directory of Open Access Journals (Sweden)

Hanna Domin

2018-04-01

Full Text Available The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.

The Absence of Pupylation (Prokaryotic Ubiquitin-Like Protein Modification) Affects Morphological and Physiological Differentiation in Streptomyces coelicolor

Czech Academy of Sciences Publication Activity Database

Boubakri, H.; Seghezzi, N.; Duchateau, M.; Gominet, M.; Kofroňová, Olga; Benada, Oldřich; Mazodier, P.; Pernodet, J.-L.

2015-01-01

Roč. 197, č. 21 (2015), s. 3388-3399 ISSN 0021-9193 Institutional support: RVO:61388971 Keywords : PROTEASOMAL ATPASE HOMOLOG * MYCOBACTERIUM-TUBERCULOSIS * 20S PROTEASOME Subject RIV: EE - Microbiology, Virology Impact factor: 3.198, year: 2015

The Role of Cognitive and Affective Empathy in Spouses' Support Interactions: An Observational Study

Science.gov (United States)

Verhofstadt, Lesley; Devoldre, Inge; Buysse, Ann; Stevens, Michael; Hinnekens, Céline; Ickes, William; Davis, Mark

2016-01-01

The present study examined how support providers’ empathic dispositions (dispositional perspective taking, empathic concern, and personal distress) as well as their situational empathic reactions (interaction-based perspective taking, empathic concern, and personal distress) relate to the provision of spousal support during observed support interactions. Forty-five committed couples provided questionnaire data and participated in two ten-minute social support interactions designed to assess behaviors when partners are offering and soliciting social support. A video-review task was used to assess situational forms of perspective taking (e.g., empathic accuracy), empathic concern and personal distress. Data were analyzed by means of the multi-level Actor-Partner Interdependence Model. Results revealed that providers scoring higher on affective empathy (i.e., dispositional empathic concern), provided lower levels of negative support. In addition, for male partners, scoring higher on cognitive empathy (i.e., situational perspective taking) was related to lower levels of negative support provision. For both partners, higher scores on cognitive empathy (i.e., situational perspective taking) correlated with more instrumental support provision. Male providers scoring higher on affective empathy (i.e., situational personal distress) provided higher levels of instrumental support. Dispositional perspective taking was related to higher scores on emotional support provision for male providers. The current study furthers our insight into the empathy-support link, by revealing differential effects (a) for men and women, (b) of both cognitive and affective empathy, and (c) of dispositional as well as situational empathy, on different types of support provision. PMID:26910769

Bottom-up and top-down human impacts interact to affect a protected coastal Chilean marsh.

Science.gov (United States)

Fariña, José M; He, Qiang; Silliman, Brian R; Bertness, Mark D

2016-03-01

Many ecosystems, even in protected areas, experience multiple anthropogenic impacts. While anthropogenic modification of bottom-up (e.g., eutrophication) and top-down (e.g., livestock grazing) forcing often co-occurs, whether these factors counteract or have additive or synergistic effects on ecosystems is poorly understood. In a Chilean bio-reserve, we examined the interactive impacts of eutrophication and illegal livestock grazing on plant growth with a 4-yr fertilization by cattle exclusion experiment. Cattle grazing generally decreased plant biomass, but had synergistic, additive, and antagonistic interactions with fertilization in the low, middle, and high marsh zones, respectively. In the low marsh, fertilization increased plant biomass by 112%, cattle grazing decreased it by 96%, and together they decreased plant biomass by 77%. In the middle marsh, fertilization increased plant biomass by 47%, cattle grazing decreased it by 37%, and together they did not affect plant biomass. In the high marsh, fertilization and cattle grazing decreased plant biomass by 81% and 92%, respectively, but together they increased plant biomass by 42%. These interactions were also found to be species specific. Different responses of plants to fertilization and cattle grazing were likely responsible for these variable interactions. Thus, common bottom-up and top-down human impacts can interact in different ways to affect communities even within a single ecosystem. Incorporating this knowledge into conservation actions will improve ecosystem management in a time when ecosystems are increasingly challenged by multiple interacting human impacts.

