WorldWideScience

Sample records for targeted protein degradation

  1. Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

    Science.gov (United States)

    Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

    2016-02-19

    Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

    Science.gov (United States)

    Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

  3. New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.

    Science.gov (United States)

    Wang, Xin; Feng, Shaozhen; Fan, Jinjin; Li, Xiaoyan; Wen, Qiong; Luo, Ning

    2016-09-15

    Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Toru, E-mail: toru@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kim, Minsoo [Division of Bacterial Infection, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Watanabe, Masato [Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Oyama, Masaaki [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Tsumoto, Kouhei [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Medical Genome Science, School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562 (Japan); Yamamoto, Tadashi, E-mail: tyamamot@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Cell Signal Unit, Okinawa Institute of Science and Technology, 1919-1 Onna-son, Kunigami, Okinawa 904-0412 (Japan)

    2011-05-27

    Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  5. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    International Nuclear Information System (INIS)

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-01-01

    Highlights: → Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. → Monoubiquitination sites of Tob are identified by mass spectrometry. → The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  6. Molecular chaperones in targeting misfolded proteins for ubiquitin-dependent degradation

    DEFF Research Database (Denmark)

    Kriegenburg, Franziska; Ellgaard, Lars; Hartmann-Petersen, Rasmus

    2012-01-01

    The accumulation of misfolded proteins presents a considerable threat to the health of individual cells and has been linked to severe diseases, including neurodegenerative disorders. Considering that, in nature, cells often are exposed to stress conditions that may lead to aberrant protein...... conformational changes, it becomes clear that they must have an efficient quality control apparatus to refold or destroy misfolded proteins. In general, cells rely on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation...... via the ubiquitin-proteasome system. The specificity of this proteolysis is generally provided by E3 ubiquitin-protein ligases, hundreds of which are encoded in the human genome. However, rather than binding the misfolded proteins directly, most E3s depend on molecular chaperones to recognize...

  7. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    Science.gov (United States)

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Chemical biology based on target-selective degradation of proteins and carbohydrates using light-activatable organic molecules.

    Science.gov (United States)

    Toshima, Kazunobu

    2013-05-01

    Proteins and carbohydrates play crucial roles in a wide range of biological processes, including serious diseases. The development of novel and innovative methods for selective control of specific proteins and carbohydrates functions has attracted much attention in the field of chemical biology. In this account article, the development of novel chemical tools, which can degrade target proteins and carbohydrates by irradiation with a specific wavelength of light under mild conditions without any additives, is introduced. This novel class of photochemical agents promise bright prospects for finding not only molecular-targeted bioprobes for understanding of the structure-activity relationships of proteins and carbohydrates but also novel therapeutic drugs targeting proteins and carbohydrates.

  9. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies.

    Science.gov (United States)

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-03-13

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

  10. A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

    Science.gov (United States)

    Troupin, Andrea; Londono-Renteria, Berlin; Conway, Michael J; Cloherty, Erin; Jameson, Samuel; Higgs, Stephen; Vanlandingham, Dana L; Fikrig, Erol; Colpitts, Tonya M

    2016-09-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Degradation of Akt using protein-catalyzed capture agents.

    Science.gov (United States)

    Henning, Ryan K; Varghese, Joseph O; Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M; Heath, James R

    2016-04-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  12. Herp enhances ER-associated protein degradation by recruiting ubiquilins

    International Nuclear Information System (INIS)

    Kim, Tae-Yeon; Kim, Eunmin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2008-01-01

    ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates

  13. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    Science.gov (United States)

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  14. The different roles of selective autophagic protein degradation in mammalian cells.

    Science.gov (United States)

    Wang, Da-wei; Peng, Zhen-ju; Ren, Guang-fang; Wang, Guang-xin

    2015-11-10

    Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases.

  15. Accelerated degradation of the D2 protein of photosystem II under ultraviolet radiation

    International Nuclear Information System (INIS)

    Jansen, M.A.K.; Edelman, M.; Greenberg, B.M.; Gaba, V.

    1996-01-01

    The D2 protein of photosystem II is relatively stable in vivo under photosynthetic active radiation, but its degradation accelerates under UVB radiation. Little is known about accelerated D2 protein degradation. We characterized wavelength dependence and sensitivity toward photosystem II inhibitors. The in vivo D2 degradation spectrum resembles the pattern for the rapidly turning over D1 protein of photosystem II, with rates being maximal in the UVB region. We propose that D2 degradation, like D1 degradation, is activated by distinct photosensitizers in the UVB and visible regions of the spectrum. In both wavelength regions, photosystem II inhibitors that are known to be targeted to the D1 protein affect D2 degradation. This suggests that degradation of the two proteins is coupled, D2 degradation being influenced by events occurring at the Q B niche on the D1 protein. (Author)

  16. 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.

  17. 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.

  18. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    Directory of Open Access Journals (Sweden)

    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.

  19. The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation.

    Science.gov (United States)

    Zhao, Dong; Zheng, Han-Qiu; Zhou, Zhongmei; Chen, Ceshi

    2010-06-01

    Fbw7 is a tumor suppressor frequently inactivated in cancers. The KLF5 transcription factor promotes breast cell proliferation and tumorigenesis through upregulating FGF-BP. The KLF5 protein degrades rapidly through the ubiquitin proteasome pathway. Here, we show that the Skp1-CUL1-Fbw7 E3 ubiquitin ligase complex (SCF(Fbw7)) targets KLF5 for ubiquitin-mediated degradation in a GSK3beta-mediated KLF5 phosphorylation-dependent manner. Mutation of the critical S303 residue in the KLF5 Cdc4 phospho-degrons motif ((303)SPPSS) abolishes the protein interaction, ubiquitination, and degradation by Fbw7. Inactivation of endogenous Fbw7 remarkably increases the endogenous KLF5 protein abundances. Endogenous Fbw7 suppresses the FGF-BP gene expression and breast cell proliferation through targeting KLF5 for degradation. These findings suggest that Fbw7 inhibits breast cell proliferation at least partially through targeting KLF5 for proteolysis. This new regulatory mechanism of KLF5 degradation may result in useful diagnostic and therapeutic targets for breast cancer and other cancers. Copyright 2010 AACR.

  20. ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

    Science.gov (United States)

    Isono, Erika; Kalinowska, Kamila

    2017-12-01

    To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  2. Protein degradation pathways in Parkinson's disease: curse or blessing.

    Science.gov (United States)

    Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J

    2012-08-01

    Protein misfolding, aggregation and deposition are common disease mechanisms in many neurodegenerative diseases including Parkinson's disease (PD). Accumulation of damaged or abnormally modified proteins may lead to perturbed cellular function and eventually to cell death. Thus, neurons rely on elaborated pathways of protein quality control and removal to maintain intracellular protein homeostasis. Molecular chaperones, the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are critical pathways that mediate the refolding or removal of abnormal proteins. The successive failure of these protein degradation pathways, as a cause or consequence of early pathological alterations in vulnerable neurons at risk, may present a key step in the pathological cascade that leads to spreading neurodegeneration. A growing number of studies in disease models and patients have implicated dysfunction of the UPS and ALP in the pathogenesis of Parkinson's disease and related disorders. Deciphering the exact mechanism by which the different proteolytic systems contribute to the elimination of pathogenic proteins, like α-synuclein, is therefore of paramount importance. We herein review the role of protein degradation pathways in Parkinson's disease and elaborate on the different contributions of the UPS and the ALP to the clearance of altered proteins. We examine the interplay between different degradation pathways and provide a model for the role of the UPS and ALP in the evolution and progression of α-synuclein pathology. With regards to exciting recent studies we also discuss the putative potential of using protein degradation pathways as novel therapeutic targets in Parkinson's disease.

  3. Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    Full Text Available Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

  4. Targeted Delivery of Protein Drugs by Nanocarriers

    Directory of Open Access Journals (Sweden)

    Antonella Battisti

    2010-03-01

    Full Text Available Recent advances in biotechnology demonstrate that peptides and proteins are the basis of a new generation of drugs. However, the transportation of protein drugs in the body is limited by their high molecular weight, which prevents the crossing of tissue barriers, and by their short lifetime due to immuno response and enzymatic degradation. Moreover, the ability to selectively deliver drugs to target organs, tissues or cells is a major challenge in the treatment of several human diseases, including cancer. Indeed, targeted delivery can be much more efficient than systemic application, while improving bioavailability and limiting undesirable side effects. This review describes how the use of targeted nanocarriers such as nanoparticles and liposomes can improve the pharmacokinetic properties of protein drugs, thus increasing their safety and maximizing the therapeutic effect.

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

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    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.

  6. 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.

  7. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

    Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

  8. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

    Directory of Open Access Journals (Sweden)

    Beatriz Orosa

    2017-01-01

    Full Text Available Hypersensitive response programmed cell death (HR-PCD is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

  9. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    Directory of Open Access Journals (Sweden)

    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.

  10. CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

    Science.gov (United States)

    Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

    2018-04-03

    The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

  11. SCF(SAP) controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Zhibiao; Li, Na; Jiang, Shan; Gonzalez, Nathalie; Huang, Xiahe; Wang, Yingchun; Inzé, Dirk; Li, Yunhai

    2016-04-06

    Control of organ size by cell proliferation and growth is a fundamental process, but the mechanisms that determine the final size of organs are largely elusive in plants. We have previously revealed that the ubiquitin receptor DA1 regulates organ size by repressing cell proliferation in Arabidopsis. Here we report that a mutant allele of STERILE APETALA (SAP) suppresses the da1-1 mutant phenotype. We show that SAP is an F-box protein that forms part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex and controls organ size by promoting the proliferation of meristemoid cells. Genetic analyses suggest that SAP may act in the same pathway with PEAPOD1 and PEAPOD2, which are negative regulators of meristemoid proliferation, to control organ size, but does so independently of DA1. Further results reveal that SAP physically associates with PEAPOD1 and PEAPOD2, and targets them for degradation. These findings define a molecular mechanism by which SAP and PEAPOD control organ size.

  12. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

    Science.gov (United States)

    Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

    2014-10-16

    Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Polyubiquitin chain assembly and organisation determine the dynamics of protein activation and degradation

    Directory of Open Access Journals (Sweden)

    Lan K. Nguyen

    2014-01-01

    Full Text Available Protein degradation via ubiquitination is a major proteolytic mechanism in cells. Once a protein is destined for degradation, it is tagged by multiple ubiquitin molecules. The synthesised polyubiquitin chains can be recognised by the 26S proteosome where proteins are degraded. These chains form through multiple ubiquitination cycles that are similar to multi-site phosphorylation cycles. As kinases and phosphatases, two opposing enzymes (E3 ligases and deubiquitinases DUBs catalyse (deubiquitination cycles. Although multi-ubiquitination cycles are fundamental mechanisms of controlling protein concentrations within a cell, their dynamics have never been explored. Here, we fill this knowledge gap. We show that under permissive physiological conditions, the formation of polyubiquitin chain of length greater than two and subsequent degradation of the ubiquitinated protein, which is balanced by protein synthesis, can display bistable, switch-like responses. Interestingly, the occurrence of bistability becomes pronounced, as the chain grows, giving rise to all-or-none regulation at the protein levels. We give predictions of protein distributions under bistable regime awaiting experimental verification. Importantly, we show for the first time that sustained oscillations can robustly arise in the process of formation of ubiquitin chain, largely due to the degradation of the target protein. This new feature is opposite to the properties of multi-site phosphorylation cycles, which are incapable of generating oscillation if the total abundance of interconverted protein forms is conserved. We derive structural and kinetic constraints for the emergence of oscillations, indicating that a competition between different substrate forms and the E3 and DUB is critical for oscillation. Our work provides the first detailed elucidation of the dynamical features brought about by different molecular setups of the polyubiquitin chain assembly process responsible for

  14. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

    International Nuclear Information System (INIS)

    Okumura, Atsushi; Alce, Tim; Lubyova, Barbora; Ezelle, Heather; Strebel, Klaus; Pitha, Paula M.

    2008-01-01

    The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication

  15. F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

    Science.gov (United States)

    Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

    2012-01-01

    Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

  16. A proteomic perspective of inbuilt viral protein regulation: pUL46 tegument protein is targeted for degradation by ICP0 during herpes simplex virus type 1 infection.

    Science.gov (United States)

    Lin, Aaron E; Greco, Todd M; Döhner, Katinka; Sodeik, Beate; Cristea, Ileana M

    2013-11-01

    Much like the host cells they infect, viruses must also regulate their life cycles. Herpes simples virus type 1 (HSV-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. Following entry into host cells, the virion-associated outer tegument proteins pUL46 and pUL47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. Because pUL46 has gone largely unstudied, we employed a hybrid mass spectrometry-based approach to determine how pUL46 exerts its functions during early stages of infection. For a spatio-temporal characterization of pUL46, time-lapse microscopy was performed in live cells to define its dynamic localization from 2 to 24 h postinfection. Next, pUL46-containing protein complexes were immunoaffinity purified during infection of human fibroblasts and analyzed by mass spectrometry to investigate virus-virus and virus-host interactions, as well as post-translational modifications. We demonstrated that pUL46 is heavily phosphorylated in at least 23 sites. One phosphorylation site matched the consensus 14-3-3 phospho-binding motif, consistent with our identification of 14-3-3 proteins and host and viral kinases as specific pUL46 interactions. Moreover, we determined that pUL46 specifically interacts with the viral E3 ubiquitin ligase ICP0. We demonstrated that pUL46 is partially degraded in a proteasome-mediated manner during infection, and that the catalytic activity of ICP0 is responsible for this degradation. This is the first evidence of a viral protein being targeted for degradation by another viral protein during HSV-1 infection. Together, these data indicate that pUL46 levels are tightly controlled and important for the temporal regulation of viral gene expression throughout the virus life cycle. The concept of a structural virion protein, pUL46, performing nonstructural roles is likely to reflect a theme common to many viruses

  17. Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

    Science.gov (United States)

    Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

    2015-01-01

    The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To

  18. 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...

  19. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

    2015-01-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

  20. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Directory of Open Access Journals (Sweden)

    Koenraad Van Doorslaer

    2015-06-01

    Full Text Available In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  1. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  2. The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation*

    Science.gov (United States)

    Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P. Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

    2012-01-01

    BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCFFBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCFFBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCFFBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCFFBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development. PMID:23086937

  3. AAA-ATPases in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Ravikiran S. Yedidi

    2017-06-01

    Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  4. AAA-ATPases in Protein Degradation.

    Science.gov (United States)

    Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

    2017-01-01

    Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  5. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

    2014-01-01

    Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

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

    Directory of Open Access Journals (Sweden)

    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.

  7. The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

    Science.gov (United States)

    Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

    2012-11-30

    BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

  8. Model for Stress-induced Protein Degradation in Lemna minor1

    Science.gov (United States)

    Cooke, Robert J.; Roberts, Keith; Davies, David D.

    1980-01-01

    Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

  9. Cyclin A degradation by primate cytomegalovirus protein pUL21a counters its innate restriction of virus replication.

    Directory of Open Access Journals (Sweden)

    Nicolas Caffarelli

    Full Text Available Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC, but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.

  10. Dissection of membrane protein degradation mechanisms by reversible inhibitors

    International Nuclear Information System (INIS)

    Hare, J.F.

    1988-01-01

    The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma membrane polypeptides in response to reversible temperature and lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were radiolabeled and left for 24 h to allow the removal of rapidly degraded proteins. Remaining trichloroacetic acid-precipitable protein was degraded (t 1/2 = 40-68 h) by an apparent first order process 60-86% sensitive to 10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by temperature reduction to 18 degrees C. Thus, membrane proteins are selected for degradation in a time-dependent manner by a system which is sensitive to both 18 degrees C and to lysosomotropic amines. When inhibitory conditions were removed after 40-48 h, degradation of 125I-labeled protein resumed at the same rate as that seen in their absence. Since membrane proteins do not exhibit accelerated degradation after removal of inhibitory conditions, there can be no marking or sorting of those proteins destined for degradation during the 40-h exposure to inhibitory conditions. Exposure to amines or 18 degrees C did not affect the position of two-dimensionally resolved labeled polypeptides. Fractionation of labeled cells on Percoll gradients after 40 h of exposure to low temperature or amines showed that labeled protein remained in the plasma membrane fractions of the gradient although shifted to a slightly lower buoyant density in the presence of amines. These results support the notion that selection of plasma membrane proteins for degradation requires their internalization into acidic vesicles. Lysosomotropic amines and reduced temperature interfere with the selection process by preventing membrane fusion events

  11. Uptake and degradation of circulating proteins by the liver.

    NARCIS (Netherlands)

    Buys, Carolus Henricus Cornelis Maria

    1976-01-01

    Circulating proteins, like all proteins in a living animal, are subject to continual replacement or turnover. This process implies both synthesis and degradation, This thesis deals with the degradative part of turnover of circulating proteins. ... Summary

  12. Protein oxidation and degradation caused by particulate matter

    Science.gov (United States)

    Lai, Ching-Huang; Lee, Chun-Nin; Bai, Kuan-Jen; Yang, You-Lan; Chuang, Kai-Jen; Wu, Sheng-Ming; Chuang, Hsiao-Chi

    2016-09-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM.

  13. Aminopeptidase-resistant peptides are targeted to lysosomes and subsequently degraded

    NARCIS (Netherlands)

    Gillis, Judith M.; Benckhuijsen, Willemien; van Veen, Henk; Sanz, Alicia Sanz; Drijfhout, Jan W.; Reits, Eric A.

    2011-01-01

    Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of

  14. Microbial degradation of dissolved proteins in seawater

    International Nuclear Information System (INIS)

    Hollibaugh, J.T.; Azam, F.

    1983-01-01

    An experimental protocol using radiolabeled proteins was developed to investigate the rates and mechanisms whereby dissolved proteins are degraded in natural marine plankton communities. The results of field observations and laboratory experiments indicate that proteins are degraded by a particle-bound, thermolabile system, presumably bacteria-associated enzymes, with an apparent half-saturation constant of ca. 25 μg bovine serum albumin (BSA) per liter. Gel permeation chromatography indicated that peptides of chain length intermediate between BSA and the final products of degradation (MW<700) do not accumulate in the medium. Competition experiments indicate that the system is relatively nonspecific. Turnover rates for the protein pool in samples collected in the Southern California Bight were of the same order of magnitude as the turnover rate of the L-leucine pool and were correlated with primary productivity, chlorophyll a concentrations, bacterial abundance and biomass, and L-leucine turnover rate. These data suggest that amino acids derived from proteins are utilized preferentially and do not completely mix with the amino acids in the bulk phase

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  16. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    Science.gov (United States)

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  17. The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

    Science.gov (United States)

    Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

    2016-10-15

    Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins

    Directory of Open Access Journals (Sweden)

    Vasko eVeljanovski

    2014-06-01

    Full Text Available Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double-membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

  19. Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

    OpenAIRE

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M.; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligas...

  20. The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

    Science.gov (United States)

    Szalmás, Anita; Tomaić, Vjekoslav; Basukala, Om; Massimi, Paola; Mittal, Suruchi; Kónya, József; Banks, Lawrence

    2017-04-01

    Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays. IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor. Copyright © 2017 American Society for Microbiology.

  1. Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

    Science.gov (United States)

    Wang, N; Kang, H S; Ahmmed, G; Khan, S A; Makarenko, V V; Prabhakar, N R; Nanduri, J

    2016-03-01

    Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH. Copyright © 2016 the American Physiological Society.

  2. 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.

  3. Light-induced protein degradation in human-derived cells.

    Science.gov (United States)

    Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

    2017-05-27

    Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Investigating ER-Associated Degradation with RNAi Screening - and Searching for Model Proteins to Do It with

    DEFF Research Database (Denmark)

    Jensen, Njal Winther

    Abstract In eukaryotes, secretory proteins are translocated into the endoplasmic reticulum (ER) for folding assistance, acquisition of posttranslational modifications and sorting. Proteins that do not obtain their native conformation are eliminated by ER-associated degradation (ERAD). ERAD...... is a sophisticated pathway that recognizes misfolded proteins and targets them for degradation by the 26S proteasome residing in the cytosol. More than 60 diseases including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have been linked to the ERAD pathway underscoring its crucial role...... for cellular homeostasis. The aim of this thesis has been to gain insight into ERAD. The experimental approach was RNAi screening, which is a fast and efficient method for initial evaluation of a large pool of genes. Since relatively few proteins routinely are used as ERAD substrates, the first goal...

  5. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    Science.gov (United States)

    Shah, Dhiral Ashwin

    functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. The same concept of naturally occurring protein-protein interactions can also be implemented to selectively bring intracellular protein targets in close proximity to proteasomal degradation machinery in cells and effect their depletion from the cellular compartments. This approach will be able to not only target entire pool of proteins to ubiquitination-mediated degradation, but also to specific sub-pools of posttranslationally modified proteins in the cell, provided peptides having distinct binding affinities are identified for posttranslational modifications. This system can then be tested for intracellular protein delivery using nanoparticle carriers to identify roles of different posttranslational modifications on the protein's activity. In future work, we propose to develop a cellular detection system, based on GFP complementation, which can be used to evaluate the efficiency of different protein delivery carriers to internalize proteins into the cell cytosol. We envision the application of nanoscale materials as intracellular protein delivery vehicles to target diverse cell signaling pathways at the posttranslational level, and subsequent metabolic manipulation, which may have interesting therapeutic properties and can potentially target stem cell fate.

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

    Science.gov (United States)

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

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. 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 ...

  9. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  10. Protein degradation and protection against misfolded or damaged proteins

    Science.gov (United States)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  11. In vitro degradation of the 32kDa PS II reaction centre protein

    International Nuclear Information System (INIS)

    Eckenswiller, L.C.; Greenberg, B.M.

    1989-01-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components

  12. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  13. Long-term aversive taste memory requires insular and amygdala protein degradation.

    Science.gov (United States)

    Rodriguez-Ortiz, Carlos J; Balderas, Israela; Saucedo-Alquicira, Fernando; Cruz-Castañeda, Paulina; Bermudez-Rattoni, Federico

    2011-03-01

    Some reports have shown that the ubiquitin-proteasome system (UPS) is necessary to degrade repressor factors to produce new proteins essential to memory consolidation. Furthermore, recent evidence suggests that memory updating also relies on protein degradation through the UPS. To evaluate whether degradation of proteins is part of the cellular events needed for long-term storage of taste aversion, we injected lactacystin--an UPS inhibitor--into the amygdala and/or insular cortex 30 min before the first or second training trials. The results revealed that degradation of proteins in either the amygdala or insular cortex suffices for long-term stabilization of first-time encounter taste aversion. On the other hand, lactacystin applied in the insula, but not in the amygdala, before the second training prevented long-term storage of updated information. Our results support that degradation of proteins by means of the UPS is required every time taste aversion is to be stored in long-term memory. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  15. BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

    Directory of Open Access Journals (Sweden)

    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.

  16. BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

    Science.gov (United States)

    Miah, Sayem; Goel, Raghuveera Kumar; Dai, Chenlu; Kalra, Natasha; Beaton-Brown, Erika; Bagu, Edward T; Bonham, Keith; Lukong, Kiven E

    2014-01-01

    Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

  17. Targeting protein-protein interactions for parasite control.

    Directory of Open Access Journals (Sweden)

    Christina M Taylor

    2011-04-01

    Full Text Available Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific orthologous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank. EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite and B. malayi (H. sapiens parasite, which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly

  18. Selectivity in protein degradation during sporulation of Bacillus subtilis

    International Nuclear Information System (INIS)

    Mitani, Takahiko; Kadota, Hajime

    1976-01-01

    The breakdown of cellular protein was investigated in Bacillus subtilis ATCC 6051 labeled with glycine-2- 3 H or L-phenylalanine-U- 14 C at the different stages of vegetative growth and sporulation. The growth of the culture was determined by measuring optical density at 660 nm. The heat-resistant spores were scored by plating after heating at 80 deg C for 10 minutes. A question whether the turnover of glycine-labeled protein is similar to that of phenylalanine-labeled protein was experimentally studied. The patterns obtained with the glycine-labeled protein were different from those of phenylalanine-labeled protein. This was not multiple turnover. The cellular protein which was labeled with glycine at an early stage of sporulation showed rapid degradation, but the degradation of the protein labeled with glycine at later stages did not occur at all. Another question whether the labeled glycine incorporated into cells at the different stages of growth and sporulation was present in the spore coat fraction of matured spores was studied. Experiment demonstrated that the glycine incorporated into cells at the late sporulation stage was mainly utilized for the biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amount of glycine is rarely subject to further degradation. (Iwakiri, K.)

  19. Human sperm degradation of zona pellucida proteins contributes to fertilization.

    Science.gov (United States)

    Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

    2015-09-02

    The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

  20. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

    OpenAIRE

    Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-01-01

    Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...

  1. Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits

    International Nuclear Information System (INIS)

    Samarel, A.M.; Parmacek, M.S.; Magid, N.M.; Decker, R.S.; Lesch, M.

    1987-01-01

    To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [ 3 H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting

  2. The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

    Science.gov (United States)

    Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

    2014-09-01

    Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  3. Are animal models predictive for human postmortem muscle protein degradation?

    Science.gov (United States)

    Ehrenfellner, Bianca; Zissler, Angela; Steinbacher, Peter; Monticelli, Fabio C; Pittner, Stefan

    2017-11-01

    A most precise determination of the postmortem interval (PMI) is a crucial aspect in forensic casework. Although there are diverse approaches available to date, the high heterogeneity of cases together with the respective postmortal changes often limit the validity and sufficiency of many methods. Recently, a novel approach for time since death estimation by the analysis of postmortal changes of muscle proteins was proposed. It is however necessary to improve the reliability and accuracy, especially by analysis of possible influencing factors on protein degradation. This is ideally investigated on standardized animal models that, however, require legitimization by a comparison of human and animal tissue, and in this specific case of protein degradation profiles. Only if protein degradation events occur in comparable fashion within different species, respective findings can sufficiently be transferred from the animal model to application in humans. Therefor samples from two frequently used animal models (mouse and pig), as well as forensic cases with representative protein profiles of highly differing PMIs were analyzed. Despite physical and physiological differences between species, western blot analysis revealed similar patterns in most of the investigated proteins. Even most degradation events occurred in comparable fashion. In some other aspects, however, human and animal profiles depicted distinct differences. The results of this experimental series clearly indicate the huge importance of comparative studies, whenever animal models are considered. Although animal models could be shown to reflect the basic principles of protein degradation processes in humans, we also gained insight in the difficulties and limitations of the applicability of the developed methodology in different mammalian species regarding protein specificity and methodic functionality.

  4. Radiation degradation of silk protein

    International Nuclear Information System (INIS)

    Pewlong, W.; Sudatis, B.; Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu

    2000-01-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated using an electron beam accelerator to investigate the application of the radiation degradation technique as a means to solubilize fibroin. The irradiation caused a significant degradation of the fiber. The tensile strength of fibroin fiber irradiated up to 2500 kGy decreased rapidly with increasing dose. The presence of oxygen in the irradiation atmosphere enhanced degradation of the tensile strength. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: a calcium chloride solution(CaCl 2 /C 2 H 5 OH/H 2 O=1:2:8 in mole ratio), a hydrochloric acid (0.5 N) and a distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water soluble proteins was extracted by a distilled water. (author)

  5. Radiation degradation of silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Pewlong, W; Sudatis, B [Office of Atomic Energy for Peace, Bangkok (Thailand); Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2000-03-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated using an electron beam accelerator to investigate the application of the radiation degradation technique as a means to solubilize fibroin. The irradiation caused a significant degradation of the fiber. The tensile strength of fibroin fiber irradiated up to 2500 kGy decreased rapidly with increasing dose. The presence of oxygen in the irradiation atmosphere enhanced degradation of the tensile strength. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: a calcium chloride solution(CaCl{sub 2}/C{sub 2}H{sub 5}OH/H{sub 2}O=1:2:8 in mole ratio), a hydrochloric acid (0.5 N) and a distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water soluble proteins was extracted by a distilled water. (author)

  6. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Su, Xiong [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Liu, Jialiu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Sundaresan, Sinju [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Stahl, Philip D., E-mail: pstahl@wustl.edu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  7. HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.

    Directory of Open Access Journals (Sweden)

    Esben G Poulsen

    Full Text Available In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1. Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

  8. Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

    Science.gov (United States)

    Nongonierma, Alice B; FitzGerald, Richard J

    2018-06-01

    Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

  9. Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

    Science.gov (United States)

    Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

    2016-12-27

    Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

  10. Properties of Protein Drug Target Classes

    Science.gov (United States)

    Bull, Simon C.; Doig, Andrew J.

    2015-01-01

    Accurate identification of drug targets is a crucial part of any drug development program. We mined the human proteome to discover properties of proteins that may be important in determining their suitability for pharmaceutical modulation. Data was gathered concerning each protein’s sequence, post-translational modifications, secondary structure, germline variants, expression profile and drug target status. The data was then analysed to determine features for which the target and non-target proteins had significantly different values. This analysis was repeated for subsets of the proteome consisting of all G-protein coupled receptors, ion channels, kinases and proteases, as well as proteins that are implicated in cancer. Machine learning was used to quantify the proteins in each dataset in terms of their potential to serve as a drug target. This was accomplished by first inducing a random forest that could distinguish between its targets and non-targets, and then using the random forest to quantify the drug target likeness of the non-targets. The properties that can best differentiate targets from non-targets were primarily those that are directly related to a protein’s sequence (e.g. secondary structure). Germline variants, expression levels and interactions between proteins had minimal discriminative power. Overall, the best indicators of drug target likeness were found to be the proteins’ hydrophobicities, in vivo half-lives, propensity for being membrane bound and the fraction of non-polar amino acids in their sequences. In terms of predicting potential targets, datasets of proteases, ion channels and cancer proteins were able to induce random forests that were highly capable of distinguishing between targets and non-targets. The non-target proteins predicted to be targets by these random forests comprise the set of the most suitable potential future drug targets, and should therefore be prioritised when building a drug development programme. PMID

  11. 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

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

    Science.gov (United States)

    Steely, Andrea M; Willoughby, Jamin A; Sundar, Shyam N; Aivaliotis, Vasiliki I; Firestone, Gary L

    2017-10-01

    Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

  13. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    International Nuclear Information System (INIS)

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-01-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([ 14 C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([ 14 C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole

  14. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

    Science.gov (United States)

    Yuan, Y; Zhang, G Q; Chai, W; Ni, M; Xu, C; Chen, J Y

    2016-10-01

    Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J

  15. Prion protein degradation by lichens of the genus Cladonia

    Science.gov (United States)

    Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

    2012-01-01

    It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

  16. Accurate microRNA target prediction correlates with protein repression levels

    Directory of Open Access Journals (Sweden)

    Simossis Victor A

    2009-09-01

    Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

  17. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  18. The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

    Science.gov (United States)

    Küberl, Andreas; Polen, Tino; Bott, Michael

    2016-04-26

    The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

  19. Ubiquitin ligase RNF123 mediates degradation of heterochromatin protein 1α and β in lamin A/C knock-down cells.

    Directory of Open Access Journals (Sweden)

    Pankaj Chaturvedi

    Full Text Available The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1. However, the pathways of proteasomal degradation have not been well characterized.To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells.Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.

  20. Protein degradation during reconsolidation as a mechanism for memory reorganization

    Directory of Open Access Journals (Sweden)

    Bong-Kiun Kaang

    2011-02-01

    Full Text Available Memory is a reference formed from a past experience that is used to respond to present situations. However, the world is dynamic and situations change, so it is important to update the memory with new information each time it is reactivated in order to adjust the response in the future. Recent researches indicate that memory may undergo a dynamic process that could work as an updating mechanism. This process which is called reconsolidation involves destabilization of the memory after it is reactivated, followed by restabilization. Recently, it has been demonstrated that the initial destabilization process of reconsolidation requires protein degradation. Using protein degradation inhibition as a method to block reconsolidation, recent researches suggest that reconsolidation, especially the protein degradation-dependent destabilization process is necessary for memory reorganization.

  1. Dma1-dependent degradation of SIN proteins during meiosis in Schizosaccharomyces pombe.

    Science.gov (United States)

    Krapp, Andrea; Simanis, Viesturs

    2014-07-15

    The Schizosaccharomyces pombe septation initiation network (SIN) is required for cytokinesis during vegetative growth and for spore formation during meiosis. Regulation of the SIN during mitosis has been studied extensively, but less is known about its meiotic regulation. Here, we show that several aspects of SIN regulation differ between mitosis and meiosis. First, the presence of GTP-bound Spg1p is not the main determinant of the timing of Cdc7p and Sid1p association with the spindle pole body (SPB) during meiosis. Second, the localisation dependencies of SIN proteins differ from those in mitotic cells, suggesting a modified functional organisation of the SIN during meiosis. Third, there is stage-specific degradation of SIN components in meiosis; Byr4p is degraded after meiosis I, whereas the degradation of Cdc7p, Cdc11p and Sid4p occurs after the second meiotic division and depends upon the ubiquitin ligase Dma1p. Finally, Dma1p-dependent degradation is not restricted to the SIN, as we show that Dma1p is needed for the degradation of Mcp6p (also known as Hrs1p) during meiosis I. Taken together, these data suggest that stage-specific targeted proteolysis plays an important role in regulating meiotic progression. © 2014. Published by The Company of Biologists Ltd.

  2. Restoring degraded land: contributing to Aichi Targets 14, 15, and beyond

    DEFF Research Database (Denmark)

    Navarro, Laetitia M.; Marques, Alexandra; Ceausu, Silvia

    2018-01-01

    Restoration ecology is gaining momentum on the international conservation scene. In particular, restoring degraded ecosystems is central to Aichi Biodiversity Targets 14 and 15 set by the Convention on Biological Diversity. Depending on the definition of degradation, from 2 to 47% of the global...... for restoration. Finally, we assess national conservation targets to examine the potential multifaceted impacts of achieving Aichi Targets 14 and 15 on biodiversity and society....... land surface could require restoration. Here, we review the range of goals and approaches to restoration, from active interventions to more passive approaches such as rewilding. We identify biodiversity offsets, payments for ecosystem services and agri-enviromental schemes as enabling policy mechanisms...

  3. Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-16-1-0615 TITLE: Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy PRINCIPAL...29 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy...infection or cigarette smoke enhanced pulmonary metastasis from breast cancer in humans and mice. Similarly, autoimmune arthritis, characterized by

  4. Radiation degradation of silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Wachiraporn Pewlong; Boonya Sudatis [Office of Atomic Energy for Peace, Bangkok (Thailand); Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2000-09-01

    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated in the dose range up to 2500 kGy using an electron beam accelerator to apply the radiation degradation technique as a means to solubilize fibroin. The tensile strength of irradiated fibroin fiber decreased with increasing dose and the presence of oxygen in the irradiation atmosphere enhanced the degradation. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: calcium chloride solution (CaCl{sub 2}/C{sub 2}H{sub 5}OH/H{sub 2}O = 1 : 2 : 8 in mole ratio), hydrochloric acid (0.5N) and distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water-soluble protein was extracted by distilled water. (author)

  5. Establishment of protein delivery systems targeting podocytes.

    Directory of Open Access Journals (Sweden)

    Wen Chih Chiang

    2010-07-01

    Full Text Available Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.

  6. Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements

    International Nuclear Information System (INIS)

    Rogers, S.W.; Rechsteiner, M.

    1988-01-01

    Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability

  7. Sequence and 3D structure based analysis of TNT degrading proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Bhattacherjee, Amrita; Mandal, Rahul Shubhra; Das, Santasabuj; Kundu, Sudip

    2014-03-01

    TNT, accidentally released at several manufacturing sites, contaminates ground water and soil. It has a toxic effect to algae and invertebrate, and chronic exposure to TNT also causes harmful effects to human. On the other hand, many plants including Arabidopsis thaliana have the ability to metabolize TNT either completely or at least to a reduced less toxic form. In A. thaliana, the enzyme UDP glucosyltransferase (UDPGT) can further conjugate the reduced forms 2-HADNT and 4-HADNT (2-hydroxylamino-4, 6- dinitrotoluene and 4-hydroxylamino-2, 6- dinitrotoluene) of TNT. Based on the experimental analysis, existing literature and phylogenetic analysis, it is evident that among 107 UDPGT proteins only six are involved in the TNT degrading process. A total of 13 UDPGT proteins including five of these TNT degrading proteins fall within the same group of phylogeny. Thus, these 13 UDPGT proteins have been classified into two groups, TNT-degrading and TNT-non-degrading proteins. To understand the differences in TNT-degrading capacities; using homology modeling we first predicted two structures, taking one representative sequence from both the groups. Next, we performed molecular docking of the modeled structure and TNT reduced form 2-hydroxylamino-4, 6- dinitrotoluene (2-HADNT). We observed that while the Trp residue located within the active site region of the TNT- degrading protein showed π-Cation interaction; such type of interaction was absent in TNT-non-degrading protein, as the respective Trp residue lay outside of the pocket in this case. We observed the conservation of this π-Cation interaction during MD simulation of TNT-degrading protein. Thus, the position and the orientation of the active site residue Trp could explain the presence and absence of TNT-degrading capacity of the UDPGT proteins.

  8. Retraction: Myostatin Induces Degradation of Sarcomeric Proteins through a Smad3 Signaling Mechanism During Skeletal Muscle Wasting

    Science.gov (United States)

    Lokireddy, Sudarsanareddy; McFarlane, Craig; Ge, Xiaojia; Zhang, Huoming; Sze, Siu Kwan; Sharma, Mridula

    2011-01-01

    Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1. PMID:21964591

  9. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

    Directory of Open Access Journals (Sweden)

    Moisés Martínez-Castillo

    2015-01-01

    Full Text Available Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C employing conditioned medium (CM and total crude extracts (TCEs of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

  10. Hot-spot analysis for drug discovery targeting protein-protein interactions.

    Science.gov (United States)

    Rosell, Mireia; Fernández-Recio, Juan

    2018-04-01

    Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

  11. Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

    2016-06-01

    Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

  12. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  13. Export-defective lamB protein is a target for translational control caused by ompC porin overexpression.

    OpenAIRE

    Click, E M; Schnaitman, C A

    1989-01-01

    Overexpression of OmpC protein from an inducible plasmid vector reduced the amount of the precursor form of LamB protein in LamB signal sequence mutants. The stability of the precursor form of LamB protein was not affected, indicating that the effect of OmpC overexpression was on the synthesis of the precursor rather than on degradation. These results indicate that a functional signal sequence is not required on an outer membrane protein for it to be a target for translational control.

  14. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    Science.gov (United States)

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  15. Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG)

    OpenAIRE

    Worby, Carolyn A.; Gentry, Matthew S.; Dixon, Jack E.

    2007-01-01

    Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual-specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PT...

  16. Protein quality control in the nucleus

    DEFF Research Database (Denmark)

    Nielsen, Sofie V.; Poulsen, Esben Guldahl; Rebula, Caio A.

    2014-01-01

    to aggregate, cells have evolved several elaborate quality control systems to deal with these potentially toxic proteins. First, various molecular chaperones will seize the misfolded protein and either attempt to refold the protein or target it for degradation via the ubiquitin-proteasome system...... to be particularly active in protein quality control. Thus, specific ubiquitin-protein ligases located in the nucleus, target not only misfolded nuclear proteins, but also various misfolded cytosolic proteins which are transported to the nucleus prior to their degradation. In comparison, much less is known about...... these mechanisms in mammalian cells. Here we highlight recent advances in our understanding of nuclear protein quality control, in particular regarding substrate recognition and proteasomal degradation....

  17. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    Muscle cell culture (L/sub 6/) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 ..mu..M compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of (/sup 3/H) leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using (/sup 3/H) leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 ..mu..M level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.

  18. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    International Nuclear Information System (INIS)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    1986-01-01

    Muscle cell culture (L 6 ) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 μM compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of [ 3 H] leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using [ 3 H] leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 μM level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle

  19. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    Directory of Open Access Journals (Sweden)

    Beatriz Alvarez-Castelao

    2012-01-01

    Full Text Available Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.

  20. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  1. Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins.

    Science.gov (United States)

    Bence, Melinda; Jankovics, Ferenc; Lukácsovich, Tamás; Erdélyi, Miklós

    2017-04-01

    Inducible protein degradation techniques have considerable advantages over classical genetic approaches, which generate loss-of-function phenotypes at the gene or mRNA level. The plant-derived auxin-inducible degradation system (AID) is a promising technique which enables the degradation of target proteins tagged with the AID motif in nonplant cells. Here, we present a detailed characterization of this method employed during the adult oogenesis of Drosophila. Furthermore, with the help of CRISPR/Cas9-based genome editing, we improve the utility of the AID system in the conditional elimination of endogenously expressed proteins. We demonstrate that the AID system induces efficient and reversible protein depletion of maternally provided proteins both in the ovary and the early embryo. Moreover, the AID system provides a fine spatiotemporal control of protein degradation and allows for the generation of different levels of protein knockdown in a well-regulated manner. These features of the AID system enable the unraveling of the discrete phenotypes of genes with highly complex functions. We utilized this system to generate a conditional loss-of-function allele which allows for the specific degradation of the Vasa protein without affecting its alternative splice variant (solo) and the vasa intronic gene (vig). With the help of this special allele, we demonstrate that dramatic decrease of Vasa protein in the vitellarium does not influence the completion of oogenesis as well as the establishment of proper anteroposterior and dorsoventral polarity in the developing oocyte. Our study suggests that both the localization and the translation of gurken mRNA in the vitellarium is independent from Vasa. © 2017 Federation of European Biochemical Societies.

  2. ORGANIC MATTER AND CRUDE PROTEIN DEGRADATION SYNCHRONY IN DIETS SELECTED BY RANGE GOATS.

    Directory of Open Access Journals (Sweden)

    Rafael Ramírez Orduña

    2010-09-01

    Full Text Available The study was carried out with the aim to asses the synchrony of organic matter and crude protein degradation in the rumen of diets selected by range goats through two years. Five esophageal cannulated adult male goats were used to collect extrusa samples during summer (August 9–13 and autumn (November 29 –December 3 of 2006, winter (February 20 – 24, spring (April 29 –May 5, summer (September 10–15 and autumn (December 4–8 of 2007 and winter (February 20 – 25 and spring (May 9 –13 of 2008. Extrusa samples were subjected to chemical analysis to determine organic matter (OM, crude protein (CP in situ and in vitro true digestibility of dry matter. OM and CP intake were estimated by total fecal collection. Effective extent of degradation of the OM and CP was calculated hourly and total 24 hours. From the hourly quantity of OM and CP degraded, a synchrony index of CP to OM was calculated, and from the total 24 hours degradation, degraded organic matter intake and crude protein intake were also estimated. Sampling date was the main effect that determined the variation of diet OM and CP degradation parameters. Degraded crude protein intake as a proportion of degraded OM was affected by sampling date and was correlated to rainfall. During winter of the first year degraded crude protein intake was below the requirements for maintenance or to promote growth for range goats weighing 40 kg. Even though, synchrony index between OM and CP degradation was affected by sampling date goats maintained a high synchrony index throughout the years.

  3. Chemical degradation of proteins in the solid state with a focus on photochemical reactions.

    Science.gov (United States)

    Mozziconacci, Olivier; Schöneich, Christian

    2015-10-01

    Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

    Directory of Open Access Journals (Sweden)

    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.

  5. Does altered protein metabolism interfere with postmortem degradation analysis for PMI estimation?

    Science.gov (United States)

    Zissler, A; Ehrenfellner, B; Foditsch, E E; Monticelli, F C; Pittner, S

    2018-03-02

    An accurate estimation of the postmortem interval (PMI) is a central aspect in forensic routine. Recently, a novel approach based on the analysis of postmortem muscle protein degradation has been proposed. However, a number of questions remain to be answered until sensible application of this method to a broad variety of forensic cases is possible. To evaluate whether altered in vivo protein metabolism interferes with postmortem degradation patterns, we conducted a comparative study. We developed a standardized animal degradation model in rats, and collected additional muscle samples from animals recovering from muscle injury and from rats with developed disuse muscle atrophy after induced spinal cord injury. All samples were analyzed by SDS-PAGE and Western blot, labeling well-characterized muscle proteins. Tropomyosin was found to be stable throughout the investigated PMI and no alterations were detected in regenerating and atrophic muscles. In contrast, significant predictable postmortem changes occurred in desmin and vinculin protein band patterns. While no significant deviations from native patterns were detected in at-death samples of disuse muscle atrophy, interestingly, samples of rats recovering from muscle injury revealed additional desmin and vinculin degradation bands that did not occur in this form in any of the examined postmortem samples regardless of PMI. It remains to be investigated whether in vivo-altered metabolism influences postmortem degradation kinetics or if such muscle samples undergo postmortem degradation in a regular fashion.

  6. Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

    Directory of Open Access Journals (Sweden)

    Jye-Lin Hsu

    2015-04-01

    Full Text Available Human cytomegalovirus (HCMV US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I. Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

  7. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    Directory of Open Access Journals (Sweden)

    Mayumi F. Kohiyama

    2015-01-01

    Full Text Available Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by a coding polyglutamine expansion in the Ataxin-1 gene ( ATXN1 , which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

  8. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Directory of Open Access Journals (Sweden)

    Hendrik Fuchs

    2016-07-01

    Full Text Available The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  9. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  10. Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1.

    Science.gov (United States)

    Csorba, Tibor; Lózsa, Rita; Hutvágner, György; Burgyán, József

    2010-05-01

    RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein-protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA-RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.