Interactive effects of trait and state affect on top-down control of attention.

Science.gov (United States)

Hur, Juyoen; Miller, Gregory A; McDavitt, Jenika R B; Spielberg, Jeffrey M; Crocker, Laura D; Infantolino, Zachary P; Towers, David N; Warren, Stacie L; Heller, Wendy

2015-08-01

Few studies have investigated how attentional control is affected by transient affective states while taking individual differences in affective traits into consideration. In this study, participants completed a color-word Stroop task immediately after undergoing a positive, neutral or negative affective context manipulation (ACM). Behavioral performance was unaffected by any ACM considered in isolation. For individuals high in trait negative affect (NA), performance was impaired by the negative but not the positive or neutral ACM. Neuroimaging results indicate that activity in primarily top-down control regions of the brain (inferior frontal gyrus and dorsal anterior cingulate cortex) was suppressed in the presence of emotional arousal (both negative and positive ACMs). This effect appears to have been exacerbated or offset by co-occurring activity in other top-down control regions (parietal) and emotion processing regions (orbitofrontal cortex, amygdala and nucleus accumbens) as a function of the valence of state affect (positive or negative) and trait affect (trait NA or trait PA). Neuroimaging results are consistent with behavioral findings. In combination, they indicate both additive and interactive influences of trait and state affect on top-down control of attention. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Motion and emotion: depression reduces psychomotor performance and alters affective movements in caregiving interactions

Directory of Open Access Journals (Sweden)

Katherine S Young

2015-02-01

Full Text Available Background: Impaired social functioning is a well-established feature of depression. Evidence to date suggests that disrupted processing of emotional cues may constitute part of this impairment. Beyond processing of emotional cues, fluent social interactions require that people physically move in synchronised, contingent ways. Disruptions to physical movements are a diagnostic feature of depression (psychomotor disturbance but have not previously been assessed in the context of social functioning. Here we investigated the impact of psychomotor disturbance in depression on physical responsive behaviour in both an experimental and observational setting.Methods: In Experiment 1, we examined motor disturbance in depression in response to salient emotional sounds, using a laboratory-based effortful motor task. In Experiment 2, we explored whether psychomotor disturbance was apparent in real-life social interactions. Using mother-infant interactions as a model affective social situation, we compared physical behaviours of mothers with and without postnatal depression (PND.Results: We found impairments in precise, controlled psychomotor performance in adults with depression relative to healthy adults (Experiment 1. Despite this disruption, all adults showed enhanced performance following exposure to highly salient emotional cues (infant cries. Examining real-life interactions, we found differences in physical movements, namely reduced affective touching, in mothers with PND responding to their infants, compared to healthy mothers (Experiment 2.Conclusions: Together, these findings suggest that psychomotor disturbance may be an important feature of depression that can impair social functioning. Future work investigating whether improvements in physical movement in depression could have a positive impact on social interactions would be of much interest.

SUMO-targeted ubiquitin ligases.

Science.gov (United States)

Sriramachandran, Annie M; Dohmen, R Jürgen

2014-01-01

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. © 2013. Published by Elsevier B.V. All rights reserved.

Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

Science.gov (United States)

Ding, Yezhang; Dommel, Matthew; Mou, Zhonglin

2016-04-01

Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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*

Science.gov (United States)

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

Urban Interaction and Affective Experience

DEFF Research Database (Denmark)

Fritsch, Jonas; Brynskov, Martin

2008-01-01

As interactive digital technologies become a still more integrated and complex part of the everyday physical, social and cultural spaces we inhabit, research into these spaces’ dynamics and struc-tures needs to formulate adequate methods of analysis and dis-course. In this position paper we argue...... approach holds potential to address important aspects of the design of such blended digital spaces, extending beyond traditional interaction design. And finally we argue for the importance of construction, i.e. actual interventions of consider-able scale....

Gamma irradiation on canola seeds affects herbivore-plant and host-parasitoid interactions

International Nuclear Information System (INIS)

Akandeh, M.; Kocheili, F.; Rasekh, A.; Soufbaf, M.