  11. The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein.

    Directory of Open Access Journals (Sweden)

    Daniel W Summers

    Full Text Available Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP. The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

  12. Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Ndoni, Sokol

    2007-01-01

    Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used w...

  13. The establishment of a protein degradability data base for dairy ...

    African Journals Online (AJOL)

    The degradability values ranged from 96,2; 91,9 and 88,7% for ... McDonald, 1979; Kristensen, et ai., 1982; Lindberg,. 1983 ... mass of 570 kg, and average production of 5000 kg ... Dicalcium phosphate'. 0,5. Commercial vitamin and trace mineral premix. 0,3 .... effects on the degradation of most protein supplements.

  14. Interplays between Sumoylation, SUMO-Targeted Ubiquitin Ligases, and the Ubiquitin-Adaptor Protein Ufd1 in Fission Yeast

    DEFF Research Database (Denmark)

    Køhler, Julie Bonne

    and the specific molecular interactions and sequence of events linking sumoylation, ubiquitylation and substrate degradation, has been largely uncovered. Using the fission yeast model organism I here present evidence for a role of the Ufd1 (ubiquitinfusion degradation 1) protein, and by extension of the Cdc48-Ufd1...... proteasome mediates direct cross-talk between the two modification systems. By contributing to the dynamic turnover of SUMO conjugated species these SUMO-targeted ubiquitin ligases (STUbLs) fulfills essential roles in both yeast and man. However, the specific sumoylated proteins affected by STUbL activity...... either in STUbL or Ufd1 function. In addition to identifying more than 900 unique sumoylated sites, these efforts revealed a number of proteins with upregulated sumoylation either in STUbL and/or Ufd1 mutant cells. These findings propose specific candidate substrates through which STUbL and Cdc48-Ufd1...

  15. Estimation of Relationship Between In Situ and In Vitro Rumen Protein Degradability of Extruded Full Fat Soybean

    Directory of Open Access Journals (Sweden)

    Arzu Erol Tunç

    2017-10-01

    Full Text Available The objectives of this study were to estimate the protein degradability of extruded full fat soybean (ESB by in situ (nylon bag and in vitro enzymatic method and to develop an equation in order predict in situ degradability from in vitro values. In the study enzymatic technique; hydrolysis after 1 h (INV1 and after 24 h (INV24 by a purified protease extracted from Streptomyces griseus in a borate-phosphate buffer at pH 8 was used as in vitro method. Relationship between in situ effective protein degradability (INSE and in vitro degradability after 1 and 24 hours incubations (INV1 and INV24 were determined. In situ protein degradability was measured at 0, 2, 4, 8, 16, 24, and 48 and at 72 h incubations in the rumen of 3 Holstein cows. In the study INSE, INV1 and INV24 were determined as 58.05, 20.24 and 41.46% respectively. Despite there were differences between in situ and in vitro protein degradability values, correlation coefficients between in situ and in vitro protein degradability of ESB were high and regression equations for estimation of in situ from in vitro were found significant. As conclusion in vitro enzymatic protein degradability (INV1 and INV24 can be used for estimation of in situ effective protein degradability of extruded full fat soybean.

  16. Protein search for multiple targets on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lange, Martin [Johannes Gutenberg University, Mainz 55122 (Germany); Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kochugaeva, Maria [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-09-14

    Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations.

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

    Science.gov (United States)

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

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. © 2015 Institute of Botany, Chinese Academy of Sciences.

  18. Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

    Directory of Open Access Journals (Sweden)

    Wang Ting

    2012-08-01

    Full Text Available Abstract Background Exposure to particulate matter (PM is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. Objectives We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC barrier integrity and enhanced cardiopulmonary dysfunction. Methods Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm. Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. Results PM exposure induced tight junction protein Zona occludens-1 (ZO-1 relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin. N-acetyl-cysteine (NAC, 5 mM reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2, in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. Conclusions These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

  19. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

  20. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

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

    Science.gov (United States)

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

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

    Directory of Open Access Journals (Sweden)

    Honghao Li

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

  3. Muscle protein degradation and amino acid metabolism during prolonged knee-extensor exercise in humans

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; Saltin, B; Wagenmakers, A J

    1999-01-01

    to a substantial increase in net muscle protein degradation, and that a lowering of the starting muscle glycogen content leads to a further increase. The carbon atoms of the branched-chain amino acids (BCAA), glutamate, aspartate and asparagine, liberated by protein degradation, and the BCAA and glutamate......The aim of this study was to investigate whether prolonged one-leg knee-extensor exercise enhances net protein degradation in muscle with a normal or low glycogen content. Net amino acid production, as a measure of net protein degradation, was estimated from leg exchange and from changes...... in the concentrations of amino acids that are not metabolized in skeletal muscle. Experiments were performed at rest and during one-leg knee-extensor exercise in six subjects having one leg with a normal glycogen content and the other with a low glycogen content. Exercise was performed for 90 min at a workload of 60...

  4. E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis.

    Science.gov (United States)

    Jung, Cho-Rok; Hwang, Kyung-Sun; Yoo, Jinsang; Cho, Won-Kyung; Kim, Jin-Man; Kim, Woo Ho; Im, Dong-Soo

    2006-07-01

    The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    Science.gov (United States)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A. A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-07-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, Pruminal protein degradation and increase (linear effect, Pruminant nutrition.

  7. A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

    Science.gov (United States)

    Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E; Lieberman, Andrew P; Osawa, Yoichi

    2014-11-01

    In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. © 2014 by the Society for Experimental Biology and Medicine.

  8. Toxicological relationships between proteins obtained from protein target predictions of large toxicity databases

    International Nuclear Information System (INIS)

    Nigsch, Florian; Mitchell, John B.O.

    2008-01-01

    The combination of models for protein target prediction with large databases containing toxicological information for individual molecules allows the derivation of 'toxiclogical' profiles, i.e., to what extent are molecules of known toxicity predicted to interact with a set of protein targets. To predict protein targets of drug-like and toxic molecules, we built a computational multiclass model using the Winnow algorithm based on a dataset of protein targets derived from the MDL Drug Data Report. A 15-fold Monte Carlo cross-validation using 50% of each class for training, and the remaining 50% for testing, provided an assessment of the accuracy of that model. We retained the 3 top-ranking predictions and found that in 82% of all cases the correct target was predicted within these three predictions. The first prediction was the correct one in almost 70% of cases. A model built on the whole protein target dataset was then used to predict the protein targets for 150 000 molecules from the MDL Toxicity Database. We analysed the frequency of the predictions across the panel of protein targets for experimentally determined toxicity classes of all molecules. This allowed us to identify clusters of proteins related by their toxicological profiles, as well as toxicities that are related. Literature-based evidence is provided for some specific clusters to show the relevance of the relationships identified

  9. The establishment of a protein degradability data base for dairy ...

    African Journals Online (AJOL)

    The establishment of a protein degradability data base for dairy cattle using the nylon bag technique. 1. Protein sources. LJ Erasmus, J Prinsloo, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals ...

  10. DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

    Science.gov (United States)

    Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

    2017-10-01

    Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

  11. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    Science.gov (United States)

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-06

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  12. Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

    Science.gov (United States)

    Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

    2009-07-20

    Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

  13. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    Energy Technology Data Exchange (ETDEWEB)

    Shawrang, P. [Agriculture, Medical and Industrial Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, P.O. Box 31485-498, Karaj (Iran, Islamic Republic of); Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of)], E-mail: parvinshawrang@yahoo.co.uk; Nikkhah, A.; Zare-Shahneh, A. [Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of); Sadeghi, A.A. [Department of Animal Science, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, P.O. Box 14515-4933, Tehran (Iran, Islamic Republic of); Raisali, G. [Radiation Applications Research School, Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, P.O. Box 11365-3486, Tehran (Iran, Islamic Republic of); Moradi-Shahrebabak, M. [Department of Animal Science, Faculty of Agriculture, Tehran University P.O. Box 4111, Karaj (Iran, Islamic Republic of)

    2008-07-15

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition.

  14. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    International Nuclear Information System (INIS)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A.A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-01-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition

  15. Fabrication of Flexible, Fully Organic, Degradable Energy Storage Devices Using Silk Proteins.

    Science.gov (United States)

    Pal, Ramendra K; Kundu, Subhas C; Yadavalli, Vamsi K

    2018-03-21

    Flexible and thin-film devices are of great interest in epidermal and implantable bioelectronics. The integration of energy storage and delivery devices such as supercapacitors (SCs) with properties such as flexibility, miniaturization, biocompatibility, and degradability are sought for such systems. Reducing e-waste and using sustainable materials and processes are additional desirable qualities. Herein, a silk protein-based biocompatible and degradable thin-film microSC (μSC) is reported. A protein carrier with the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and reduced graphene oxide dopant is used as a photopatternable biocomposite ink. Active electrodes are fabricated using photolithography under benign conditions, using only water as the solvent. These electrodes are printed on flexible protein sheets to form degradable, organic devices with a benign agarose-NaCl gel electrolyte. High capacitance, power density, cycling stability over 500 cycles, and the ability to power a light-emitting diode are shown. The device is flexible, can sustain cyclic mechanical stresses over 450 cycles, and retain capacitive properties over several days in liquid. Significantly, the μSCs are cytocompatible and completely degraded over the period of ∼1 month. By precise control of the device configuration, these silk protein-based, all-polymer organic devices can be designed to be tunably transient and provide viable alternatives for powering flexible and implantable bioelectronics.

  16. Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

    Directory of Open Access Journals (Sweden)

    Tsubasa Munakata

    2007-09-01

    Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

  17. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Halawani Dalia

    2007-10-01

    Full Text Available Abstract Background HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER-associated protein degradation (ERAD. Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub-proteasome degradative system through an interaction with β-TrCP, a component of the SCFβ-TrCP E3 Ub ligase complex. Results Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFβ-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. Conclusion Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFβ-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

  18. Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

    DEFF Research Database (Denmark)

    Gretzmeier, Christine; Eiselein, Sven; Johnson, Gregory R.

    2017-01-01

    , unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending...... on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways...

  19. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    International Nuclear Information System (INIS)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-01

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  20. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ronghai [Department of Urology, Linzi District People' s Hospital, Zibo, 255400 (China); Zhang, Ping, E-mail: zpskx001@163.com [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Li, Jinhang [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Guan, Hongzai [Laboratory Department, School of Medicine, Qingdao University, Qingdao, 266071 (China); Shi, Guangjun, E-mail: qdmhshigj@yahoo.com [Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao, 266071 (China)

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  1. Protein-protein interactions and cancer: targeting the central dogma.

    Science.gov (United States)

    Garner, Amanda L; Janda, Kim D

    2011-01-01

    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  2. Biochemistry of cellulose degradation and cellulose utilization for feeds and for protein

    Energy Technology Data Exchange (ETDEWEB)

    Sadara, J C; Lachke, A H; Shewale, J G

    1979-01-01

    A review discussing production of single-cell protein, fuel, and glucose from cellulose decomposition; surface or solid fermentations of single-cell protein; production of cellulases; and the biochemistry of cellulose degradation was presented.

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

    Science.gov (United States)

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

    1998-01-01

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

  4. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

    Science.gov (United States)

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-20

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  5. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2017-01-01

    Full Text Available Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  6. Protein Degradation in Normal and Beige (Chediak-Higashi) Mice

    Science.gov (United States)

    Lyons, Robert T.; Pitot, Henry C.

    1978-01-01

    The beige mouse, C57BL/6 (bg/bg), is an animal model for the Chediak-Higashi syndrome in man, a disease characterized morphologically by giant lysosomes in most cell types. Half-lives for the turnover of [14C]bicarbonate-labeled total soluble liver protein were determined in normal and beige mice. No significant differences were observed between the normal and mutant strain for both rapidly and slowly turning-over classes of proteins. Glucagon treatment during the time-course of protein degradation had similar effects on both normal and mutant strains and led to the conclusion that the rate of turnover of endogenous intracellular protein in the beige mouse liver does not differ from normal. The rates of uptake and degradation of an exogenous protein were determined in normal and beige mice by intravenously injecting 125I-bovine serum albumin and following, in peripheral blood, the loss with time of phosphotungstic acid-insoluble bovine serum albumin and the parallel appearance of phosphotungstic acid-soluble (degraded) material. No significant differences were observed between beige and normal mice in the uptake by liver lysosomes of 125I-bovine serum albumin (t½ = 3.9 and 2.8 h, respectively). However, it was found that lysosomes from livers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slower than normal (t½ = 6.8 and 3.1 h, respectively). This defect in beige mice could be corrected by chronic administration of carbamyl choline (t½ = 3.5 h), a cholinergic agonist which raises intracellular cyclic GMP levels. However, no significant differences between normal and beige mice were observed either in the ability of soluble extracts of liver and kidney to bind [3H]cyclic GMP in vitro or in the basal levels of cyclic AMP in both tissues. The relevance of these observations to the presumed biochemical defect underlying the Chediak-Higashi syndrome is discussed. PMID:202611

  7. Effect of Electron Beam Irradiation on Degradability Coefficients and Ruminalpostruminal Digestibility of Dry Matter and Crude Protein of some Plant Protein Sources

    Directory of Open Access Journals (Sweden)

    gasem tahan

    2016-06-01

    Full Text Available Effect of electron beam irradiation on degradability coefficients and ruminal- postruminal digestibility of dry matter and crude protein of soybean meal, canola meal and Lathyrus sativus seed, irradiated at doses of 50, 100 and 150 kGy was investigated. Ruminal degradability of dry matter and crude protein was determined by in situ method using two cannulated Holstein heifers. Ruminal- postruminal digestibility of dry matter and crude protein was determined by in situ (nylon bag-in vitro (daisy digestor techniques. Data analyzed using SAS software as randomized completely design and the treatment means were compared using Tukey test. The results indicated that irradiation had no effect on dry matter, ether extract and ash content of feeds. In soybean meal, washout fraction and potentially degradable fraction of dry matter and crude protein was higher and lower at dose of 150 kGy irradiation than other treatments, respectively, and degradation rate constant and ruminal effective degradability of dry matter and crude protein was lower at all doses of irradiation than untreated soybean meal. In canola meal, irradiation at doses of 50 and 100 kGy decreased washout fraction and increased potentially degradable fraction of crude protein compared with untreated canola meal. In Lathyrus sativus seed, only potentially degradable fraction of dry matter and crude protein was lower at dose of 150 kGy irradiation than untreated Lathyrus sativus seed. Ruminal digestibility of crude protein decreased in soybean meal at doses of 100 and 150 kGy irradiation and for canola meal at all doses of irradiation than untreated samples. Total tract digestibility of crude protein decreased in soybean meal at dose of 150 kGy irradiation and for canola meal at all doses of irradiation than untreated samples. In Lathyrus sativus seed, ruminal-postruminal digestibility and total tract digestibility of dry matter increased at doses of 100 and 150 kGy irradiation than untreated

  8. Effect of protein degradability on milk production of dairy ewes.

    Science.gov (United States)

    Mikolayunas-Sandrock, C; Armentano, L E; Thomas, D L; Berger, Y M

    2009-09-01

    The objective of this experiment was to determine the effect of protein degradability of dairy sheep diets on milk yield and protein utilization across 2 levels of milk production. Three diets were formulated to provide similar energy concentrations and varying concentrations of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP): 12% RDP and 4% RUP (12-4) included basal levels of RDP and RUP, 12% RDP and 6% RUP (12-6) included additional RUP, and 14% RDP and 4% RUP (14-4) included additional RDP. Diets were composed of alfalfa-timothy cubes, whole and ground corn, whole oats, dehulled soybean meal, and expeller soybean meal (SoyPlus, West Central, Ralston, IA). Estimates of RDP and RUP were based on the Small Ruminant Nutrition System model (2008) and feed and orts were analyzed for Cornell N fractions. Eighteen multiparous dairy ewes in midlactation were divided by milk yield (low and high) into 2 blocks of 9 ewes each and were randomly assigned within block (low and high) to 3 pens of 3 ewes each. Dietary treatments were arranged in a 3 x 3 Latin square within each block and applied to pens for 14-d periods. We hypothesized that pens consuming high-RUP diets (12-6) would produce more milk and milk protein than the basal diet (12-4) and pens consuming high-RDP diets (14-4) would not produce more milk than the basal diet (12-4). Ewes in the high-milk-yield square consumed more dry matter and produced more milk, milk fat, and milk protein than ewes in the low-milk-yield square. There was no effect of dietary treatment on dry matter intake. Across both levels of milk production, the 12-6 diet increased milk yield by 14%, increased milk fat yield by 14%, and increased milk protein yield by 13% compared with the 14-4 and 12-4 diets. Gross N efficiency (milk protein N/intake protein N) was 11 and 15% greater in the 12-6 and 12-4 diets, respectively, compared with the 14-4 diet. Milk urea N concentration was greater in the 12-6 diet and tended to be

  9. In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

    Science.gov (United States)

    Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

    2013-11-01

    Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

  10. Dosimetric effects of rotational output variation and x-ray target degradation on helical tomotherapy plans

    International Nuclear Information System (INIS)

    Staton, Robert J.; Langen, Katja M.; Kupelian, Patrick A.; Meeks, Sanford L.

    2009-01-01

    In this study, two potential sources of IMRT delivery error have been identified for helical tomotherapy delivery using the HiART system (TomoTherapy, Inc., Madison, WI): Rotational output variation and target degradation. The HiArt system is known to have output variation, typically about ±2%, due to the absence of a dose servo system. On the HiArt system, x-ray target replacement is required approximately every 10-12 months due to target degradation. Near the end of target life, the target thins and causes a decrease in the beam energy and a softening of the beam profile at the lateral edges of the beam. The purpose of this study is to evaluate the dosimetric effects of rotational output variation and target degradation by modeling their effects and incorporating them into recalculated treatment plans for three clinical scenarios: Head and neck, partial breast, and prostate. Models were created to emulate both potential sources of error. For output variation, a model was created using a sine function to match the amplitude (±2%), frequency, and phase of the measured rotational output variation data. A second model with a hypothetical variation of ±7% was also created to represent the largest variation that could exist without violating the allowable dose window in the delivery system. A measured beam profile near the end of target life was used to create a modified beam profile model for the target degradation. These models were then incorporated into the treatment plan by modifying the leaf opening times in the delivery sinogram. A new beam model was also created to mimic the change in beam energy seen near the end of target life. The plans were then calculated using a research version of the PLANNED ADAPTIVE treatment planning software from TomoTherapy, Inc. Three plans were evaluated in this study: Head and neck, partial breast, and prostate. The D 50 of organs at risk, the D 95 for planning target volumes (PTVs), and the local dose difference were used to

  11. Context Memory Formation Requires Activity-Dependent Protein Degradation in the Hippocampus

    Science.gov (United States)

    Cullen, Patrick K.; Ferrara, Nicole C.; Pullins, Shane E.; Helmstetter, Fred J.

    2017-01-01

    Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel…

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

    Directory of Open Access Journals (Sweden)

    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.

  13. The protein micro-crystallography beamlines for targeted protein research program

    International Nuclear Information System (INIS)

    Hirata, Kunio; Yamamoto, Masaki; Matsugaki, Naohiro; Wakatsuki, Soichi

    2010-01-01

    In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data collection system should be designed to provide high signal-to noise ratio in diffraction images. SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted Proteins Research Program by MEXT of Japan. The program aims to reveal the structure and function of proteins that are difficult to solve but have great importance in both academic research and industrial application. At SPring-8, a new 1-micron beam beamline for protein micro-crystallography, RIKEN Targeted Proteins Beamline (BL32XU), is developed. At KEK-PF a new low energy micro-beam beamline, BL-1A, is dedicated for SAD micro-crystallography. The two beamlines will start operation in the end of 2010. The present status of the research and development for protein micro-crystallography will be presented. (author)

  14. Degradation of Crude Protein in Groundnut Cake, Guinea Grass ...

    African Journals Online (AJOL)

    Three West African dwarf rams fitted with rumen cannula, were used in a completely randomized design for degradation of crude protein (CP) of groundnut cake (GNC), Panicum maximum, rumen epithelial scraping (RES), and diets containing increasing levels of RES. Concentrate diets were formulated such that 0% (A), ...

  15. Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

    Science.gov (United States)

    Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

    2017-02-01

    Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

  16. Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

    Science.gov (United States)

    McDowell, Gary S; Philpott, Anna

    2018-05-16

    The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

  17. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

    Science.gov (United States)

    Russo, F.; Johnson, C.J.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2009-01-01

    Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

  19. How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: the early history of ERAD.

    Science.gov (United States)

    Needham, Patrick G; Brodsky, Jeffrey L

    2013-11-01

    All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation. What was less clear was how secreted and integral membrane proteins, which in eukaryotes enter the endoplasmic reticulum (ER), were subject to quality control decisions. In this review, we highlight early studies that ultimately led to the discovery that secreted and integral membrane proteins also utilize several components that constitute the cytoplasmic quality control machinery. This component of the cellular quality control pathway is known as ER associated degradation, or ERAD. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Degradation of the disease-associated prion protein by a serine protease from lichens

    Science.gov (United States)

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J.C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.; Bartz, Jason C.

    2011-01-01

    The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  1. Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

    Science.gov (United States)

    Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

    2017-01-01

    Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

  2. A STRUCTURAL OVERVIEW OF GH61 PROTEINS – FUNGAL CELLULOSE DEGRADING POLYSACCHARIDE MONOOXYGENASES

    Directory of Open Access Journals (Sweden)

    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  3. A structural overview of GH61 proteins – fungal cellulose degrading polysaccharide monooxygenases

    Directory of Open Access Journals (Sweden)

    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  4. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    Science.gov (United States)

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

  5. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    International Nuclear Information System (INIS)

    Jessup, W.; Dean, R.T.

    1983-01-01

    Normal rates of pinocytosis of [ 3 H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  6. Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

    Science.gov (United States)

    Zhao, Hui; Mitsuiki, Shinji; Takasugi, Mikako; Sakai, Masashi; Goto, Masatoshi; Kanouchi, Hiroaki; Oka, Tatsuzo

    2012-04-01

    Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

  7. TRANGE: computer code to calculate the energy beam degradation in target stack

    International Nuclear Information System (INIS)

    Bellido, Luis F.

    1995-07-01

    A computer code to calculate the projectile energy degradation along a target stack was developed for an IBM or compatible personal microcomputer. A comparison of protons and deuterons bombarding uranium and aluminium targets was made. The results showed that the data obtained with TRANGE were in agreement with other computers code such as TRIM, EDP and also using Williamsom and Janni range and stopping power tables. TRANGE can be used for any charged particle ion, for energies between 1 to 100 MeV, in metal foils and solid compounds targets. (author). 8 refs., 2 tabs

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation

    International Nuclear Information System (INIS)

    Smith, C.B.; Deibler, G.E.; Eng, N.; Schmidt, K.; Sokoloff, L.

    1988-01-01

    A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1- 14 C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account

  10. HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

    DEFF Research Database (Denmark)

    Poulsen, Esben G; Steinhauer, Cornelia; Lees, Michael

    2012-01-01

    In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized...... in degradation of the UFD substrate Ub(G76V)-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD...... substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1). Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V)-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1...

  11. degradable protein sources on performance of high-producing dairy ...

    African Journals Online (AJOL)

    with high-quality, low·degradable protein sources prOViding47% UDP is advocated for ... saliva and through the rumen wall (Waldo, 1968). Based on this type of ... of the feed industry, but is based on very little solid evidence. (Huber, 1984). Chalupa ...... of rumen fermentation in relation to ammonia concentration. Br. J. Nutr.

  12. Protein interactions in genome maintenance as novel antibacterial targets.

    Directory of Open Access Journals (Sweden)

    Aimee H Marceau

    Full Text Available Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  13. Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

    Directory of Open Access Journals (Sweden)

    Tobias Fromme

    2018-02-01

    Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

  14. Protein degradation in preimplantation mouse embryos and the lethality of tritiated amino acids

    International Nuclear Information System (INIS)

    Wielbold, J.L.

    1982-01-01

    The role of protein degradation in preimplantation development in the mouse was studied. Proteins of morulae and blastocysts (M and B) cultured in vitro after labeling for 1 hour (h) in 3 H-leucine exhibit a mean half-life (t 1 / 2 ) of 8.1 h. The t 1 / 2 tends to increase (9.5 h) when 10% fetal calf serum is added to the chase medium. This decrease in protein degradation in the presence of serum is associated with an increase in the percentage of B that are hatching (P 3 H-leucine in their proteins than did Day 4 embryos remaining in culture (P<0.02), while Day 4 embryos in a Day 3 uterus retained the same amount of radioactivity as did Day 4 embryos in culture. This differential effect of uterine environment was also seen when Day 4 embryos were transferred to recipients. More fetuses developed to term when the recipient was in Day 3 of PSP (50.8%) than when the recipient was in Day 4 PSP (25.9%, P<0.001), regardless of the age of the recipient. Age of the recipient does affect the percentage of transferred embryos developing to term. Thus, protein degradation may vary with the stage of embryo development and the conditions to which the embryos are exposed. However, even low levels of incorporated tritiated leucine can have lethal effects on the embryos and compromise the validity of the protein half-lives determined

  15. Copper-mediated oxidative degradation of catecholamines and oxidative damage of protein

    Energy Technology Data Exchange (ETDEWEB)

    Goncalves, P.R.; Harria, M.I.N.; Felix, J.M.; Hoffmann, M.E. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Biologia

    1997-12-31

    Full text. Degradative oxidation of catecholamines has been a matter of large interest in recent years due to the evidences associating their autoxidation with the etiology of neurotoxic and cardiotoxic processes. In this work we present data on the degradative oxidation of catecholamines of physiological importance: isoproterenol (IP), epinephrine (EP), norepinephrine (NEP), deoxyepinephrine (DEP) and dopamine (DA). The degradative oxidation of the catecholamines was followed by measurement of spectral changes and oxygen consumption by neutral aqueous solutions. The data show that Cu{sup 2+} strongly accelerated the rate of catecholamine oxidation, following the decreasing order; EP>DEP>IP>NEP>DA. The production of superoxide anion radical during catecholamine oxidation was very slow, even in the presence of Cu{sup 2+}. The ability of IP to induce damages on bovine serum albumin (BSA) was determined by measuring the formation of carbonyl-groups in the protein, detected by reduction with tritiated Na BH{sub 4}. The incubation of BSA with IP (50-500{mu}M), in the presence of 100{mu}M Cu{sup 2+} leaded to an increased and dose dependent {sup 3} H-incorporation by the oxidized protein. The production of oxidative damage by IP/Cu{sup 2+} was accompanied by marked BSA fragmentation, detected by SDS-polyacrylamide gel dependent (25-400{mu}M IP) des appearance of the original BSA band and appearance of smaller fragments spread in the gel, when incubation has been done in the presence of 100{mu}M Cu{sup 2+}. These results suggest that copper-catalysed oxidative degradation of proteins induced by catecholamines might be critically involved in the toxic action of these molecules

  16. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

    Directory of Open Access Journals (Sweden)

    Yunwon Moon

    Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

  17. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor--1 alpha protein in hypoxic conditions.

    Science.gov (United States)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG-HIF-1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

  19. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

    Science.gov (United States)

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

    1998-01-01

    Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

  20. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    International Nuclear Information System (INIS)

    Brun, Emilie; Duchambon, Patricia; Blouquit, Yves; Keller, Gerard; Sanche, Leon; Sicard-Roselli, Cecile

    2009-01-01

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10 -4 and with the higher X-ray energy of 49 keV

  1. Cellular Chaperones As Therapeutic Targets in ALS to Restore Protein Homeostasis and Improve Cellular Function

    Directory of Open Access Journals (Sweden)

    Bernadett Kalmar

    2017-09-01

    Full Text Available Heat shock proteins (Hsps are ubiquitously expressed chaperone proteins that enable cells to cope with environmental stresses that cause misfolding and denaturation of proteins. With aging this protein quality control machinery becomes less effective, reducing the ability of cells to cope with damaging environmental stresses and disease-causing mutations. In neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS, such mutations are known to result in protein misfolding, which in turn results in the formation of intracellular aggregates cellular dysfunction and eventual neuronal death. The exact cellular pathology of ALS and other neurodegenerative diseases has been elusive and thus, hindering the development of effective therapies. However, a common scheme has emerged across these “protein misfolding” disorders, in that the mechanism of disease involves one or more aspects of proteostasis; from DNA transcription, RNA translation, to protein folding, transport and degradation via proteosomal and autophagic pathways. Interestingly, members of the Hsp family are involved in each of these steps facilitating normal protein folding, regulating the rate of protein synthesis and degradation. In this short review we summarize the evidence that suggests that ALS is a disease of protein dyshomeostasis in which Hsps may play a key role. Overwhelming evidence now indicates that enabling protein homeostasis to cope with disease-causing mutations might be a successful therapeutic strategy in ALS, as well as other neurodegenerative diseases. Novel small molecule co-inducers of Hsps appear to be able to achieve this aim. Arimoclomol, a hydroxylamine derivative, has shown promising results in cellular and animal models of ALS, as well as other protein misfolding diseases such as Inclusion Body Myositis (IBM. Initial clinical investigations of Arimoclomol have shown promising results. Therefore, it is possible that the long series of

  2. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

    Science.gov (United States)

    Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

    2007-01-01

    Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

  3. Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

    Science.gov (United States)

    Fernie, Alisdair R.; Stitt, Mark

    2015-01-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

  4. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    OpenAIRE

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit t...

  5. Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications.

    Science.gov (United States)

    Kobayashi, Hiroki; Harada, Hiroko; Nakamura, Masaomi; Futamura, Yushi; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-Ichiro; Shin-Ya, Kazuo; Doi, Takayuki; Takahashi, Takashi; Natsume, Tohru; Imoto, Masaya; Sakakibara, Yasubumi

    2012-04-05

    Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

  6. Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications

    Directory of Open Access Journals (Sweden)

    Kobayashi Hiroki

    2012-04-01

    Full Text Available Abstract Background Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. Results We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system. As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. Conclusions This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

  7. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  8. Effects of Supplementation of Branched-Chain Amino Acids to Reduced-Protein Diet on Skeletal Muscle Protein Synthesis and Degradation in the Fed and Fasted States in a Piglet Model

    Directory of Open Access Journals (Sweden)

    Liufeng Zheng

    2016-12-01

    Full Text Available Supplementation of branched-chain amino acids (BCAA has been demonstrated to promote skeletal muscle mass gain, but the mechanisms underlying this observation are still unknown. Since the regulation of muscle mass depends on a dynamic equilibrium (fasted losses–fed gains in protein turnover, the aim of this study was to investigate the effects of BCAA supplementation on muscle protein synthesis and degradation in fed/fasted states and the related mechanisms. Fourteen 26- (Experiment 1 and 28-day-old (Experiment 2 piglets were fed reduced-protein diets without or with supplemental BCAA. After a four-week acclimation period, skeletal muscle mass and components of anabolic and catabolic signaling in muscle samples after overnight fasting were determined in Experiment 1. Pigs in Experiment 2 were implanted with carotid arterial, jugular venous, femoral arterial and venous catheters, and fed once hourly along with the intravenous infusion of NaH13CO3 for 2 h, followed by a 6-h infusion of [1-13C]leucine. Muscle leucine kinetics were measured using arteriovenous difference technique. The mass of most muscles was increased by BCAA supplementation. During feeding, BCAA supplementation increased leucine uptake, protein synthesis, protein degradation and net transamination. The greater increase in protein synthesis than in protein degradation resulted in elevated protein deposition. Protein synthesis was strongly and positively correlated with the intramuscular net production of α-ketoisocaproate (KIC and protein degradation. Moreover, BCAA supplementation enhanced the fasted-state phosphorylation of protein translation initiation factors and inhibited the protein-degradation signaling of ubiquitin-proteasome and autophagy-lysosome systems. In conclusion, supplementation of BCAA to reduced-protein diet increases fed-state protein synthesis and inhibits fasted-state protein degradation, both of which could contribute to the elevation of skeletal muscle

  9. Application of green fluorescent protein for monitoring phenol-degrading strains

    Directory of Open Access Journals (Sweden)

    Ana Milena Valderrama F.

    2001-07-01

    Full Text Available Several methods have been developed for detecting microorganisms in environmental samples. Some systems for incorporating reporter genes, such as lux or the green fluorescent protein (GFP gene, have been developed recently This study describes gfp gene marking of a phenol degrading strain, its evaluation and monitoring in a bioreactor containing refinery sour water. Tagged strains were obtained having the same physiological and metabolic characteristics as the parent strain. Fluorescent expression was kept stable with no selection for more than 50 consecutive generations and tagged strains were recovered from the bioreactor after forty-five days of phenol-degradation treatment.

  10. 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.

  11. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    Directory of Open Access Journals (Sweden)

    Ping Zhou

    Full Text Available Dietary protein levels and cysteamine (CS supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP levels (14% or 10% and CS supplemental levels (0 or 700 mg/kg. The low-protein (LP diets (10% CP were supplemented with enough essential amino acids (EAA to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS (P<0.01 and plasma urea nitrogen (PUN (P<0.001, while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001 and lean percentage (P<0.05, and decreased the feed conversion ratio (P<0.05 and back fat (P<0.05. CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1 (P<0.001, and reduced the concentrations of leptin, SS, and PUN (P<0.001. Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001 and decreased mRNA abundance of Forkhead Box O (FOXO 4 (P<0.01 and muscle atrophy F-box (P<0.001 were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target of

  12. Filling and mining the reactive metabolite target protein database.

    Science.gov (United States)

    Hanzlik, Robert P; Fang, Jianwen; Koen, Yakov M

    2009-04-15

    The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible (http://tpdb.medchem.ku.edu:8080/protein_database/) resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of "-omics" studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites.

  13. PDTD: a web-accessible protein database for drug target identification

    Directory of Open Access Journals (Sweden)

    Gao Zhenting

    2008-02-01

    Full Text Available Abstract Background Target identification is important for modern drug discovery. With the advances in the development of molecular docking, potential binding proteins may be discovered by docking a small molecule to a repository of proteins with three-dimensional (3D structures. To complete this task, a reverse docking program and a drug target database with 3D structures are necessary. To this end, we have developed a web server tool, TarFisDock (Target Fishing Docking http://www.dddc.ac.cn/tarfisdock, which has been used widely by others. Recently, we have constructed a protein target database, Potential Drug Target Database (PDTD, and have integrated PDTD with TarFisDock. This combination aims to assist target identification and validation. Description PDTD is a web-accessible protein database for in silico target identification. It currently contains >1100 protein entries with 3D structures presented in the Protein Data Bank. The data are extracted from the literatures and several online databases such as TTD, DrugBank and Thomson Pharma. The database covers diverse information of >830 known or potential drug targets, including protein and active sites structures in both PDB and mol2 formats, related diseases, biological functions as well as associated regulating (signaling pathways. Each target is categorized by both nosology and biochemical function. PDTD supports keyword search function, such as PDB ID, target name, and disease name. Data set generated by PDTD can be viewed with the plug-in of molecular visualization tools and also can be downloaded freely. Remarkably, PDTD is specially designed for target identification. In conjunction with TarFisDock, PDTD can be used to identify binding proteins for small molecules. The results can be downloaded in the form of mol2 file with the binding pose of the probe compound and a list of potential binding targets according to their ranking scores. Conclusion PDTD serves as a comprehensive and

  14. In situ ruminal degradability of protein feeds with distinct physical ...

    African Journals Online (AJOL)

    Journal Home > Vol 47, No 1 (2017) > ... of protein feeds with different physical forms (meal versus grain) through a meta-analysis study. ... grains with the meal forms of soybean, peanut, sunflower, cottonseed, and corn gluten. The degradation parameters of dry matter did not differ significantly between meals and grains.

  15. Establishment of a ruminal protein degradation data base for dairy ...

    African Journals Online (AJOL)

    Establishment of a ruminal protein degradation data base for dairy cattle using the in situ polyester bag technique. 2. Energy sources. LJ Erasmus, J Prinsloo, PM Botha, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  16. Establishment of a ruminal protein degradation data base for dairy ...

    African Journals Online (AJOL)

    Establishment of a ruminal protein degradation data base for dairy cattle using the in situ polyester bag technique. 3. Roughages. LJ Erasmus, J Prinsloo, PM Botha, HH Meissner. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  17. Targeting IAP proteins in combination with radiotherapy

    International Nuclear Information System (INIS)

    Fulda, Simone

    2015-01-01

    The efficacy of radiotherapy critically depends on the activation of intrinsic cell death programs in cancer cells. This implies that evasion of cell death, a hallmark of human cancers, can contribute to radioresistance. Therefore, novel strategies to reactivate cell death programs in cancer cells are required in order to overcome resistance to radiotherapy. Since Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in multiple cancers and block cell death induction at a central point, therapeutic targeting of IAP proteins represents a promising approach to potentiate the efficacy of radiotherapy. The current review discusses the concept of targeting IAP proteins in combination with radiotherapy

  18. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  19. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2015-08-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  20. Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs.

    Directory of Open Access Journals (Sweden)

    Athanasia Stathopoulou

    Full Text Available BACKGROUND: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. METHODOLOGY/PRINCIPAL FINDING: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. CONCLUSION/SIGNIFICANCE: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

  1. Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

    Science.gov (United States)

    Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

    2018-01-01

    This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

  2. Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation.

    Science.gov (United States)

    Stylli, Stanley S; Stacey, T T I; Verhagen, Anne M; Xu, San San; Pass, Ian; Courtneidge, Sara A; Lock, Peter

    2009-08-01

    Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.

  3. Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

    Directory of Open Access Journals (Sweden)

    Hanzlik Robert P

    2009-06-01

    Full Text Available Abstract Background Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity. Results Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein. Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine content and protein instability index (i.e., rate of turnover in vivo were among the features most important to determining susceptibility. Conclusion As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.

  4. Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Brun, Emilie [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France); Duchambon, Patricia; Blouquit, Yves [INSERM U759, Imagerie Integrative, Campus Universitaire d' Orsay, Bat. 112, Institut Curie, Centre de Recherche, Laboratoire R. Latarjet, Campus Universitaire d' Orsay, 91405 Orsay Cedex (France); Keller, Gerard [UMR CNRS 8612, Physico-Chimie-Pharmacotechnie-Biopharmacie, Universite Paris 11, Faculte de Pharmacie, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry (France); Sanche, Leon [Groupe en Sciences des Radiations, Departement de Medecine Nucleaire et Radiobiologie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 (Canada); Sicard-Roselli, Cecile [Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud 11, Bat. 350, 91405 Orsay Cedex (France)], E-mail: cecile.sicard@u-psud.fr

    2009-03-15

    In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2x10{sup -4} and with the higher X-ray energy of 49 keV.

  5. Arsenic trioxide (AT) is a novel human neutrophil pro-apoptotic agent: effects of catalase on AT-induced apoptosis, degradation of cytoskeletal proteins and de novo protein synthesis.

    Science.gov (United States)

    Binet, François; Cavalli, Hélène; Moisan, Eliane; Girard, Denis

    2006-02-01

    The anti-cancer drug arsenic trioxide (AT) induces apoptosis in a variety of transformed or proliferating cells. However, little is known regarding its ability to induce apoptosis in terminally differentiated cells, such as neutrophils. Because neutropenia has been reported in some cancer patients after AT treatment, we hypothesised that AT could induce neutrophil apoptosis, an issue that has never been investigated. Herein, we found that AT-induced neutrophil apoptosis and gelsolin degradation via caspases. AT did not increase neutrophil superoxide production and did not induce mitochondrial generation of reactive oxygen species. AT-induced apoptosis in PLB-985 and X-linked chronic granulomatous disease (CGD) cells (PLB-985 cells deficient in gp91(phox) mimicking CGD) at the same potency. Addition of catalase, an inhibitor of H2O2, reversed AT-induced apoptosis and degradation of the cytoskeletal proteins gelsolin, alpha-tubulin and lamin B1. Unexpectedly, AT-induced de novo protein synthesis, which was reversed by catalase. Cycloheximide partially reversed AT-induced apoptosis. We conclude that AT induces neutrophil apoptosis by a caspase-dependent mechanism and via de novo protein synthesis. H2O2 is of major importance in AT-induced neutrophil apoptosis but its production does not originate from nicotinamide adenine dinucleotide phosphate dehydrogenase activation and mitochondria. Cytoskeletal structures other than microtubules can now be considered as novel targets of AT.

  6. Rate and extent of ruminal degradation of crude protein from ...

    African Journals Online (AJOL)

    Predicted crude protein degradation was calculated at rate constants for outflow of 0.04 and 0.06/h respect- ively. ... as buffers, an ionophore and an antibiotic according to general .... the non-bird resistant ('sweet') varieties. Ruminal .... have been affected by both the particle type and the math- ematical model we used.

  7. Preparation, radioiodination and in vitro evaluation of a nido-carborane-dextran conjugate, a potential residualizing label for tumor targeting proteins and peptides

    International Nuclear Information System (INIS)

    Tolmachev, V.; Bruskin, A.; Uppsala University; Sjoeberg, S.; Carlsson, J.; Lundqvist, H.

    2004-01-01

    Polysaccharides are not degradable by proteolytic enzymes in lysosomes and do not diffuse through cellular membranes. Thus, attached to an internalizing, targeting protein, such polysaccharide linkers, will remain intracellularly after protein degradation. They can be labeled with halogens and provide then a so called residualizing label. Such an approach improves tumor-to-non-tumor radioactivity ratio and, consequently, the results of radionuclide diagnostics and therapy. A new approach to obtain a stable halogenation of the polysaccharide dextran using 7-(3-amino-propyl)-7,8-dicarba-nido-undecaborate (-) (ANC) is presented. Dextran T10 was partially oxidized by metaperiodate, and ANC was coupled to dextran by reductive amination. The conjugate was then labeled with 125 I using either Chloramine-T or IodoGen as oxidants. Labeling efficiency was 69-85%. Stability of the label was evaluated in rat liver homogenates. Under these conditions, the ANC-dextran conjugate was found to be more stable than labeled albumin, which was used as a control protein. (author)

  8. Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

    Science.gov (United States)

    Ito, Mikako; Ohno, Kinji

    2018-02-20

    Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

  9. 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…

  10. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  11. Effect of Rapid Chilling on Beef Quality and Cytoskeletal Protein Degradation in of Chinese Yellow Crossbred Bulls

    Directory of Open Access Journals (Sweden)

    Yanwei Mao

    2012-08-01

    Full Text Available The objective of this study was to investigate the effect of rapid chilling (RC on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM. The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness.

  12. HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

    Directory of Open Access Journals (Sweden)

    Zhaobo Li

    2017-06-01

    Full Text Available Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

  13. Collagen targeting using multivalent protein-functionalized dendrimers

    NARCIS (Netherlands)

    Breurken, M.; Lempens, E.H.M.; Temming, R.P.; Helms, B.A.; Meijer, E.W.; Merkx, M.

    2011-01-01

    Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic

  14. Proteins interacting with the 26S proteasome

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Gordon, C

    2004-01-01

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

  15. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    Science.gov (United States)

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  16. Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway.

    Science.gov (United States)

    Dissmeyer, Nico; Rivas, Susana; Graciet, Emmanuelle

    2018-05-01

    Contents Summary 929 I. conservation and diversity of N-end rule pathways 929 II. Defensive functions of the N-end rule pathway in plants 930 III. Proteases and degradation by the N-end rule pathway 930 IV. New proteomics approaches for the identification of N-end rule substrates 932 V. Concluding remarks 932 Acknowledgements 934 References 934 SUMMARY: The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  17. Neuroprotective mechanism of Kai Xin San: upregulation of hippocampal insulin-degrading enzyme protein expression and acceleration of amyloid-beta degradation

    Directory of Open Access Journals (Sweden)

    Na Wang

    2017-01-01

    Full Text Available Kai Xin San is a Chinese herbal formula composed of Radix Ginseng , Poria , Radix Polygalae and Acorus Tatarinowii Rhizome . It has been used in China for many years for treating amnesia. Kai Xin San ameliorates amyloid-β (Aβ-induced cognitive dysfunction and is neuroprotective in vivo , but its precise mechanism remains unclear. Expression of insulin-degrading enzyme (IDE, which degrades Aβ, is strongly correlated with cognitive function. Here, we injected rats with exogenous Aβ42 (200 μM, 5 μL into the hippocampus and subsequently administered Kai Xin San (0.54 or 1.08 g/kg/d intragastrically for 21 consecutive days. Hematoxylin-eosin and Nissl staining revealed that Kai Xin San protected neurons against Aβ-induced damage. Furthermore, enzyme-linked immunosorbent assay, western blot and polymerase chain reaction results showed that Kai Xin San decreased Aβ42 protein levels and increased expression of IDE protein, but not mRNA, in the hippocampus. Our findings reveal that Kai Xin San facilitates hippocampal Aβ degradation and increases IDE expression, which leads, at least in part, to the alleviation of hippocampal neuron injury in rats.

  18. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  19. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    Science.gov (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  20. Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

    Science.gov (United States)

    White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

    2014-08-01

    The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

  1. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

    Science.gov (United States)

    Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

    2014-11-24

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

  2. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    Directory of Open Access Journals (Sweden)

    Jaume Torres

    2015-06-01

    Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  3. Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

    Science.gov (United States)

    An, Chuanjing; Gao, Yuefang; Li, Jinyu; Liu, Xiaomin; Gao, Fuli; Gao, Hongbo

    2017-07-01

    A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

  4. Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides.

    Science.gov (United States)

    Lohse, Jonas; Swier, Lotteke J Y M; Oudshoorn, Ruben C; Médard, Guillaume; Kuster, Bernhard; Slotboom, Dirk-Jan; Witte, Martin D

    2017-04-19

    In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.