2017-01-01

As an agricultural modernization, gamma irradiation is an important method for enhancing crop yield and quality. Nevertheless, its use can alter other plant traits such as nutrition and resistance to different biotic/abiotic stresses that consequently affect plant-insect interactions. A tritrophic system was utilized based on two canola mutant lines produced through gamma irradiation (RGS 8-1 and Talaye 8-3). Plutella xylostella (L.), as a worldwide pest of Brassicaceae and Cotesia vestalis (Holiday) as a key biocontrol agent of P. xylostella were examined for the potential indirect effects of canola seed irradiation on the experimental insects' performance when acting on the respective mutant lines. This study showed that physical mutation did not affect plant nitrogen and herbivore-damaged total phenolics; however, phenolic compounds showed greater concentration in damaged leaves than undamaged leaves of both mutant and control plants. The relative growth rate and pupal weight of P. xylostella reared on RGS 8-1 were significantly higher than those reared on the control RGS. There was no significant difference by performance parameters of the parasitoid, C. vestalis, including total pre-oviposition period, adult longevity, adult fresh body weight of males and females, pupal weight, forewing area, and total longevity of both sexes on tested canola cultivars in comparison with their mutant lines. Life table parameters of C. vestalis on mutant lines of both cultivars, RGS and Talaye, were not significantly different from their control treatments. Comprehensive studies should be conducted to find out the mechanisms under which gamma rays affect plant-insect interactions. (author)

Gamma irradiation on canola seeds affects herbivore-plant and host-parasitoid interactions

Energy Technology Data Exchange (ETDEWEB)

Akandeh, M.; Kocheili, F.; Rasekh, A. [Dept. of Entomology, Shahid Chamran Univ of Ahvaz (Iran, Islamic Republic of); Soufbaf, M., E-mail: msoufbaf@nrcam.org [Agricultural, Medical and Industrial Research School, Karaj (Iran, Islamic Republic of)

2017-06-15

As an agricultural modernization, gamma irradiation is an important method for enhancing crop yield and quality. Nevertheless, its use can alter other plant traits such as nutrition and resistance to different biotic/abiotic stresses that consequently affect plant-insect interactions. A tritrophic system was utilized based on two canola mutant lines produced through gamma irradiation (RGS 8-1 and Talaye 8-3). Plutella xylostella (L.), as a worldwide pest of Brassicaceae and Cotesia vestalis (Holiday) as a key biocontrol agent of P. xylostella were examined for the potential indirect effects of canola seed irradiation on the experimental insects' performance when acting on the respective mutant lines. This study showed that physical mutation did not affect plant nitrogen and herbivore-damaged total phenolics; however, phenolic compounds showed greater concentration in damaged leaves than undamaged leaves of both mutant and control plants. The relative growth rate and pupal weight of P. xylostella reared on RGS 8-1 were significantly higher than those reared on the control RGS. There was no significant difference by performance parameters of the parasitoid, C. vestalis, including total pre-oviposition period, adult longevity, adult fresh body weight of males and females, pupal weight, forewing area, and total longevity of both sexes on tested canola cultivars in comparison with their mutant lines. Life table parameters of C. vestalis on mutant lines of both cultivars, RGS and Talaye, were not significantly different from their control treatments. Comprehensive studies should be conducted to find out the mechanisms under which gamma rays affect plant-insect interactions. (author)

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

DEFF Research Database (Denmark)

Andersen, Katrine M; Klausen, Louise Kjær; Prag, Søren

2009-01-01

in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26S proteasome. eEF1A1 is therefore a likely physiological substrate....... In response to knock-down of Txnl1, ubiquitin-protein conjugates were moderately stabilised. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms....

Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease

DEFF Research Database (Denmark)

Aziz, N A; Jurgens, C K; Landwehrmeyer, G B

2009-01-01

OBJECTIVE: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). We aimed to assess whether interaction between CAG repeat sizes in the mutant and normal allele could affect disease severity and progression. METHODS: Using...... with less severe symptoms and pathology. CONCLUSIONS: Increasing CAG repeat size in normal HTT diminishes the association between mutant CAG repeat size and disease severity and progression in Huntington disease. The underlying mechanism may involve interaction of the polyglutamine domains of normal...

Network Regulation and Support Schemes - How Policy Interactions Affect the Integration of Distributed Generation

DEFF Research Database (Denmark)

Ropenus, Stephanie; Jacobsen, Henrik; Schröder, Sascha Thorsten

2011-01-01

This article seeks to investigate the interactions between the policy dimensions of support schemes and network regulation and how they affect distributed generation. Firstly, the incentives of distributed generators and distribution system operators are examined. Frequently there exists a trade......-off between the incentives for these two market agents to facilitate the integration of distributed generation. Secondly, the interaction of these policy dimensions is analyzed, including case studies based on five EU Member States. Aspects of operational nature and investments in grid and distributed...

Maternal interaction style in affective disordered, physically ill, and normal women.

Science.gov (United States)

Hamilton, E B; Jones, M; Hammen, C

1993-09-01

Affective style (AS) and communication deviance (CD) have been suggested as markers of dysfunctional family environments that may be associated with psychiatric illness. Studies have focused mainly on parental responses during family interactions when an offspring is the identified patient. The present study is unique in examining AS and CD in mothers with unipolar depression, bipolar disorder, or chronic physical illness, and in normal controls. The sample consisted of 64 mothers with children ages 8 to 16. Unipolar mothers were more likely to show negative AS than were any other maternal group. There were no group differences for CD. Chronic stress, few positive life events, and single parenting were associated with AS. CD was associated solely with lower socioeconomic status. Results suggest that dysfunctional interactions are determined not only by maternal psychopathology, but also by an array of contextual factors that are related to the quality of the family environment.

[Chlamydia trachomatis proteasome protein as one of the significant pathogenicity factors of exciter].

Science.gov (United States)

Davydov, D Iu; Zigangirova, N A

2014-01-01

Sex-related infections are a global problem. Such infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. Protein CPAF is leading factor of pathogenesis. This protein inhibits the signaling pathways of host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review general properties of the proteasome Chlamydia protein CPAF, its functions, and role in pathology. The role of protein CPAF in the anti-chlamydia immune reaction is discussed. The prospects of the development of promising anti-chlamydia vaccine, as well as new effective anti-chlamydia drugs are also discussed.

User Experience of Mobile Interactivity: How Do Mobile Websites Affect Attitudes and Relational Outcomes?

Science.gov (United States)

Dou, Xue

2013-01-01

Mobile media offer new opportunities for fostering communications between individuals and companies. Corporate websites are being increasingly accessed via smart phones and companies are scrambling to offer a mobile-friendly user experience on their sites. However, very little is known about how interactivity in the mobile context affects user…

How does real affect affect affect recognition in speech?

NARCIS (Netherlands)

Truong, Khiet Phuong

2009-01-01

The automatic analysis of affect is a relatively new and challenging multidisciplinary research area that has gained a lot of interest over the past few years. The research and development of affect recognition systems has opened many opportunities for improving the interaction between man and

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo

Science.gov (United States)

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

Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

Directory of Open Access Journals (Sweden)

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.

Novel green synthesis of gold nanoparticles using Citrullus lanatus rind and investigation of proteasome inhibitory activity, antibacterial, and antioxidant potential

Directory of Open Access Journals (Sweden)