  5. Mast cell chymase degrades the alarmins heat shock protein 70, biglycan, HMGB1, and interleukin-33 (IL-33) and limits danger-induced inflammation.

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-03

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W(sash))-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.

  6. Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan, HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation*

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M.; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-01

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(Wsash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation. PMID:24257755

  7. Effect of conventional and extrusion pelleting on in situ ruminal degradability of starch, protein, and fibre in cattle

    DEFF Research Database (Denmark)

    Razzaghi, Ali; Larsen, Mogens; Lund, Peter

    2016-01-01

    +50% sugar beet pulp (SBP), or 50% maize+50% SBP. Meals were pelleted by either conventional pelleting, or by cooking extrusion using two distinct settings giving pellets with either high density (HD) or low density (LD). Ruminal degradation of starch, crude protein (CP) and NDF, and intestinal...... affected ruminal degradability of starch, protein, and NDF differently depending on both type of cereal and composition of the concentrate mixture.......>Pelleting>Meal). In contradiction, ESD for pure wheat and wheat mixtures was reduced, though differences were minor. Conventional pelleting reduced the effective protein degradability (EPD) for pure wheat, but extrusion did not further affect the EPD. In contrast, the most intense processing with extrusion LD increased EPD...

  8. In vitro estimation of rumen protein degradability using 35S to label the bacterial mass

    International Nuclear Information System (INIS)

    Khristov, A.; Aleksandrov, S.; Aleksiev, I.

    1994-01-01

    An experiment was carried out in order to simplify a previously developed 15 N-method for in vitro estimation of rumen protein degradability. Casein (Cas), whole soybeans (Sb) heated at 120 o C for 20 min (SbTherm) and sunflower (Sfl) were incubated at 39 o C for 4 hours in a water bathshaker with the following media: McDougall's buffer, strained and enriched with particle associated bacteria rumen fluid (2:1), rapidly (maltose, sucrose, glucose) and more slowly (pectin, soluble starch) degradable carbohydrates with final concentration of 815 mg/100 ml and 21.7 μCi/100 ml of 35 S (from Na 2 35 SO 4 ). After the incubation had been ceased, a bacterial fraction was isolated through differential centrifugation and specific activity of bacterial (Bac) and high speed total solids (TS) nitrogen was measured. The ratio was used to calculate bacterial mass in TS and through the Kjeldahl nitrogen concentration in TS - the net bacterial growth (against control vessels without protein). The level of ammonia-N in the supernate after blank correction was used to find the ammonia-N released from protein degradation. The data showed that the rate (and extend) of degradation for the Cas (as a standard protein) was lower compared to those obtained through the 15 N-method but it was higher than the rate derived through another in vitro method. The Cas equivalent of the Sb was higher than the figure we found in a previous experiment with solvent extracted soybean meal suggesting that the 35 S-method underestimated the degradability of the Cas. After being tested on a wider range of foodstuffs, the proposed 35 S-method might be considered as an alternative procedure which is less laborous than the 15 N-method. (author)

  9. NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

    Science.gov (United States)

    Rapino, F; Abhari, B A; Jung, M; Fulda, S

    2015-03-12

    Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

  10. Degradation and de novo synthesis of D1 protein and psbA ...

    Indian Academy of Sciences (India)

    This shows that synthesis of D1 protein is not the only component involved in the recovery process. Our events, which ... transcript levels in the green alga Chlamydomonas reinhardtii in ..... and Gaba V 1996 Accelerated degradation of the D2 ...

  11. Plant pathology: monitoring a pathogen-targeted host protein.

    Science.gov (United States)

    Ellis, Jeff; Dodds, Peter

    2003-05-13

    A plant protein RIN4 is targeted and modified by bacterial pathogens as part of the disease process. At least two host resistance proteins monitor this pathogen interference and trigger the plant's defence responses.

  12. Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks

    Science.gov (United States)

    2012-09-21

    Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks Paulo Shakarian1*, J. Kenneth Wickiser2 1 Paulo Shakarian...significantly attacked. Citation: Shakarian P, Wickiser JK (2012) Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks...to 00-00-2012 4. TITLE AND SUBTITLE Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks 5a. CONTRACT NUMBER 5b

  13. WSB1 overcomes oncogene-induced senescence by targeting ATM for degradation

    Science.gov (United States)

    Kim, Jung Jin; Lee, Seung Baek; Yi, Sang-Yeop; Han, Sang-Ah; Kim, Sun-Hyun; Lee, Jong-Min; Tong, Seo-Yun; Yin, Ping; Gao, Bowen; Zhang, Jun; Lou, Zhenkun

    2017-01-01

    Oncogene-induced senescence (OIS) or apoptosis through the DNA-damage response is an important barrier of tumorigenesis. Overcoming this barrier leads to abnormal cell proliferation, genomic instability, and cellular transformation, and finally allows cancers to develop. However, it remains unclear how the OIS barrier is overcome. Here, we show that the E3 ubiquitin ligase WD repeat and SOCS box-containing protein 1 (WSB1) plays a role in overcoming OIS. WSB1 expression in primary cells helps the bypass of OIS, leading to abnormal proliferation and cellular transformation. Mechanistically, WSB1 promotes ATM ubiquitination, resulting in ATM degradation and the escape from OIS. Furthermore, we identify CDKs as the upstream kinase of WSB1. CDK-mediated phosphorylation activates WSB1 by promoting its monomerization. In human cancer tissue and in vitro models, WSB1-induced ATM degradation is an early event during tumorigenic progression. We suggest that WSB1 is one of the key players of early oncogenic events through ATM degradation and destruction of the tumorigenesis barrier. Our work establishes an important mechanism of cancer development and progression in premalignant lesions. PMID:27958289

  14. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    LENUS (Irish Health Repository)

    Toomey, David

    2009-01-01

    BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins\\/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and\\/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY\\/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS\\/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under \\'change-of-application\\' patents.

  15. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    Directory of Open Access Journals (Sweden)

    David Toomey

    Full Text Available BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i homologous to previously crystallized proteins or (ii targets of known drugs, but are (iii not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under 'change-of-application' patents.

  16. The reactive metabolite target protein database (TPDB)--a web-accessible resource.

    Science.gov (United States)

    Hanzlik, Robert P; Koen, Yakov M; Theertham, Bhargav; Dong, Yinghua; Fang, Jianwen

    2007-03-16

    The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins. The Reactive Metabolite Target Protein Database (TPDB) is a comprehensive, curated, searchable, documented compilation of publicly available information on the protein targets of reactive metabolites of 18 well-studied chemicals and drugs of known toxicity. TPDB software enables i) string searches for author names and proteins names/synonyms, ii) more complex searches by selecting chemical compound, animal species, target tissue and protein names/synonyms from pull-down menus, and iii) commonality searches over multiple chemicals. Tabulated search results provide information, references and links to other databases. The TPDB is a unique on-line compilation of information on the covalent modification of cellular proteins by reactive metabolites of chemicals and drugs. Its comprehensiveness and searchability should facilitate the elucidation of mechanisms of reactive metabolite toxicity. The database is freely available at http://tpdb.medchem.ku.edu/tpdb.html.

  17. Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions

    International Nuclear Information System (INIS)

    Greenberg, B.M.; Gaba, V.; Canaani, O.; Malkin, S.; Mattoo, A.K.; Edelman, M.

    1989-01-01

    A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance

  18. A Role of Protein Degradation in Memory Consolidation after Initial Learning and Extinction Learning in the Honeybee ("Apis mellifera")

    Science.gov (United States)

    Felsenberg, Johannes; Dombrowski, Vincent; Eisenhardt, Dorothea

    2012-01-01

    Protein degradation is known to affect memory formation after extinction learning. We demonstrate here that an inhibitor of protein degradation, MG132, interferes with memory formation after extinction learning in a classical appetitive conditioning paradigm. In addition, we find an enhancement of memory formation when the same inhibitor is…

  19. Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein.

    Directory of Open Access Journals (Sweden)

    Guangzhi Zhang

    Full Text Available The Gag protein of the mouse mammary tumor virus (MMTV is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV, assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

  20. Protein and Peptide in Drug Targeting and its Therapeutic Approach

    Directory of Open Access Journals (Sweden)

    Raj K. Keservani

    2015-09-01

    Full Text Available Aim: The main aim of this review article is to provide information like advantages of protein and peptides via different routes of drug administration, targeted to a particular site and its implication in drug delivery system. Methods: To that aim, from the web sites of PubMed, HCAplus, Thomson, and Registry were used as the main sources to perform the search for the most significant research articles published on the subject. The information was then carefully analyzed, highlighting the most important results in the development of protein and peptide drug targeting as well as its therapeutic activity. Results: In recent years many researchers use protein and peptide as a target site of drug by a different delivery system. Proteins and peptides are used as specific and effective therapeutic agents, due to instability and side effects their use is complicated. Protein kinases are important regulators of most, if not all, biological processes. Abnormal activity of proteins and peptides has been implicated in many human diseases, such as diabetes, cancer and neurodegenerative disorders. Conclusions: It is concluded that the protein and peptide were used in drug targeting to specific site and also used in different diseased states like cancer, diabetes, immunomodulating, neurodegenerative effects and antimicrobial activity.

  1. The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves[OPEN

    Science.gov (United States)

    Feike, Doreen; Seung, David; Graf, Alexander; Bischof, Sylvain; Ellick, Tamaryn; Coiro, Mario; Soyk, Sebastian; Eicke, Simona; Mettler-Altmann, Tabea; Lu, Kuan Jen; Trick, Martin; Zeeman, Samuel C.

    2016-01-01

    To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. PMID:27207856

  2. An assessment of differences in the ruminal degradability and intestinal digestibility of crude protein in brewer’s grains and maize draff

    Directory of Open Access Journals (Sweden)

    Vladimír Majer

    2012-01-01

    Full Text Available The submitted thesis aims to assess the differences between the ruminal degradability and intestinal digestibility of crude protein contained in brewer’s grains (BG and maize draff (AMG. The effectiveness of ruminal degradability was tested using the “in sacco” method on 3 dry Holstain cows fitted with rumen cannulas. The dynamics of ruminal degradability of crude protein (CP was detected after 0, 4, 8, 16, and 24 hours of samples incubation in the rumen. The intestinal digestibility of crude protein undegradable in the rumen was determined using the “mobile bag” method on 3 dry Holstain cows fitted with duodenal cannulas. The crude protein degradability of BG was detected in the above-mentioned hours (%: 4.06; 18.16; 32.40; 38.56, and 50.70; crude protein degradability of AMG: 42.04; 63.56; 84.47; 85.16, and 87.19. The effectiveness of rumen degradability of BG crude protein at the rate of passage of rumen content 6 % per hour was calculated at 35.33 % and that of AMG, at 76.29 %. Intestinal digestibility of BG crude protein and dry matter at the rate of passage of intestinal content 6 % per hour was calculated at 79.41 % and 22.84 %, respectively, and that of AMG, at 57.01 % and 11.33 %, respectively. The differences between the indicators of both feedstuffs were significant (P < 0.05. The results show that BG are mostly a source of crude protein with higher intestinal digestibility than AMG.

  3. Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

    Directory of Open Access Journals (Sweden)

    Dickson Dennis W

    2009-10-01

    Full Text Available Abstract Background Insulin-degrading enzyme (IDE is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs targeting natively folded human and rodent IDE. Results Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

  4. Rumen Degradability and Post-ruminal Digestion of Dry Matter, Nitrogen and Amino Acids of Three Protein Supplements.

    Science.gov (United States)

    Gao, Wei; Chen, Aodong; Zhang, Bowen; Kong, Ping; Liu, Chenli; Zhao, Jie

    2015-04-01

    This study evaluated the in situ ruminal degradability, and subsequent small intestinal digestibility (SID) of dry matter, crude protein (CP), and amino acids (AA) of cottonseed meal (CSM), sunflower seed meal (SFSM) and distillers dried grains with solubles (DDGS) by using the modified three-step in vitro procedure. The ruminal degradability and subsequent SID of AA in rumen-undegradable protein (RUP-AA) varied among three protein supplements. The result show that the effective degradability of DM for SFSM, CSM, and DDGS was 60.8%, 56.4%, and 41.0% and their ruminal fermentable organic matter was 60.0%, 55.9%, and 39.9%, respectively. The ruminal degradable protein (RDP) content in CP for SFSM, CSM, and DDGS was 68.3%, 39.0%, and 32.9%, respectively, at the ruminal solid passage rate of 1.84%/h. The SFSM is a good source of RDP for rumen micro-organisms; however, the SID of RUP of SFSM was lower. The DDGS and CSM are good sources of RUP for lambs to digest in the small intestine to complement ruminal microbial AA of growing lambs. Individual RUP-AA from each protein source was selectively removed by the rumen micro-organisms, especially for Trp, Arg, His, and Lys (p<0.01). The SID of individual RUP-AA was different within specific RUP origin (p<0.01). Limiting amino acid was Leu for RUP of CSM and Lys for both RUP of SFSM and DDGS, respectively. Therefore, different protein supplements with specific limitations should be selected and combined carefully in growing lambs ration to optimize AA balance.

  5. Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

    Science.gov (United States)

    Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

    2018-01-15

    The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

  6. Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Anowara Khatun

    2018-04-01

    Full Text Available The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1 depolarization responses induced by paxilline (PAX, a specific KCa1.1 blocker and (2 PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in

  7. Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

    Directory of Open Access Journals (Sweden)

    Ruchi Bansal

    collagen and α-SMA expression and potentiating collagen degradation. IFNγ also induced reduction in fibrogenesis but showed significant decrease in platelet counts, which was restored with targeted proteins. These results suggest that these rationally designed proteins can be further developed as novel anti-fibrotic therapeutics.

  8. Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

    Directory of Open Access Journals (Sweden)

    Eduardo Cebollero

    2012-01-01

    Full Text Available During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

  9. Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy

    International Nuclear Information System (INIS)

    Safa, Ahmad R.; Pollok, Karen E.

    2011-01-01

    Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIP L ), short (c-FLIP S ), and c-FLIP R splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIP L and c-FLIP S are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIP L in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIP L and c-FLIP S splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function

  10. Wnt Signaling Translocates Lys48-Linked Polyubiquitinated Proteins to the Lysosomal Pathway

    Directory of Open Access Journals (Sweden)

    Hyunjoon Kim

    2015-05-01

    Full Text Available Cellular proteins are degraded in either proteasomes or lysosomes depending on the types of ubiquitin chains that covalently modify them. It is not known whether the choice between these two pathways is physiologically regulated. The Lys48-polyubiquitin chain is the major signal directing proteins for degradation in proteasomes. Here, we report the unexpected finding that canonical Wnt signaling translocates some K48-linked polyubiquitinated proteins to the endolysosomal pathway. Proteasomal target proteins, such as β-catenin, Smad1, and Smad4, were targeted into endolysosomes in a process dependent on GSK3 activity. Relocalization was also dependent on Axin1 and the multivesicular body (MVB proteins HRS/Vps27 and Vps4. The Wnt-induced accumulation of K48-linked polyubiquitinated proteins in endolysosomal organelles was accompanied by a transient decrease in cellular levels of free mono-ubiquitin, which may contribute to Wnt-regulated stabilization of proteins (Wnt/STOP. We conclude that Wnt redirects Lys48-polyubiquitinated proteins that are normally degraded in proteasomes to endolysosomes.

  11. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    Directory of Open Access Journals (Sweden)

    Kessler Felix

    2007-01-01

    Full Text Available Abstract Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34 as the carrier. Similar to adipocyte differentiation related protein (ADRP in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.

  12. Targeting protein biotinylation enhances tuberculosis chemotherapy.

    Science.gov (United States)

    Tiwari, Divya; Park, Sae Woong; Essawy, Maram M; Dawadi, Surendra; Mason, Alan; Nandakumar, Madhumitha; Zimmerman, Matthew; Mina, Marizel; Ho, Hsin Pin; Engelhart, Curtis A; Ioerger, Thomas; Sacchettini, James C; Rhee, Kyu; Ehrt, Sabine; Aldrich, Courtney C; Dartois, Véronique; Schnappinger, Dirk

    2018-04-25

    Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual drugs and the regimens to which they contribute. Biologically and chemically validated targets capable of replacing individual components of current TB chemotherapy are a major unmet need in TB drug development. We demonstrate that chemical inhibition of the bacterial biotin protein ligase (BPL) with the inhibitor Bio-AMS (5'-[ N -(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine) killed Mycobacterium tuberculosis ( Mtb ), the bacterial pathogen causing TB. We also show that genetic silencing of BPL eliminated the pathogen efficiently from mice during acute and chronic infection with Mtb Partial chemical inactivation of BPL increased the potency of two first-line drugs, rifampicin and ethambutol, and genetic interference with protein biotinylation accelerated clearance of Mtb from mouse lungs and spleens by rifampicin. These studies validate BPL as a potential drug target that could serve as an alternate frontline target in the development of new drugs against Mtb . Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  13. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    Science.gov (United States)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  14. Degradation kinetics of fisetin and quercetin in solutions affected by medium pH, temperature and co-existed proteins

    Directory of Open Access Journals (Sweden)

    Wang Jing

    2016-01-01

    Full Text Available Impacts of medium pH, temperature and coexisted proteins on the degradation of two flavonoids fisetin and quercetin were assessed by spectroscopic method in the present study. Based on the measured degradation rate constants (k, fisetin was more stable than quercetin in all cases. Increasing medium pH from 6.0 to 7.5 at 37°C enhanced respective k values of fisetin and quercetin from 8.30x10−3 and 2.81x10−2 to 0.202 and 0.375 h-1 (P<0.05. In comparison with their degradation at 37°C, fisetin and quercetin showed larger k values at higher temperature (0.124 and 0.245 h−1 at 50°C, or 0.490 and 1.42 h−1 at 65°C. Four protein products in medium could stabilize the two flavonoids (P<0.05, as these proteins at 0.10 g L-1 decreased respective k values of fisetin and quercetin to 2.28x10−2-2.98x10−2 and 4.37´10−2-5.97x10−2 h−1. Hydrophobic interaction between the proteins and the two flavonoids was evidenced responsible for the stabilization, as sodium dodecyl sulfate could destroy the stabilization significantly (P<0.05. Casein and soybean protein provided greater stabilization than whey protein isolate. It is thus concluded that higher temperature and alkaline pH can enhance flavonoid loss, whereas coexisted proteins as flavonoid stabilizers can inhibit flavonoid degradation.

  15. Generation of Oxygen Free Radicals by Proflavine: Implication in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Mansour K.M. Gatasheh

    2017-07-01

    Full Text Available Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS. These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II is included in the reaction. Bathocuproine, a specific Cu (I sequestering agent, when present in the reaction mixture containing Cu (II, was found to inhibit the protein degradation, showing that Cu (I is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins.

  16. Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

    Science.gov (United States)

    Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

    2014-01-01

    We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

  17. Identifiability study of the proteins degradation model, based on ADM1, using simultaneous batch experiments

    DEFF Research Database (Denmark)

    Flotats, X.; Palatsi, J.; Ahring, Birgitte Kiær

    2006-01-01

    are not inhibiting the hydrolysis process. The ADM1 model adequately expressed the consecutive steps of hydrolysis and acidogenesis, with estimated kinetic values corresponding to a fast acidogenesis and slower hydrolysis. The hydrolysis was found to be the rate limiting step of anaerobic degradation. Estimation...... of yield coefficients based on the relative initial slopes of VFA profiles obtained in a simple batch experiment produced satisfactory results. From the identification study, it was concluded that it is possible to determine univocally the related kinetic parameter values for protein degradation...... if the evolution of amino acids is measured in simultaneous batch experiments, with different initial protein and amino acids concentrations....

  18. Rumen Degradability and Post-ruminal Digestion of Dry Matter, Nitrogen and Amino Acids of Three Protein Supplements

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2015-04-01

    Full Text Available This study evaluated the in situ ruminal degradability, and subsequent small intestinal digestibility (SID of dry matter, crude protein (CP, and amino acids (AA of cottonseed meal (CSM, sunflower seed meal (SFSM and distillers dried grains with solubles (DDGS by using the modified three-step in vitro procedure. The ruminal degradability and subsequent SID of AA in rumen-undegradable protein (RUP-AA varied among three protein supplements. The result show that the effective degradability of DM for SFSM, CSM, and DDGS was 60.8%, 56.4%, and 41.0% and their ruminal fermentable organic matter was 60.0%, 55.9%, and 39.9%, respectively. The ruminal degradable protein (RDP content in CP for SFSM, CSM, and DDGS was 68.3%, 39.0%, and 32.9%, respectively, at the ruminal solid passage rate of 1.84%/h. The SFSM is a good source of RDP for rumen micro-organisms; however, the SID of RUP of SFSM was lower. The DDGS and CSM are good sources of RUP for lambs to digest in the small intestine to complement ruminal microbial AA of growing lambs. Individual RUP-AA from each protein source was selectively removed by the rumen micro-organisms, especially for Trp, Arg, His, and Lys (p<0.01. The SID of individual RUP-AA was different within specific RUP origin (p<0.01. Limiting amino acid was Leu for RUP of CSM and Lys for both RUP of SFSM and DDGS, respectively. Therefore, different protein supplements with specific limitations should be selected and combined carefully in growing lambs ration to optimize AA balance.

  19. 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.

  20. GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

    Science.gov (United States)

    Negahdar, Maria; Aukrust, Ingvild; Johansson, Bente B; Molnes, Janne; Molven, Anders; Matschinsky, Franz M; Søvik, Oddmund; Kulkarni, Rohit N; Flatmark, Torgeir; Njølstad, Pål Rasmus; Bjørkhaug, Lise

    2012-11-01

    GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Protease-activatable collagen targeting based on protein cyclization

    NARCIS (Netherlands)

    Breurken, M.; Lempens, E.H.M.; Merkx, M.

    2010-01-01

    Threading collagen through a protein needle: The collagen-binding protein CNA35 operates by wrapping itself around the collagen triple helix. By connecting the N and C termini through an MMP recognition sequence, a dual-specific MMP-sensitive collagen-targeting ligand is obtained that can be used

  2. Chromatin-regulating proteins as targets for cancer therapy

    International Nuclear Information System (INIS)

    Oike, Takahiro; Ogiwara, Hideaki; Kohno, Takashi; Amornwichet, Napapat; Nakano, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. (author)

  3. 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

  4. Amyloid-degrading ability of nattokinase from Bacillus subtilis natto.

    Science.gov (United States)

    Hsu, Ruei-Lin; Lee, Kung-Ta; Wang, Jung-Hao; Lee, Lily Y-L; Chen, Rita P-Y

    2009-01-28

    More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

  5. The use of a tannin crude extract from Cistus ladanifer L. to protect soya-bean protein from degradation in the rumen.

    Science.gov (United States)

    Dentinho, M T P; Moreira, O C; Pereira, M S; Bessa, R J B

    2007-06-01

    Cistus ladanifer L. (CL) is a perennial shrub abundant in dry woods and dry land of Mediterranean zone, with high level of tannins. Tannins bind to protein, preventing its degradation in the digestive compartments. This tannin/protein complex may be advantageous when partially protecting good-quality feed protein from excessive rumen protein degradation. The objective of this trial was to use a CL phenol crude extract to prevent excessive rumen degradation of soya-bean meal protein. The phenolic compounds were extracted using an acetone/water solution (70:30, v/v). Soya-bean meal was then treated with this crude CL extract, containing 640 g of total phenols (TP) per kg of dry matter (DM), in order to obtain mixtures with 0, 12.5, 25, 50, 100 and 150 g of TP per kg DM. Three rumen-cannulated rams were used to assess in sacco rumen degradability of DM and nitrogen (N). The three-step in vitro procedure was used to determine intestinal digestibility. Increasing extract concentrations quadratically decreased the N-soluble fraction a (R2 = 0.96, P = 0.0001) and increased the non-soluble degradable fraction b (R2 = 0.92, P = 0.005). The rate of degradation c linearly decreased with CL extract doses (R2 = 0.44, P = 0.0065). For the effective rumen degradability of N, a linear reduction (R2 = 0.94, P < 0.0001) was observed. The in vitro intestinal digestibility of protein (ivID) quadratically decreased (R2 = 0.99, P < 0.0001) with TP inclusion and the rumen undegradable protein (RUP) showed a quadratic increase (R2 = 0.94, P = 0.0417). Total intestinal protein availability, computed from the RUP and ivID, linearly decreased with TP inclusion level (R2 = 0.45, P = 0.0033).

  6. Ruminal degradation and intestinal digestibility of protein and amino acids in high-protein feedstuffs commonly used in dairy diets.

    Science.gov (United States)

    Paz, H A; Klopfenstein, T J; Hostetler, D; Fernando, S C; Castillo-Lopez, E; Kononoff, P J

    2014-10-01

    A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio 1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Fungal lectin MpL enables entry of protein drugs into cancer cells and their subcellular targeting.

    Science.gov (United States)

    Å Urga, Simon; Nanut, Milica Perišić; Kos, Janko; Sabotič, Jerica

    2017-04-18

    Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin α3β1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initially in the Golgi apparatus and, finally, in the lysosomes. For effective binding and internalization a functional binding site on the α-repeat is needed. To test the potential of MpL as a carrier for delivering protein drugs to cancer cells we constructed fusion proteins consisting of MpL and the cysteine peptidase inhibitors cystatin C and clitocypin. The fused proteins followed the same endocytic route as the unlinked MpL. Peptidase inhibitor-MpL fusions impaired both the intracellular degradation of extracellular matrix and the invasiveness of cancer cells. MpL is thus shown in vitro to be a lectin that can enable protein drugs to enter cancer cells, enhance their internalization and sort them to lysosomes and the Golgi apparatus.

  8. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Targeted proteomics as a tool for porcine acute phase proteins measurements

    DEFF Research Database (Denmark)

    Marco-Ramell, Anna; Bassols, Anna; Bislev, Stine Lønnerup

    2013-01-01

    . Selected reaction monitoring (SRM), a targeted quantitative proteomic technique, may be used as an alternative to commercial kits for the measurement and validation of target proteins. Acute phase proteins (APPs) are widely recognized inflammation and infection biomarkers (Eckersall, 2010...

  10. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  11. Comparison of protein degradation, protein oxidation, and μ-calpain activation between pale, soft, and exudative and red, firm, and nonexudative pork during postmortem aging.

    Science.gov (United States)

    Yin, Y; Zhang, W G; Zhou, G H; Guo, B

    2014-08-01

    The objective of this study was to investigate the differences in protein modifications between pale, soft, and exudative (PSE) and red, firm, and nonexudative (RFN) pork during postmortem (PM) aging. Longissimus dorsi (LD) including 8 PSE and 8 RFN muscles were individually removed from 16 carcasses. These 16 LD muscles were vacuum packaged at 24 h after slaughter and stored at 4°C for 1, 3, and 5 d. The centrifugation loss, drip loss, color, protein solubility, protein oxidation, protein degradation including desmin, troponin T, and integrin, and μ-calpain activation were determined. The pH of PSE samples was significantly lower than that of RFN samples at both 1 and 24 h PM (P 0.05). In addition, PSE pork presented a lower solubility of sarcoplasmic protein, myofibrillar protein, and total protein than RFN pork except the solubility of myofibrillar protein at d 1 (P firm, and nonexudative pork presented lower intensity of intact 80 kDa calpain and greater intensity of autolyzed 76 kDa product compared to PSE pork (P < 0.01). The results indicate that the degree of μ-calpain activation, the extent of protein degradation including desmin and integrin, and the level of protein solubility in PSE pork could contribute to its low water holding capacity during PM storage.

  12. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  13. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

    Science.gov (United States)

    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs. © 2016 Bersuker et al.

  14. Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins.

    Science.gov (United States)

    Maher, Michael P; Matta, Jose A; Gu, Shenyan; Seierstad, Mark; Bredt, David S

    2017-12-06

    Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

    Science.gov (United States)

    Hu, Zhenyu; Li, Guang; Wang, Jiong-Wei; Chong, Suet Yen; Yu, Dejie; Wang, Xiaoyuan; Soon, Jia Lin; Liang, Mui Cheng; Wong, Yuk Peng; Huang, Na; Colecraft, Henry M; Liao, Ping; Soong, Tuck Wah

    2018-04-12

    Background -L-type Ca V 1.2 channels play crucial roles in regulation of blood pressure. Galectin-1 (Gal-1), has been reported to bind to the I-II loop of Ca V 1.2 channels to reduce their current density. However, the mechanistic understanding for the down-regulation of Ca V 1.2 channels by Gal-1, and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. Methods - In vitro experiments involving co-IP, western blot, patch-clamp recordings, immunohistochemistry and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 down-regulates Ca V 1.2 channel in transfected HEK 293 cells, smooth muscle cells, arteries from Lgasl1 -/- mice, rat and human patients. In vivo experiments involving delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting Ca V 1.2-Gal-1 interaction on blood pressure monitored by tail cuff or telemetry methods. Results -Our study reveals that Gal-1 is a key regulator for proteasomal degradation of Ca V 1.2 channels. Gal-1 competed allosterically with Ca V β subunit for binding to the I-II loop of Ca V 1.2 channel. This competitive disruption of Ca V β binding led to Ca V 1.2 degradation by exposing the channels to poly-ubiquitination. Notably, we demonstrated that the inverse relationship of reduced Gal-1 and increased Ca V 1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice due to up-regulated Ca V 1.2 protein level in arteries. To directly regulate blood pressure by targeting the Ca V 1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1, by a mini-osmotic pump and this specific disruption of Ca V 1.2-Gal-1 coupling increased smooth muscle Ca V 1.2 currents, induced larger arterial contraction and caused hypertension in rats. In contrasting experiments, over-expression of Gal-1 in smooth muscle by a

  16. The reactive metabolite target protein database (TPDB – a web-accessible resource

    Directory of Open Access Journals (Sweden)

    Dong Yinghua

    2007-03-01

    Full Text Available Abstract Background The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins. Description The Reactive Metabolite Target Protein Database (TPDB is a comprehensive, curated, searchable, documented compilation of publicly available information on the protein targets of reactive metabolites of 18 well-studied chemicals and drugs of known toxicity. TPDB software enables i string searches for author names and proteins names/synonyms, ii more complex searches by selecting chemical compound, animal species, target tissue and protein names/synonyms from pull-down menus, and iii commonality searches over multiple chemicals. Tabulated search results provide information, references and links to other databases. Conclusion The TPDB is a unique on-line compilation of information on the covalent modification of cellular proteins by reactive metabolites of chemicals and drugs. Its comprehensiveness and searchability should facilitate the elucidation of mechanisms of reactive metabolite toxicity. The database is freely available at http://tpdb.medchem.ku.edu/tpdb.html

  17. Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

    Science.gov (United States)

    Ponomarenko, Natalia A; Durova, Oxana M; Vorobiev, Ivan I; Belogurov, Alexey A; Kurkova, Inna N; Petrenko, Alexander G; Telegin, Georgy B; Suchkov, Sergey V; Kiselev, Sergey L; Lagarkova, Maria A; Govorun, Vadim M; Serebryakova, Marina V; Avalle, Bérangère; Tornatore, Pete; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

  18. Orally active-targeted drug delivery systems for proteins and peptides.

    Science.gov (United States)

    Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

    2014-09-01

    In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

  19. FBXW7 regulates glucocorticoid response in T-cell acute lymphoblastic leukaemia by targeting the glucocorticoid receptor for degradation.

    Science.gov (United States)

    Malyukova, A; Brown, S; Papa, R; O'Brien, R; Giles, J; Trahair, T N; Dalla Pozza, L; Sutton, R; Liu, T; Haber, M; Norris, M D; Lock, R B; Sangfelt, O; Marshall, G M

    2013-04-01

    Loss of function mutation in FBXW7, an E3 ubiquitin ligase, is associated with good prognosis and early glucocorticoid treatment response in childhood T-cell acute lymphoblastic leukemia (T-ALL) by unknown mechanisms. Here, we show that FBXW7 targets the glucocorticoid receptor α (GRα) for ubiquitylation and proteasomal degradation in a manner dependent on glycogen synthase kinase 3 β-mediated phsophorylation. FBXW7 inactivation caused elevated GRα levels, and enhanced the transcriptional response to glucocorticoids. There was significant enhancement of GR transcriptional responses in FBXW7-deficient cell lines and primary T-ALL samples, in particular, for those pro-apoptotic regulatory proteins, BIM and PUMA. Reduced FBXW7 expression or function promoted glucocorticoid sensitivity, but not sensitivity to other chemotherapeutic agents used in T-ALL. Moreover, this was a general feature of different cancer cell types. Taken together, our work defines GRα as a novel FBXW7 substrate and demonstrates that favorable patient prognosis in T-ALL is associated with FBXW7 mutations due to enhanced GRα levels and steroid sensitivity. These findings suggest that inactivation of FBXW7, a putative tumor suppressor protein, may create a synthetic lethal state in the presence of specific anticancer therapies.

  20. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  1. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  2. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  3. Pu-Erh Tea Extract Induces the Degradation of FET Family Proteins Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Yang Yu

    2014-01-01

    Full Text Available FET family proteins consist of fused in sarcoma/translocated in liposarcoma (FUS/TLS, Ewing's sarcoma (EWS, and TATA-binding protein-associated factor 15 (TAF15. Mutations in the copper/zinc superoxide dismutase (SOD1, TAR DNA-binding protein 43 (TDP-43, and FET family proteins are associated with the development of amyotrophic lateral sclerosis (ALS, a fatal neurodegenerative disease. There is currently no cure for this disease and few effective treatments are available. Epidemiological studies indicate that the consumption of tea is associated with a reduced risk of developing neurodegenerative diseases. The results of this study revealed that components of a pu-erh tea extract (PTE interacted with FET family proteins but not with TDP-43 or SOD1. PTE induced the degradation of FET family proteins but had no effects on TDP-43 or SOD1. The most frequently occurring ALS-linked FUS/TLS mutant protein, R521C FUS/TLS, was also degraded in the presence of PTE. Furthermore, ammonium chloride, a lysosome inhibitor, but not lactacystin, a proteasome inhibitor, reduced the degradation of FUS/TLS protein by PTE. PTE significantly reduced the incorporation of R521C FUS/TLS into stress granules under stress conditions. These findings suggest that PTE may have beneficial health effects, including preventing the onset of FET family protein-associated neurodegenerative diseases and delaying the progression of ALS by inhibiting the cytoplasmic aggregation of FET family proteins.

  4. Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

    Science.gov (United States)

    Hinrichsen, Inga; Weßbecher, Isabel M; Huhn, Meik; Passmann, Sandra; Zeuzem, Stefan; Plotz, Guido; Biondi, Ricardo M; Brieger, Angela

    2017-12-01

    MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1 L749P and MLH1 Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα. © 2017 Wiley Periodicals, Inc.

  5. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Finnie, Christine

    2006-01-01

    Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure...... of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays...... a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares...

  7. Similar pathogen targets in Arabidopsis thaliana and homo sapiens protein networks.

    Directory of Open Access Journals (Sweden)

    Paulo Shakarian

    Full Text Available We study the behavior of pathogens on host protein networks for humans and Arabidopsis - noting striking similarities. Specifically, we preform [Formula: see text]-shell decomposition analysis on these networks - which groups the proteins into various "shells" based on network structure. We observe that shells with a higher average degree are more highly targeted (with a power-law relationship and that highly targeted nodes lie in shells closer to the inner-core of the network. Additionally, we also note that the inner core of the network is significantly under-targeted. We show that these core proteins may have a role in intra-cellular communication and hypothesize that they are less attacked to ensure survival of the host. This may explain why certain high-degree proteins are not significantly attacked.

  8. The drug-minded protein interaction database (DrumPID) for efficient target analysis and drug development.

    Science.gov (United States)

    Kunz, Meik; Liang, Chunguang; Nilla, Santosh; Cecil, Alexander; Dandekar, Thomas

    2016-01-01

    The drug-minded protein interaction database (DrumPID) has been designed to provide fast, tailored information on drugs and their protein networks including indications, protein targets and side-targets. Starting queries include compound, target and protein interactions and organism-specific protein families. Furthermore, drug name, chemical structures and their SMILES notation, affected proteins (potential drug targets), organisms as well as diseases can be queried including various combinations and refinement of searches. Drugs and protein interactions are analyzed in detail with reference to protein structures and catalytic domains, related compound structures as well as potential targets in other organisms. DrumPID considers drug functionality, compound similarity, target structure, interactome analysis and organismic range for a compound, useful for drug development, predicting drug side-effects and structure-activity relationships.Database URL:http://drumpid.bioapps.biozentrum.uni-wuerzburg.de. © The Author(s) 2016. Published by Oxford University Press.

  9. Protection of a protein against irradiation-induced degradation by additives in the solid state

    International Nuclear Information System (INIS)

    Shalaev, E.; Reddy, R.; Kimball, R.N.; Weinschenk, M.F.; Guinn, M.; Margulis, L.

    2003-01-01

    The impact of ionizing radiation on a globular protein (porcine somatotropin, pST) in the solid state was studied using rate of dissolution, high-performance liquid chromatography, and Electron spin resonance (ESR) in the presence of different additives. o-Vanillin stabilized pST against irradiation-induced degradation whereas effects of trolox and isopropyl alcohol were less significant. Stabilization effect of o-vanillin has been related to the energy transfer from pST molecules to the additive which was facilitated by formation of covalent bonds between o-vanillin and pST molecules. Anticorrelation between the level of free radicals and chemical degradation (i.e. degradation increased with decrease in a free radical level) was observed in the presence of o-vanillin

  10. 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

  11. Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

    Science.gov (United States)

    Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

    2015-01-01

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

  12. Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

    Science.gov (United States)

    Medraño-Fernandez, Iria; Fagioli, Claudio; Mezghrani, Alexandre; Otsu, Mieko; Sitia, Roberto

    2014-04-01

    To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

  13. Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

    International Nuclear Information System (INIS)

    Montrose, Kristopher; Krissansen, Geoffrey W.

    2014-01-01

    Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC

  14. Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

    Energy Technology Data Exchange (ETDEWEB)

    Montrose, Kristopher; Krissansen, Geoffrey W., E-mail: gw.krissansen@auckland.ac.nz

    2014-10-31

    Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC.

  15. Differential expression in Phanerochaete chrysosporium of membrane- associated proteins relevant to lignin degradation

    Science.gov (United States)

    Semarjit Shary; Alexander N. Kapich; Ellen A. Panisko; Jon K. Magnuson; Daniel Cullen; Kenneth E. Hammel

    2008-01-01

    Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew...

  16. Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

    Science.gov (United States)

    Rampello, Anthony J; Glynn, Steven E

    2017-03-24

    The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  18. Downregulation of chloroplast-targeted beta-amylase leads to a starch-excess phenotype in leaves

    DEFF Research Database (Denmark)

    Scheidig, A.; Fröhlich, A.; Schulze, S.

    2002-01-01

    showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch......A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast-targeted 5-amylase of Arabidopsis. Expression of the gene in E. coli...

  19. Targeting low-density lipoprotein receptors with protein-only nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhikun [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Céspedes, María Virtudes [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Unzueta, Ugutz [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Álamo, Patricia [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Pesarrodona, Mireia [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Mangues, Ramón [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Vázquez, Esther; Villaverde, Antonio, E-mail: antoni.villaverde@uab.cat; Ferrer-Miralles, Neus, E-mail: neus.ferrer@uab.cat [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain)

    2015-03-15

    Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood–brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases.

  20. Targeting low-density lipoprotein receptors with protein-only nanoparticles

    International Nuclear Information System (INIS)

    Xu, Zhikun; Céspedes, María Virtudes; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2015-01-01

    Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood–brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases

  1. Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

    Directory of Open Access Journals (Sweden)

    Anbarasu Kumarasamy

    2008-09-01

    Full Text Available Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH and betaL-crystallin as target proteins, was increased in alphaA(1-172 and decreased in alphaA(1-168 and alphaA(1-162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k for ADH and alphaA(1-172 was nearly the same as that of ADH and alphaA-wt, alphaA(1-168 had lower and alphaA(1-162 had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172 had slightly higher k value than alphaA-wt and alphaA(1-168 and alphaA(1-162 had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172 was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168 was similar to that of alphaA-wt and alphaA(1-162 had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

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

    Science.gov (United States)

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

    2011-06-03

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

  3. The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins.

    Directory of Open Access Journals (Sweden)

    Louise Madsen

    Full Text Available The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.

  4. Massively parallel de novo protein design for targeted therapeutics

    KAUST Repository

    Chevalier, Aaron

    2017-09-26

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

  5. Massively parallel de novo protein design for targeted therapeutics

    KAUST Repository

    Chevalier, Aaron; Silva, Daniel-Adriano; Rocklin, Gabriel J.; Hicks, Derrick R.; Vergara, Renan; Murapa, Patience; Bernard, Steffen M.; Zhang, Lu; Lam, Kwok-Ho; Yao, Guorui; Bahl, Christopher D.; Miyashita, Shin-Ichiro; Goreshnik, Inna; Fuller, James T.; Koday, Merika T.; Jenkins, Cody M.; Colvin, Tom; Carter, Lauren; Bohn, Alan; Bryan, Cassie M.; Ferná ndez-Velasco, D. Alejandro; Stewart, Lance; Dong, Min; Huang, Xuhui; Jin, Rongsheng; Wilson, Ian A.; Fuller, Deborah H.; Baker, David

    2017-01-01

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

  6. Massively parallel de novo protein design for targeted therapeutics

    Science.gov (United States)

    Chevalier, Aaron; Silva, Daniel-Adriano; Rocklin, Gabriel J.; Hicks, Derrick R.; Vergara, Renan; Murapa, Patience; Bernard, Steffen M.; Zhang, Lu; Lam, Kwok-Ho; Yao, Guorui; Bahl, Christopher D.; Miyashita, Shin-Ichiro; Goreshnik, Inna; Fuller, James T.; Koday, Merika T.; Jenkins, Cody M.; Colvin, Tom; Carter, Lauren; Bohn, Alan; Bryan, Cassie M.; Fernández-Velasco, D. Alejandro; Stewart, Lance; Dong, Min; Huang, Xuhui; Jin, Rongsheng; Wilson, Ian A.; Fuller, Deborah H.; Baker, David

    2018-01-01

    De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing. PMID:28953867

  7. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows

    DEFF Research Database (Denmark)

    Tayyab, Usama; Novoa-Garrido, Margarita; Roleda, Michael Y.

    2016-01-01

    The use of seaweeds in animal diets is not new. However, little is known about the feed value of seaweed, both in terms of chemical composition and protein digestibility, and regarding variation between species and season. In this study, eight seaweed species of the genus Acrosiphonia, Alaria......, Laminaria, Mastocarpus, Palmaria, Pelvetia, Porphyra, and Ulva were sampled in spring (March) and autumn (October and November) 2014 at the coast of Bodø in Northern Norway, and were analysed for chemical composition, in situ rumen degradability and total tract crude protein (CP) digestibility. Ash content...... for Pelvetia (90 g/kg DM). Spring samples were higher in CP than autumn samples. The effective degradability estimated at 5% rumen passage rate (ED5) of CP varied between species (P Ulva (240 g...

  8. Protection of protein from ruminal degradation by alkali-induced oxidation of chlorogenic acid in sunflower meal.

    Science.gov (United States)

    Bongartz, V; Böttger, C; Wilhelmy, N; Schulze-Kaysers, N; Südekum, K-H; Schieber, A

    2018-02-01

    Lactating ruminants require an adequate supply of absorbable amino acids for the synthesis of milk protein from two sources, that is crude protein (CP) synthesized microbially in the rumen and ruminally undegraded CP (RUP) from feed which can both be digested in the small intestine. Several chemical and physical methods have been identified as being effective in increasing the proportion of RUP of total CP of a feedstuff, yet there is a continuing need for developing and establishing methods which protect feed protein from ruminal degradation with acceptable expenditure of labour and other costs. The objective of this study was to identify and quantify effects of and interactions between chlorogenic acid and protein in solvent-extracted sunflower meal (SFM) as induced by alkali treatment. Response surface methodology was employed to investigate the influence of pH, reaction time and drying temperature on the resulting SFM and, subsequently, its protein value for ruminants estimated from laboratory values. For this purpose, alkali-treated SFM was subjected to a fractionation of feed CP according to the Cornell net carbohydrate and protein system as a basis for estimating RUP at different assumed ruminal passage rates (K p ). To estimate the intestinal digestibility of the treated SFM and its RUP, a three-step enzymatic in vitro procedure was applied. Alkaline treatment of SFM increased RUP values with factors ranging from approximately 3 (K p =.08/hr) to 12 (K p =.02/hr). Furthermore, the intestinal digestibility of the alkali-treated SFM was enhanced by approximately 10% compared to untreated SFM. Increasing pH and reaction time led to both increasing RUP values and intestinal digestibility. In conclusion, a targeted alkaline treatment of naturally occurring compounds in feedstuffs might be a promising approach to provide high-RUP feeds for ruminants which, at the same time, have improved intestinal digestibility values. © 2017 Blackwell Verlag GmbH.

  9. Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

    Science.gov (United States)

    2015-11-01

    Preparation of the Lysosomes A673 cells were treated with 100 pM chloroquine for 12 h or left untreated. Lysosomes were prepared using the Lysosome...were treated with 100 JlM chloroquine fur 12 h or left tmtreated, and the luciferase activity was determined using the same arnotmt of protein...TFEB levels or by activating TFEB using mTORC1 kinase inhibitor, torin 1. Additionally, we determined that the same approach can be used to target

  10. TALE-PvuII fusion proteins--novel tools for gene targeting.

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  11. Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules.