Patra JK

2015-12-01

Full Text Available Jayanta Kumar Patra, Kwang-Hyun Baek School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, Republic of Korea Abstract: Biological synthesis of nanoparticles using nontoxic, eco-friendly approaches is gaining importance owing to their fascinating biocompatibility and environmentally benign nature. This study describes the green synthesis approach for synthesis of gold nanoparticles (ANPs using aqueous extract of the rind of watermelon as a fruit waste and evaluate its biopotential in terms of proteasome inhibitory activity, antibacterial, and antioxidant potential. The synthesized ANPs were characterized using UV–vis spectroscopy, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra of ANPs were obtained at 560 nm. Scanning electron microscopy image revealed that particles had a spherical shape and have a size distribution of 20–140 nm, followed by the elemental analysis by energy-dispersive X-ray spectroscopy. X-ray diffraction analysis confirmed the crystallite nature of the ANPs and Fourier-transform infrared spectroscopy revealed the involvement of bioactive compounds from watermelon rind in the synthesis, capping, and stabilization of ANPs. ANPs exhibited potential antibacterial activity against five different foodborne pathogenic bacteria with diameter of inhibition zones ranged between 9.23 and 11.58 mm. They also displayed strong synergistic antibacterial activity together with kanamycin (11.93–21.08 mm inhibition zones and rifampicin (10.32–24.84 mm inhibition zones. ANPs displayed strong antioxidant activity in terms of DPPH radical scavenging (24.69%, nitric oxide scavenging (25.62%, ABTS scavenging (29.42%, and reducing power. Significantly high proteasome inhibitory potential of the ANPs (28.16% could be highly useful for cancer treatment and targeted cancer drug delivery. Overall, results highlight a

An Affect-Responsive Interactive Photo Frame

NARCIS (Netherlands)

Dibeklioğlu, H.; Kosunen, I.; Ortega Hortas, M.; Salah, A.A.; Zuzánek, P.; Salah, A.A.; Gevers, T.

2010-01-01

We develop an interactive photo-frame system in which a series of videos of a single person are automatically segmented and a response logic is derived to interact with the user in real-time. The system is composed of five modules. The first module analyzes the uploaded videos and prepares segments

Prefrontocortical dopamine loss in rats delays long-term extinction of contextual conditioned fear, and reduces social interaction without affecting short-term social interaction memory.

Science.gov (United States)

Fernandez Espejo, Emilio

2003-03-01

Prefrontal dopamine loss delays extinction of cued fear conditioning responses, but its role in contextual fear conditioning has not been explored. Medial prefrontal lesions also enhance social interaction in rats, but the role of prefrontal dopamine loss on social interaction memory is not known. Besides, a role for subcortical accumbal dopamine on mnesic changes after prefrontal dopamine manipulation has been proposed but not explored. The objective was to study the involvement of dopaminergic neurotransmission in the medial prefrontal cortex (mPFC) and nucleus accumbens in two mnesic tasks: contextual fear conditioning and social interaction memory. For contextual fear conditioning, short- and long-term freezing responses after an electric shock were studied, as well as extinction retention. Regarding social interaction memory, the recognition of a juvenile, a very sensitive short-term memory test, was used. Dopamine loss was carried out by injection of 6-hydroxydopamine, and postmortem catecholamine levels were analyzed by high-performance liquid chromatography. Prefrontocortical dopamine loss (>76%) led to a reactive enhancement of accumbal dopamine content (ploss. In lesioned rats, long-term extinction of contextual fear conditioning was significantly delayed and extinction retention was impaired without changes in acquisition and short-term contextual fear conditioning and, on the other hand, acquisition and short-term social interaction memory were not affected, although time spent on social interaction was significantly reduced. Added dopamine loss in the nucleus accumbens (>76%) did not alter these behavioral changes. In summary, the results of the present study indicate that the dopaminergic network in the mPFC (but not in the nucleus accumbens) coordinates the normal long-term extinction of contextual fear conditioning responses without affecting their acquisition, and it is involved in time spent on social interaction, but not acquisition and short

Protein: FBA2 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FBA2 19S regulatory particles(RP) Rpn11 yip5 26S proteasome non-ATPase regulatory subunit 14 26S pr...oteasome regulatory complex subunit p37B, 26S proteasome regulatory subunit rpn11, Yippee-interacting protein 5 7227 Drosophila melanogaster Q9V3H2 Q9V3H2 19075009 ...