    Science.gov (United States)

    Chapman, Daniel C; Stocki, Pawel; Williams, David B

    2015-01-01

    Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

  12. Organic matter degradation in Chilean sediments - following nature's own degradation experiment

    DEFF Research Database (Denmark)

    Langerhuus, Alice Thoft; Niggemann, Jutta; Lomstein, Bente Aagaard

    ORGANIC MATTER DEGRADATION IN CHILEAN SEDIMENTS – FOLLOWING NATURE’S OWN DEGRADATION EXPERIMENT Degradation of sedimentary organic matter was studied at two stations from the shelf of the Chilean upwelling region. Sediment cores were taken at 1200 m and 800 m water depth and were 4.5 m and 7.5 m...... in length, respectively. The objective of this study was to assess the degradability of the organic matter from the sediment surface to the deep sediments. This was done by analysing amino acids (both L- and D-isomers) and amino sugars in the sediment cores, covering a timescale of 15.000 years. Diagenetic...... indicators (percentage of carbon and nitrogen present as amino acid carbon and nitrogen, the ratio between a protein precursor and its non-protein degradation product and the percentage of D-amino acids) revealed ongoing degradation in these sediments, indicating that microorganisms were still active in 15...

  13. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    Science.gov (United States)

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Failure of protein quality control in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Durham, Heather D

    2006-01-01

    The protein chaperoning and ubiquitin-proteasome systems perform many homeostatic functions within cells involving protein folding, transport and degradation. Of paramount importance is ridding cells of mutant or post-translationally modified proteins that otherwise tend to aggregate into insoluble complexes and form inclusions. Such inclusions are characteristic of many neurodegenerative diseases and implicate protein misfolding and aggregation as common aspects of pathogenesis. In the most common familial form of ALS, mutations in SOD1 promote misfolding of the protein and target it for degradation by proteasomes. Although proteasomes can degrade the mutant proteins efficiently, altered solubility and aggregation of mutant SOD1 are features of the disease and occur most prominently in the most vulnerable cells and tissues. Indeed, lumbar spinal cord of mutant SOD1 transgenic mice show early reduction in their capacity for protein chaperoning and proteasome-mediated hydrolysis of substrates, and motor neurons are particularly vulnerable to aggregation of mutant SOD1. A high threshold for upregulating key pathways in response to the stress of added substrate load may contribute to this vulnerability. The broad spectrum neuroprotective capability and efficacy of some chaperone-based therapies in preclinical models makes these pathways attractive as targets for therapy in ALS, as well as other neurodegenerative diseases. A better understanding of the mechanisms governing the regulation of protein chaperones and UPS components would facilitate development of treatments that upregulate these pathways in a coordinated manner in neural tissue without long term toxicity.

  15. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes

    Science.gov (United States)

    Majumdar, Amitabha; Capetillo-Zarate, Estibaliz; Cruz, Dana; Gouras, Gunnar K.; Maxfield, Frederick R.

    2011-01-01

    Incomplete lysosomal acidification in microglia inhibits the degradation of fibrillar forms of Alzheimer's amyloid β peptide (fAβ). Here we show that in primary microglia a chloride transporter, ClC-7, is not delivered efficiently to lysosomes, causing incomplete lysosomal acidification. ClC-7 protein is synthesized by microglia but it is mistargeted and appears to be degraded by an endoplasmic reticulum–associated degradation pathway. Activation of microglia with macrophage colony-stimulating factor induces trafficking of ClC-7 to lysosomes, leading to lysosomal acidification and increased fAβ degradation. ClC-7 associates with another protein, Ostm1, which plays an important role in its correct lysosomal targeting. Expression of both ClC-7 and Ostm1 is increased in activated microglia, which can account for the increased delivery of ClC-7 to lysosomes. Our findings suggest a novel mechanism of lysosomal pH regulation in activated microglia that is required for fAβ degradation. PMID:21441306

  17. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Tacchi, Jessica L; Raymond, Benjamin B A; Haynes, Paul A; Berry, Iain J; Widjaja, Michael; Bogema, Daniel R; Woolley, Lauren K; Jenkins, Cheryl; Minion, F Chris; Padula, Matthew P; Djordjevic, Steven P

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. © 2016 The Authors.

  18. Targeted amino-terminal acetylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Matthew Johnson

    2010-12-01

    Full Text Available One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

  19. Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation.

    Science.gov (United States)

    Tomassen, Monic M M; Barrett, Diane M; van der Valk, Henry C P M; Woltering, Ernst J

    2007-01-01

    An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.

  20. Postmortem muscle protein degradation in humans as a tool for PMI delimitation.

    Science.gov (United States)

    Pittner, Stefan; Ehrenfellner, Bianca; Monticelli, Fabio C; Zissler, Angela; Sänger, Alexandra M; Stoiber, Walter; Steinbacher, Peter

    2016-11-01

    Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

  1. The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

    Science.gov (United States)

    Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

    2007-09-18

    Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

  2. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    Directory of Open Access Journals (Sweden)

    Jérôme eLallemand

    2015-02-01

    Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  3. A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

    Science.gov (United States)

    Pickering, Andrew. M.; Davies, Kelvin. J. A.

    2014-01-01

    Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

  4. Reverse screening methods to search for the protein targets of chemopreventive compounds

    Science.gov (United States)

    Huang, Hongbin; Zhang, Guigui; Zhou, Yuquan; Lin, Chenru; Chen, Suling; Lin, Yutong; Mai, Shangkang; Huang, Zunnan

    2018-05-01

    This article is a systematic review of reverse screening methods used to search for the protein targets of chemopreventive compounds or drugs. Typical chemopreventive compounds include components of traditional Chinese medicine, natural compounds and Food and Drug Administration (FDA)-approved drugs. Such compounds are somewhat selective but are predisposed to bind multiple protein targets distributed throughout diverse signaling pathways in human cells. In contrast to conventional virtual screening, which identifies the ligands of a targeted protein from a compound database, reverse screening is used to identify the potential targets or unintended targets of a given compound from a large number of receptors by examining their known ligands or crystal structures. This method, also known as in silico or computational target fishing, is highly valuable for discovering the target receptors of query molecules from terrestrial or marine natural products, exploring the molecular mechanisms of chemopreventive compounds, finding alternative indications of existing drugs by drug repositioning, and detecting adverse drug reactions and drug toxicity. Reverse screening can be divided into three major groups: shape screening, pharmacophore screening and reverse docking. Several large software packages, such as Schrödinger and Discovery Studio; typical software/network services such as ChemMapper, PharmMapper, idTarget and INVDOCK; and practical databases of known target ligands and receptor crystal structures, such as ChEMBL, BindingDB and the Protein Data Bank (PDB), are available for use in these computational methods. Different programs, online services and databases have different applications and constraints. Here, we conducted a systematic analysis and multilevel classification of the computational programs, online services and compound libraries available for shape screening, pharmacophore screening and reverse docking to enable non-specialist users to quickly learn and

  5. Parkinson's disease proteins: Novel mitochondrial targets for cardioprotection

    OpenAIRE

    Mukherjee, Uma A.; Ong, Sang-Bing; Ong, Sang-Ging; Hausenloy, Derek J.

    2015-01-01

    Ischemic heart disease (IHD) is the leading cause of death and disability worldwide. Therefore, novel therapeutic targets for protecting the heart against acute ischemia/reperfusion injury (IRI) are required to attenuate cardiomyocyte death, preserve myocardial function, and prevent the onset of heart failure. In this regard, a specific group of mitochondrial proteins, which have been linked to familial forms of Parkinson's disease (PD), may provide novel therapeutic targets for cardioprotect...

  6. ANP32B is a nuclear target of henipavirus M proteins.

    Directory of Open Access Journals (Sweden)

    Anja Bauer

    Full Text Available Membrane envelopment and budding of negative strand RNA viruses (NSVs is mainly driven by viral matrix proteins (M. In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.

  7. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  8. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

    Energy Technology Data Exchange (ETDEWEB)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko [Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan); Umezawa, Kazuo, E-mail: umezawa@aichi-med-u.ac.jp [Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195 (Japan)

    2016-08-05

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.

  9. pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

    Science.gov (United States)

    Koetting, Michael Clinton; Guido, Joseph Frank; Gupta, Malvika; Zhang, Annie; Peppas, Nicholas A

    2016-01-10

    Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 μg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 μg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

    Science.gov (United States)

    Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

    2017-03-17

    Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

  11. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  12. Hepatitis B core protein as a therapeutic target.

    Science.gov (United States)

    Mak, Lung-Yi; Wong, Danny Ka-Ho; Seto, Wai-Kay; Lai, Ching-Lung; Yuen, Man Fung

    2017-12-01

    Chronic hepatitis B virus (HBV) infection is difficult to cure, due to the presence of covalently-closed-circular DNA and virus-mediated blunting of host immune response. Existing therapies with nucleos(t)ide analogue or pegylated-interferon are not sufficient to achieve a high rate of HBV surface antigen seroclearance, a more desirable treatment outcome. Novel therapeutic agents targeting alternative viral replication steps are being developed. In this review, we will discuss the hepatitis B core antigen (HBcAg) as a therapeutic target. Areas covered: The basic structure and fundamental functions of HBcAg including nucleocapsid assembly, pre-genomic RNA encapsidation, reverse transcription, virion formation, cccDNA amplification, immune response regulation, and HBx protein interaction will be reviewed. Most of these are identified as therapeutic targets and tested in in vitro and in vivo studies, although clinical trials are scanty. Among the different components, the core protein allosteric modulators (CpAM) have been most widely investigated and appear promising in clinical trials. Expert opinion: The multiple and essential functions of HBcAg for HBV life cycle are important and attractive targets for HBV therapeutic interventions. Controlled trials involving CpAM are awaited. Apart from CpAM, drugs directed against different functions of HBcAg may be further explored to maximize the chance of cure.

  13. RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

    Science.gov (United States)

    Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

    2017-05-02

    RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

  14. BACE is degraded via the lysosomal pathway.

    Science.gov (United States)

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

    2005-09-16

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

  15. Cellular degradation activity is maintained during aging in long-living queen bees.

    Science.gov (United States)

    Hsu, Chin-Yuan; Qiu, Jiantai Timothy; Chan, Yu-Pei

    2016-11-01

    Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

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

    Science.gov (United States)

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

    2000-01-01

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

  17. Neutron scattering with deuterium labeling reveals the nature of complexes formed by Ca{sup 2+}-binding proteins and their regulatory targets

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Small-angle neutron scattering with deuterium labeling is extremely useful for studying the structures of complex biomolecular assemblies in solution. The different neutron scattering properties of their isotopes of hydrogen combines with the ability to uniformly label biomolecules with deuterium allow one to characterize the structures and relative dispositions of the individual components of an assembly using methods of {open_quotes}contrast variation.{close_quotes} We have applied these techniques to studies of the evolutionarily related dumbbell-shaped Ca{sup 2+}-binding proteins calmodulin and troponin C and their interactions with the target proteins whose activities they regulate. Ca{sup 2+} is one of the simplest of nature`s messengers used in the communication pathways between physiological stimulus and cellular response. The signaling mechanism generally involves Ca{sup 2+} binding to a protein and inducing a conformational change that transmits a signal to modify the activity of a specific target protein. Ca{sup 2+} is thus important in the regulation of a diverse array of intracellular responses, including neurotransmitter release, muscle contraction, the degradation of glycogen to glucose to generate energy, microtubule assembly, membrane phosphorylation, etc. It is the conformational language of the Ca{sup 2+} induced signal transduction that we have sought to understand because of its central importance to biochemical regulation and, hence, to healthy cellular function.

  18. Cheliensisin A (Chel A) induces apoptosis in human bladder cancer cells by promoting PHLPP2 protein degradation.

    Science.gov (United States)

    Zhang, Ruowen; Che, Xun; Zhang, Jingjie; Li, Yang; Li, Jingxia; Deng, Xu; Zhu, Junlan; Jin, Honglei; Zhao, Qinshi; Huang, Chuanshu

    2016-10-11

    Cheliensisin A (Chel A), a styryl-lactone compound extracted from Goniothalamus cheliensis, is reported to have significant anti-cancer effects in various cancer cells. Here we demonstrated that Chel A treatment resulted in apoptosis and an inhibition of anchorage-independent growth in human bladder cancer T24, T24T and U5637 cells. Mechanistic studies showed that such effect is mediated by PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein. Chel A treatment led to PHLPP2 degradation and subsequently increased in c-Jun phosphorylation. Moreover PHLPP2 degradation could be attenuated by inhibition of autophagy, which was mediated by Beclin 1. Collectively, we discover that Chel A treatment induces Beclin-dependent autophagy, consequently mediates PHLPP2 degradation and JNK/C-Jun phosphorylation and activation, further in turn contributing to apoptosis in human bladder cancer cells. Current studies provide a significant insight into understanding of anticancer effect of Chel A in treatment of human bladder cancer.

  19. Human immune cell targeting of protein nanoparticles - caveospheres

    Science.gov (United States)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  20. A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X.; Sun, Q; Kini, R; Sivaraman, J

    2008-01-01

    The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.

  1. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    Science.gov (United States)

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001. Copyright © 2014, Cai et al.

  2. Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia

    Science.gov (United States)

    Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation...

  3. siRNAs Targeting Viral Protein 5: The Major Capsid Protein of ...

    African Journals Online (AJOL)

    Purpose: To investigate whether siRNA targeting viral protein 5 (VP5) can become a new treatment for herpes simplex virus type 1 (HSV-1). Methods: Flow cytometry was performed to determine the ratio of siRNA and lipo2000 to reach the highest transfection efficiency. Western blot and q-PCR were performed to determine ...

  4. Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

    Science.gov (United States)

    Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

    2015-10-01

    This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Protein degradation in skeletal muscle during experimental hyperthyroidism in rats and the effect of beta-blocking agents.

    Science.gov (United States)

    Angerås, U; Hasselgren, P O

    1987-04-01

    beta-Blocking agents are increasingly used in the management of hyperthyroid patients. The effect of this treatment on increased muscle protein breakdown in the hyperthyroid state is not known. In the present study, experimental hyperthyroidism was induced in rats by daily ip injections of T3 (100 micrograms/100 g BW) during a 10-day period. Control animals received corresponding volumes of solvent. In groups of rats the selective beta-1-blocking agent metoprolol or the nonselective beta-blocker propranolol was infused by miniosmotic pumps implanted sc on the backs of the animals. Protein degradation was measured in incubated intact soleus and extensor digitorum longus muscles by determining tyrosine release into the incubation medium. The protein degradation rate in incubated extensor digitorum longus and soleus muscles was increased by 50-60% during T3 treatment. Metoprolol or propranolol did not influence muscle protein breakdown in either T3-treated or control animals. The results suggest that T3-induced increased muscle proteolysis is not mediated by beta-receptors, and muscle weakness and wasting in hyperthyroidism might not be affected by beta-blockers.

  6. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  7. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Science.gov (United States)

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  8. The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression.

    Science.gov (United States)

    Ferrara, Patrizia; Andermarcher, Elisabetta; Bossis, Guillaume; Acquaviva, Claire; Brockly, Frédérique; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2003-03-13

    c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and

  9. Alkylation damage by lipid electrophiles targets functional protein systems.

    Science.gov (United States)

    Codreanu, Simona G; Ullery, Jody C; Zhu, Jing; Tallman, Keri A; Beavers, William N; Porter, Ned A; Marnett, Lawrence J; Zhang, Bing; Liebler, Daniel C

    2014-03-01

    Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

  10. Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

    Science.gov (United States)

    Codreanu, Simona G.; Ullery, Jody C.; Zhu, Jing; Tallman, Keri A.; Beavers, William N.; Porter, Ned A.; Marnett, Lawrence J.; Zhang, Bing; Liebler, Daniel C.

    2014-01-01

    Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions. PMID:24429493

  11. Urea transporter proteins as targets for small-molecule diuretics.

    Science.gov (United States)

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

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

    Directory of Open Access Journals (Sweden)

    Scaife Jes R

    2008-02-01

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

  13. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

    2018-01-01

    In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

  14. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    NARCIS (Netherlands)

    van der Goot, Annemieke T.; Zhu, Wentao; Vazquez-Manrique, Rafael P.; Seinstra, Renee I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Silva, Mariana Ruiz; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a

  15. Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

    Science.gov (United States)

    Sitte, N; Merker, K; von Zglinicki, T; Grune, T

    2000-03-01

    One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

  16. TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

  17. Anti-nutritional Factors and Ruminal Dry Matter and Crude Protein Degradability of Gamma and Microwave Irradiated Native Rapeseed

    Directory of Open Access Journals (Sweden)

    sayyed roohollah ebrahimimahmoudabad

    2016-08-01

    Full Text Available Introduction Native rapeseed (NRS is planted in some parts of Iran because of climatic condition. The consumption of NRS in animal nutrition is limited by anti-nutritional such as phytic acid and glucosinolate. Moreover, the protein of NRS is highly degraded by rumen microorganisms. Several processing methods have been used to enhance the nutritive value of whole oilseeds, including extrusion, roasting, toasting and Jet-Sploding. However, most heat processing methods adversely affect protein digestibility in the small intestine. Recently, other processing methods such as processing by gamma and microwave irradiation have been noticed. Therefore, this research was carried out to evaluate the effects of gamma irradiation (15, 30 and 45 kGy and microwave irradiation (800 W for 2, 4 and 6 min on ruminal dry matter (DM and crude protein (CP degradability, in vitro CP digestibility, anti-nutritional factors (glucosinolate and phytic acid and chemical composition of NRS. Materials and Methods Chemical composition (DM, CP, EE and Ash of untreated and irradiated NRS was determined by AOAC methods. Then, sufficient water was added to the sample to increase the moisture content to 250 g/kg. Gamma irradiation was completed by using a cobalt-60 irradiator at 20 ºC. The dose rate determined by Fricke dosimetry was 0.36 Gy/s. Another three samples (500 g each were subjected to microwave irradiation at a power of 800 W for 2, 4 and 6 min. Phytic acid and glucosinolate contents of untreated and irradiated samples were determined by standard methods. Degradation kinetics of DM or CP were determined according to in situ procedure. Six grams of untreated or irradiated NRS were incubated in the rumen of three ruminally fistulated Taleshi bulls for 0, 2, 4, 8, 16, 24 and 48 h. Bags were placed in the rumen just before the bulls were offered their first meal. After retrieval from the rumen, bags were thoroughly washed with tap water until the rinsing water was clear

  18. Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

    Science.gov (United States)

    Sánchez-Gómez, F J; González-Morena, J M; Vida, Y; Pérez-Inestrosa, E; Blanca, M; Torres, M J; Pérez-Sala, D

    2017-03-01

    Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

    Science.gov (United States)

    Mallik, Aritra; Yammani, Raghunatha R

    2018-07-20

    Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

    Directory of Open Access Journals (Sweden)

    Yuhan Kong

    2013-11-01

    Full Text Available Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP. In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

  1. Blocking Breast Cancer Metastasis by Targeting RNA-Binding Protein HuR

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-16-1-0730 TITLE: Blocking Breast Cancer Metastasis by Targeting RNA-Binding Protein HuR PRINCIPAL INVESTIGATOR: Danny Welch...NUMBER Blocking Breast Cancer Metastasis by Targeting RNA-Binding Protein HuR 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...increased aggressiveness in breast cancer , the primary objective of this proposal is to assess whether HuR (or analogs) prevent and/or treat metastasis and/or

  2. Effective rumen degradation of dry matter, crude protein and neutral detergent fibre in forage determined by near infrared reflectance spectroscopy

    DEFF Research Database (Denmark)

    Ohlsson, C; Houmøller, L P; Weisbjerg, Martin Riis

    2007-01-01

    The objective of the present study was to examine if near infrared reflectance spectroscopy (NIRS) could be used to predict degradation parameters and effective degradation from scans of original forage samples. Degradability of dry matter (DM), crude protein (CP) and neutral detergent fibre (NDF......) of 61 samples of perennial ryegrass (Lolium perenne L.) and orchardgrass (Dactylis glomerata L.) was tested by using the in situ technique. The grass samples were harvested at three different stages, early vegetative growth, early reproductive growth and late reproductive growth. Degradability...

  3. Identification of novel human damage response proteins targeted through yeast orthology.

    Directory of Open Access Journals (Sweden)

    J Peter Svensson

    Full Text Available Studies in Saccharomyces cerevisiae show that many proteins influence cellular survival upon exposure to DNA damaging agents. We hypothesized that human orthologs of these S. cerevisiae proteins would also be required for cellular survival after treatment with DNA damaging agents. For this purpose, human homologs of S. cerevisiae proteins were identified and mapped onto the human protein-protein interaction network. The resulting human network was highly modular and a series of selection rules were implemented to identify 45 candidates for human toxicity-modulating proteins. The corresponding transcripts were targeted by RNA interference in human cells. The cell lines with depleted target expression were challenged with three DNA damaging agents: the alkylating agents MMS and 4-NQO, and the oxidizing agent t-BuOOH. A comparison of the survival revealed that the majority (74% of proteins conferred either sensitivity or resistance. The identified human toxicity-modulating proteins represent a variety of biological functions: autophagy, chromatin modifications, RNA and protein metabolism, and telomere maintenance. Further studies revealed that MMS-induced autophagy increase the survival of cells treated with DNA damaging agents. In summary, we show that damage recovery proteins in humans can be identified through homology to S. cerevisiae and that many of the same pathways are represented among the toxicity modulators.

  4. Degradation of p53 by human Alphapapillomavirus E6 proteins shows a stronger correlation with phylogeny than oncogenicity.

    Directory of Open Access Journals (Sweden)

    Leiping Fu

    2010-09-01

    Full Text Available Human Papillomavirus (HPV E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types to include members of all known HPV species groups within the Alphapapillomavirus genus.We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71 were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.These data suggest that the alpha-HPV E6 proteins' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.

  5. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

    Science.gov (United States)

    Helmerhorst, Eva J; Wei, Guoxian

    2014-05-05

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  6. Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

    Science.gov (United States)

    Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

    1998-11-01

    Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location

  7. Protein degradation systems in the skeletal muscles of parr and smolt Atlantic salmon Salmo salar L. and brown trout Salmo trutta L.