Regulation of repp86 stability by human Siah2

International Nuclear Information System (INIS)

Szczepanowski, Monika; Adam-Klages, Sabine; Kruse, Marie-Luise; Pollmann, Marc; Klapper, Wolfram; Parwaresch, Reza; Heidebrecht, Hans-Juergen

2007-01-01

Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132-a specific inhibitor of the proteasome-blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation

Situational Motivation and Perceived Intensity: Their Interaction in Predicting Changes in Positive Affect from Physical Activity

OpenAIRE

Eva Guérin; Michelle S. Fortier

2012-01-01

There is evidence that affective experiences surrounding physical activity can contribute to the proper self-regulation of an active lifestyle. Motivation toward physical activity, as portrayed by self-determination theory, has been linked to positive affect, as has the intensity of physical activity, especially of a preferred nature. The purpose of this experimental study was to examine the interaction between situational motivation and intensity [i.e., ratings of perceived exertion (RPE)] i...

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

Science.gov (United States)

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-10-21

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

Science.gov (United States)

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-01-01

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

Energy Technology Data Exchange (ETDEWEB)

Wu, Di, E-mail: DiWu@mail.nankai.edu.cn; Wu, Shian

2013-04-19

Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

The role of negative maternal affective states and infant temperament in early interactions between infants with cleft lip and their mothers.

Science.gov (United States)

Montirosso, Rosario; Fedeli, Claudia; Murray, Lynne; Morandi, Francesco; Brusati, Roberto; Perego, Guenda Ghezzi; Borgatti, Renato

2012-03-01

The study examined the early interaction between mothers and their infants with cleft lip, assessing the role of maternal affective state and expressiveness and differences in infant temperament. Mother-infant interactions were assessed in 25 2-month-old infants with cleft lip and 25 age-matched healthy infants. Self-report and behavioral observations were used to assess maternal depressive symptoms and expressions. Mothers rated infant temperament. Infants with cleft lip were less engaged and their mothers showed more difficulty in interaction than control group dyads. Mothers of infants with cleft lip displayed more negative affectivity, but did not report more self-rated depressive symptoms than control group mothers. No group differences were found in infant temperament. In order to support the mother's experience and facilitate her ongoing parental role, findings highlight the importance of identifying maternal negative affectivity during early interactions, even when they seem have little awareness of their depressive symptoms.

Age Differences in Affective and Cardiovascular Responses to a Negative Social Interaction: The Role of Goals, Appraisals, and Emotion Regulation

Science.gov (United States)

Luong, Gloria; Charles, Susan T.

2014-01-01

Older adults often report less affective reactivity to interpersonal tensions than younger individuals, but few studies have directly investigated mechanisms explaining this effect. The current study examined whether older adults’ differential endorsement of goals, appraisals, and emotion regulation strategies (i.e., conflict avoidance/de-escalation, self-distraction) during a controlled negative social interaction may explain age differences in affective and cardiovascular responses to the conflict discussion. Participants (N=159; 80 younger adults, 79 older adults) discussed hypothetical dilemmas with disagreeable confederates. Throughout the laboratory session, participants’ subjective emotional experience, blood pressure, and pulse rate were assessed. Older adults generally exhibited less reactivity (negative affect reactivity, diastolic blood pressure reactivity, and pulse rate reactivity) to the task, and more pronounced positive and negative affect recovery following the task, than did younger adults. Older adults appraised the task as more enjoyable and the confederate as more likeable, and more strongly endorsed goals to perform well on the task, which mediated age differences in negative affect reactivity, pulse rate reactivity, and positive affect recovery (i.e., increases in post-task positive affect), respectively. In addition, younger adults showed increased negative affect reactivity with greater use of self-distraction, whereas older adults did not. Together, findings suggest that older adults respond less negatively to unpleasant social interactions than younger adults, and these responses are explained in part by older adults’ pursuit of different motivational goals, less threatening appraisals of the social interaction, and more effective use of self-distraction, compared to younger adults. PMID:24773101

A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95

International Nuclear Information System (INIS)

Sugiura, Takeyuki; Yamaguchi, Aya; Miyamoto, Kentaro

2008-01-01

RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95