    Science.gov (United States)

    Kantserova, Nadezda P; Lysenko, Liudmila A; Veselov, Alexey E; Nemova, Nina N

    2017-08-01

    Although protein degradation limits the rate of muscle growth in fish, the role of proteolytic systems responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The study herein aims to evaluate the role of calpains (calcium-activated proteases) and proteasomes (ATP-dependent proteases) in mediating muscle protein turnover at different life stages in wild salmonids. Protease activities were estimated in Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) parr and smolts from the Indera River (Kola Peninsula, Russia). Calpain and proteasome activities in Atlantic salmon skeletal muscles were lower in smolts as compared with parr. Reduced muscle protein degradation accompanying Atlantic salmon parr-smolt transformation appeared to provide intense muscle growth essential for a minimum threshold size achievement that is required for smoltification. Calpain and proteasome activities in brown trout parr and smolts at age 3+ did not significantly differ. However, calpain activity was higher in smolts brown trout 4+ as compared with parr, while proteasome activity was lower. Results suggest that brown trout smoltification does not correspond with intense muscle growth and is more facultative and plastic in comparison with Atlantic salmon smoltification. Obtained data on muscle protein degradation capacity as well as length-weight parameters of fish reflect differences between salmon and trout in growth and smoltification strategies.

  8. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    International Nuclear Information System (INIS)

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-01-01

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  9. In situ ruminal crude protein degradability of by-products from cereals, oilseeds and animal origin

    NARCIS (Netherlands)

    Habib, G.; Khan, N.A.; Ali, M.; Bezabih, M.

    2013-01-01

    The aim of this study was to establish a database on in situ ruminal crude protein (CP) degradability characteristics of by-products from cereal grains, oilseeds and animal origin commonly fed to ruminants in Pakistan and South Asian Countries. The oilseed by-products were soybean meal, sunflower

  10. Hydrothermal degradation of cellulosic matter to sugars and their fermentative conversion to protein

    International Nuclear Information System (INIS)

    Bobleter, O.; Niesner, R.; Roehr, M.

    1976-01-01

    For the hydrothermal degradation of cellulosic matter, an apparatus was developed in which water is used as extraction medium. Samples, 0.15 g each, of pure cellulose (filter paper), natural straw, and 14 C-labeled straw were treated at temperatures of between 200 and 275 0 C. Of the inserted cellulose, 65.7 percent was recovered at the optimum temperature as sugars and hydroxymethylfurfural. It was possible to degrade the straw selectively: at lower temperatures, the hemicellulose part of the plant matter was converted to xylose and arabinose; and then at higher temperatures, the cellulose was converted to glucose and cellobiose. At the same time, a certain amount of the sugars was transformed to furfural compounds. The growth behavior of the yeast Candida utilis (strain Weissenbach) was analyzed, using cellobiose, xylose, and glucose (standard) as carbon sources. The growth curves applying cellobiose were nearly identical to those of glucose. Xylose showed lower productivity than the hexoses. The main products of the hydrothermal degradation can, therefore, be used favorably as nutritive substances for this protein-producing yeast

  11. Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

    Science.gov (United States)

    Shi, Pu

    Cytotoxicity, low water solubility, rapid clearance from circulation, and offtarget side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or nonpolymeric. This chapter summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. This chapter explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for

  12. Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases.

    Science.gov (United States)

    Victoria, Guiliana Soraya; Zurzolo, Chiara

    2015-09-02

    Several neurodegenerative diseases such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's diseases are caused by the conversion of cellular proteins to a pathogenic conformer. Despite differences in the primary structure and subcellular localization of these proteins, which include the prion protein, α-synuclein and amyloid precursor protein (APP), striking similarity has been observed in their ability to seed and convert naïve protein molecules as well as transfer between cells. This review aims to cover what is known about the intracellular trafficking of these proteins as well as their degradation mechanisms and highlight similarities in their movement through the endocytic pathway that could contribute to the pathogenic conversion and seeding of these proteins which underlies the basis of these diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Chemical composition and ruminal degradation kinetics of crude protein and amino acids, and intestinal digestibility of amino acids from tropical forages

    Directory of Open Access Journals (Sweden)

    Lidia Ferreira Miranda

    2012-03-01

    Full Text Available The objective of this research was to determine the chemical composition and ruminal degradation of the crude protein (CP, total and individual amino acids of leaves from tropical forages: perennial soybean (Neonotonia wightii, cassava (Manihot esculenta, leucaena (Leucaena leucocephala and ramie (Boehmeria nivea, and to estimate the intestinal digestibility of the rumen undegradable protein (RUDP and individual amino acids of leaves from the tropical forages above cited, but including pigeon pea (Cajanus cajan. Three nonlactating Holstein cows were used to determine the in situ ruminal degradability of protein and amino acids from leaves (6, 18 and 48 hours of ruminal incubation. For determination of the intestinal digestibility of RUDP, the residue from ruminal incubation of the materials was used for 18 hours. A larger concentration of total amino acids for ramie and smaller for perennial soybean were observed; however, they were very similar in leucaena and cassava. Leucine was the essential amino acid of greater concentration, with the exception of cassava, which exhibited a leucine concentration 40.45% smaller. Ramie showed 14.35 and 22.31% more lysine and methionine, respectively. The intestinal digestibility of RUDP varied from 23.56; 47.87; 23.48; 25.69 and 10.86% for leucaena, perennial soybean, cassava, ramie and pigeon pea, respectively. The individual amino acids of tropical forage disappeared in different extensions in the rumen. For the correct evaluation of those forages, one should consider their composition of amino acids, degradations and intestinal digestibility, once the amino acid composition of the forage does not reflect the amino acid profiles that arrived in the small intestine. Differences between the degradation curves of CP and amino acids indicate that degradation of amino acids cannot be estimated through the degradation curve of CP, and that amino acids are not degraded in a similar degradation profile.

  14. In Vitro Ruminal Degradability of Soybean Meal Protein Protected with Natural Tannin

    Science.gov (United States)

    Prasetiyono, B. W. H. E.; Subrata, A.; Tampoebolon, B. I. M.; Surono; Widiyanto

    2018-02-01

    The influence of tannin from tea waste and gambier as natural tannin sources on ruminal protein degradability was studied in this investigation. The soybean meal was used as protein source in this investigation. There were three treatments in this investigation mainly without protection (NT); protection with tea waste (Tt); and protection with gambier (Tg). The measured parameters consisted of in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), and the ruminal fermentation characteristics. Results of this experiment showed that protection with tannin from tea waste as well as gambier increased (pRUP) in Tt and Tg group was higher than that in NT group (66.29 and 69.20 vs 51.10%). The ruminal protozoa population decreased (pRUP. The ruminal protozoa population and ammonia concentration, on the other hand, were decreased by tannin protection from those tannin natural sources. The natural tannin from gambier was the most effective protection agent for soybean meal protein.

  15. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

    Science.gov (United States)

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

    2016-07-01

    Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs

  16. A critical role for protein degradation in the nucleus accumbens core in cocaine reward memory.

    Science.gov (United States)

    Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

    2013-04-01

    The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization.

  17. Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

    Science.gov (United States)

    Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

    2017-11-17

    With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

  18. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

    Directory of Open Access Journals (Sweden)

    Lian Jin

    2016-09-01

    Full Text Available The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB protein and an auxin/indole-3-acetic acid (Aux/IAA protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV, a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.

  19. Targeting the OB-Folds of Replication Protein A with Small Molecules

    Directory of Open Access Journals (Sweden)

    Victor J. Anciano Granadillo

    2010-01-01

    Full Text Available Replication protein A (RPA is the main eukaryotic single-strand (ss DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

  20. Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

    Science.gov (United States)

    Huang, Tengfang; Jander, Georg

    2017-10-01

    Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

  1. 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.

  2. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows

    DEFF Research Database (Denmark)

    Tayyab, Usama; Novoa-Garrido, Margarita; Roleda, Michael Y.

    2016-01-01

    , Laminaria, Mastocarpus, Palmaria, Pelvetia, Porphyra, and Ulva were sampled in spring (March) and autumn (October and November) 2014 at the coast of Bodø in Northern Norway, and were analysed for chemical composition, in situ rumen degradability and total tract crude protein (CP) digestibility. Ash content......The use of seaweeds in animal diets is not new. However, little is known about the feed value of seaweed, both in terms of chemical composition and protein digestibility, and regarding variation between species and season. In this study, eight seaweed species of the genus Acrosiphonia, Alaria....../kg CP). Digestible rumen escape protein (DEP) varied significantly between species (P Laminaria, Mastocarpus and Palmaria can supply...

  3. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

    Science.gov (United States)

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-04-15

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

  4. Quercetin targets cysteine string protein (CSPalpha and impairs synaptic transmission.

    Directory of Open Access Journals (Sweden)

    Fenglian Xu

    2010-06-01

    Full Text Available Cysteine string protein (CSPalpha is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPalpha is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPalpha in mice results in knockout mice that are normal for the first 2-3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPalpha prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPalpha represents a promising therapeutic target for the prevention of neurodegenerative disorders.Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPalpha-CSPalpha dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPalpha dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPalpha function. Quercetin's action on CSPalpha is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPalpha:Hsc70 units (70kDa heat shock cognate protein.Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s of action has been unclear. In view of the therapeutic promise of upregulation of CSPalpha and the undesired consequences of CSPalpha dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPalpha.

  5. Restoration of longitudinal laser tomography target image from inhomogeneous medium degradation under common conditions.

    Science.gov (United States)

    Yi, WenJun; Wang, Ping; Fu, MeiCheng; Tan, JiChun; Zhu, Jubo; Li, XiuJian

    2017-07-10

    In order to overcome the shortages of the target image restoration method for longitudinal laser tomography using self-calibration, a more general restoration method through backscattering medium images associated with prior parameters is developed for common conditions. The system parameters are extracted from pre-calibration, and the LIDAR ratio is estimated according to the medium types. Assisted by these prior parameters, the degradation caused by inhomogeneous turbid media can be established with the backscattering medium images, which can further be used for removal of the interferences of turbid media. The results of simulations and experiments demonstrate that the proposed image restoration method can effectively eliminate the inhomogeneous interferences of turbid media and achieve exactly the reflectivity distribution of targets behind inhomogeneous turbid media. Furthermore, the restoration method can work beyond the limitation of the previous method that only works well under the conditions of localized turbid attenuations and some types of targets with fairly uniform reflectivity distributions.

  6. 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...

  7. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

    Directory of Open Access Journals (Sweden)

    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  8. Lysosomal degradation of membrane lipids.

    Science.gov (United States)

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    OpenAIRE

    Huang, Hao; He, Yuehan; Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biologi...

  10. Large-scale prediction of drug–target interactions using protein sequences and drug topological structures

    International Nuclear Information System (INIS)

    Cao Dongsheng; Liu Shao; Xu Qingsong; Lu Hongmei; Huang Jianhua; Hu Qiannan; Liang Yizeng

    2012-01-01

    Highlights: ► Drug–target interactions are predicted using an extended SAR methodology. ► A drug–target interaction is regarded as an event triggered by many factors. ► Molecular fingerprint and CTD descriptors are used to represent drugs and proteins. ► Our approach shows compatibility between the new scheme and current SAR methodology. - Abstract: The identification of interactions between drugs and target proteins plays a key role in the process of genomic drug discovery. It is both consuming and costly to determine drug–target interactions by experiments alone. Therefore, there is an urgent need to develop new in silico prediction approaches capable of identifying these potential drug–target interactions in a timely manner. In this article, we aim at extending current structure–activity relationship (SAR) methodology to fulfill such requirements. In some sense, a drug–target interaction can be regarded as an event or property triggered by many influence factors from drugs and target proteins. Thus, each interaction pair can be represented theoretically by using these factors which are based on the structural and physicochemical properties simultaneously from drugs and proteins. To realize this, drug molecules are encoded with MACCS substructure fingerings representing existence of certain functional groups or fragments; and proteins are encoded with some biochemical and physicochemical properties. Four classes of drug–target interaction networks in humans involving enzymes, ion channels, G-protein-coupled receptors (GPCRs) and nuclear receptors, are independently used for establishing predictive models with support vector machines (SVMs). The SVM models gave prediction accuracy of 90.31%, 88.91%, 84.68% and 83.74% for four datasets, respectively. In conclusion, the results demonstrate the ability of our proposed method to predict the drug–target interactions, and show a general compatibility between the new scheme and current SAR

  11. Large-scale prediction of drug-target interactions using protein sequences and drug topological structures

    Energy Technology Data Exchange (ETDEWEB)

    Cao Dongsheng [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China); Liu Shao [Xiangya Hospital, Central South University, Changsha 410008 (China); Xu Qingsong [School of Mathematical Sciences and Computing Technology, Central South University, Changsha 410083 (China); Lu Hongmei; Huang Jianhua [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China); Hu Qiannan [Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071 (China); Liang Yizeng, E-mail: yizeng_liang@263.net [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China)

    2012-11-08

    Highlights: Black-Right-Pointing-Pointer Drug-target interactions are predicted using an extended SAR methodology. Black-Right-Pointing-Pointer A drug-target interaction is regarded as an event triggered by many factors. Black-Right-Pointing-Pointer Molecular fingerprint and CTD descriptors are used to represent drugs and proteins. Black-Right-Pointing-Pointer Our approach shows compatibility between the new scheme and current SAR methodology. - Abstract: The identification of interactions between drugs and target proteins plays a key role in the process of genomic drug discovery. It is both consuming and costly to determine drug-target interactions by experiments alone. Therefore, there is an urgent need to develop new in silico prediction approaches capable of identifying these potential drug-target interactions in a timely manner. In this article, we aim at extending current structure-activity relationship (SAR) methodology to fulfill such requirements. In some sense, a drug-target interaction can be regarded as an event or property triggered by many influence factors from drugs and target proteins. Thus, each interaction pair can be represented theoretically by using these factors which are based on the structural and physicochemical properties simultaneously from drugs and proteins. To realize this, drug molecules are encoded with MACCS substructure fingerings representing existence of certain functional groups or fragments; and proteins are encoded with some biochemical and physicochemical properties. Four classes of drug-target interaction networks in humans involving enzymes, ion channels, G-protein-coupled receptors (GPCRs) and nuclear receptors, are independently used for establishing predictive models with support vector machines (SVMs). The SVM models gave prediction accuracy of 90.31%, 88.91%, 84.68% and 83.74% for four datasets, respectively. In conclusion, the results demonstrate the ability of our proposed method to predict the drug-target

  12. Exosomal protein interactors as emerging therapeutic targets in urothelial bladder cancer

    International Nuclear Information System (INIS)

    Kumari, N.; Saxena, S.; Agrawal, U.

    2015-01-01

    Background: Exosomes are rich sources of biological material (proteins and nucleic acids) secreted by both tumor and normal cells, and found in urine of urinary bladder cancer patients. Objective: The objective of the study was to identify interacting exosomal proteins in bladder cancer for future use in targeted therapy. Methods: The Exocarta database (www.exocarta.org) was mined for urinary bladder cancer specific exosomal proteins. The urinary bladder cancer specific exosomal proteins (n = 248) were analyzed to identify enriched pathways by Onto-tool Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress). Results: Enriched pathways included cellular architecture, motility, cell to cell adhesion, tumorigenesis and metastasis. Proteins in the 9 top-ranked pathways included CTNNA1 (alpha-catenin), CTNNB1 (beta-catenin), VSAP, ITGA4, PAK1, DDR1, CDC42, RHOA, NRAS, RHO, PIK3AR1, MLC1, MMRN1, and CTTNBP2 and network analysis revealed 10 important hub proteins and identified inferred interactor NF2. Conclusions: The importance of identifying interactors is that that they can be used as targets for therapy, for example, using Bevacizumab (avastin - an angiogenesis inhibitor) against NF2 to inhibit protein-protein interactions will inhibit tumor growth and progression by hindering the exosome biogenesis

  13. Y-Trap Cancer Immunotherapy Drug Targets Two Proteins

    Science.gov (United States)

    Two groups of researchers, working independently, have fused a TGF-beta receptor to a monoclonal antibody that targets a checkpoint protein. The result, this Cancer Currents blog describes, is a single hybrid molecule called a Y-trap that blocks two pathways used by tumors to evade the immune system.

  14. GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Wang, Xuerong; Owonikoko, Taofeek K; Ramalingam, Suresh S; Khuri, Fadlo R; Sun, Shi-Yong

    2015-04-20

    The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

  15. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  16. Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

    Directory of Open Access Journals (Sweden)

    Hirotaka Takahashi

    Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

  17. TargetCrys: protein crystallization prediction by fusing multi-view features with two-layered SVM.

    Science.gov (United States)

    Hu, Jun; Han, Ke; Li, Yang; Yang, Jing-Yu; Shen, Hong-Bin; Yu, Dong-Jun

    2016-11-01

    The accurate prediction of whether a protein will crystallize plays a crucial role in improving the success rate of protein crystallization projects. A common critical problem in the development of machine-learning-based protein crystallization predictors is how to effectively utilize protein features extracted from different views. In this study, we aimed to improve the efficiency of fusing multi-view protein features by proposing a new two-layered SVM (2L-SVM) which switches the feature-level fusion problem to a decision-level fusion problem: the SVMs in the 1st layer of the 2L-SVM are trained on each of the multi-view feature sets; then, the outputs of the 1st layer SVMs, which are the "intermediate" decisions made based on the respective feature sets, are further ensembled by a 2nd layer SVM. Based on the proposed 2L-SVM, we implemented a sequence-based protein crystallization predictor called TargetCrys. Experimental results on several benchmark datasets demonstrated the efficacy of the proposed 2L-SVM for fusing multi-view features. We also compared TargetCrys with existing sequence-based protein crystallization predictors and demonstrated that the proposed TargetCrys outperformed most of the existing predictors and is competitive with the state-of-the-art predictors. The TargetCrys webserver and datasets used in this study are freely available for academic use at: http://csbio.njust.edu.cn/bioinf/TargetCrys .

  18. Transient degradation of NF-κB proteins in macrophages after interaction with mast cell granules

    Directory of Open Access Journals (Sweden)

    Noriko Ito

    1998-01-01

    Full Text Available The exposure of the macrophage cell line, J774 to mast cell granules (MCG led to the form ation of altered nuclear transcription factor proteins (NFκBx, which had faster electrophoretic mobility than the p50 homodimer of NF-κB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-κBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBα remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of m ouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-κB proteins. The alterations in NF-κB proteins did not affect the expression of inducible nitric oxide synthase (iNOS or TNF-α mRNA in J774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-κB proteins with associated inhibition of iNOS or TNF-α mRNA expression.

  19. Systematic analysis of FKBP inducible degradation domain tagging strategies for the human malaria parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Mauro Ferreira de Azevedo

    Full Text Available Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA at its C-terminus. The tagged protein demonstrated an important modulation of its

  20. Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness.

    Directory of Open Access Journals (Sweden)

    Nicole Brimer

    2017-12-01

    Full Text Available Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A, by binding to an LXXLL peptide (ELTLQELLGEE displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs and cetacean (porpoises and dolphins hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution.

  1. Degradation of some representative polycyclic aromatic hydrocarbons by the water-soluble protein extracts from Zea mays L. cv PR32-B10.

    Science.gov (United States)

    Barone, Roberto; de Biasi, Margherita-Gabriella; Piccialli, Vincenzo; de Napoli, Lorenzo; Oliviero, Giorgia; Borbone, Nicola; Piccialli, Gennaro

    2016-10-01

    The ability of the water-soluble protein extracts from Zea mais L. cv. PR32-B10 to degrade some representative polycyclic aromatic hydrocarbons (PAHs), has been evaluated. Surface sterilized seeds of corn (Zea mais L. Pioneer cv. PR32-B10) were hydroponically cultivated in a growth chamber under no-stressful conditions. The water-soluble protein extracts isolated from maize tissues showed peroxidase, polyphenol oxidase and catalase activities. Incubation of the extracts with naphthalene, fluorene, phenanthrene and pyrene, led to formation of oxidized and/or degradation products. GC-MS and TLC monitoring of the processes showed that naphthalene, phenanthrene, fluorene and pyrene underwent 100%, 78%, 92% and 65% oxidative degradation, respectively, after 120 min. The chemical structure of the degradation products were determined by (1)H NMR and ESI-MS spectrometry. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Protein degradation and post-deboning tenderization in broiler breast meat with different degrees of muscle shortening

    Science.gov (United States)

    Deboning broiler breast fillets prior to rigor mortis negatively influences tenderness due to sarcomere shortening. The effects of sarcomere shortening on muscle protein degradation and breast meat tenderization during post-deboning aging are not well understood. The objective of this study was to m...

  3. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  4. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

    International Nuclear Information System (INIS)

    Campos, Samuel K.; Barry, Michael A.

    2006-01-01

    The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

  5. Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

    Directory of Open Access Journals (Sweden)

    Datis Kharrazian

    2017-01-01

    Full Text Available Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw and modified (cooked and roasted foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease.

  6. CUP promotes deadenylation and inhibits decapping of mRNA targets

    Science.gov (United States)

    Igreja, Catia; Izaurralde, Elisa

    2011-01-01

    CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1–CCR4–NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs. PMID:21937713

  7. Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

    Directory of Open Access Journals (Sweden)

    Emmanuel Obishakin

    Full Text Available Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa, costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa, antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392 targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1 of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392 that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

  8. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Science.gov (United States)

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

  9. Therapeutic targeting strategies using endogenous cells and proteins.

    Science.gov (United States)

    Parayath, Neha N; Amiji, Mansoor M

    2017-07-28

    Targeted drug delivery has become extremely important in enhancing efficacy and reducing the toxicity of therapeutics in the treatment of various disease conditions. Current approaches include passive targeting, which relies on naturally occurring differences between healthy and diseased tissues, and active targeting, which utilizes various ligands that can recognize targets expressed preferentially at the diseased site. Clinical translation of these mechanisms faces many challenges including the immunogenic and toxic effects of these non-natural systems. Thus, use of endogenous targeting systems is increasingly gaining momentum. This review is focused on strategies for employing endogenous moieties, which could serve as safe and efficient carriers for targeted drug delivery. The first part of the review involves cells and cellular components as endogenous carriers for therapeutics in multiple disease states, while the second part discusses the use of endogenous plasma components as endogenous carriers. Further understanding of the biological tropism with cells and proteins and the newer generation of delivery strategies that exploits these endogenous approaches promises to provide better solutions for site-specific delivery and could further facilitate clinical translations. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4 signaling pathway.

    Science.gov (United States)

    Zheng, Xin; Zhao, Feng-Chao; Pang, Yong; Li, Dong-Ya; Yao, Sheng-Cheng; Sun, Shao-Song; Guo, Kai-Jin

    2017-06-01

    Osteoarthritis (OA) is characterized by degradation of chondrocyte extracellular matrix (ECM). Accumulating evidence suggests that microRNAs (miRNAs) are associated with OA, but little is known of their function in chondrocyte ECM degradation. The objective of this study was to investigate the expression and function of miRNAs in OA. miRNA expression profile was determined in OA cartilage tissues and controls, employing Solexa sequencing and reverse transcription quantitative PCR (RT-qPCR). According to a modified Mankin scale, cartilage degradation was evaluated. Functional analysis of the miRNAs on chondrocyte ECM degradation was performed after miRNA transfection and IL-1β treatment. Luciferase reporter assays and western blotting were employed to determine miRNA targets. Expression of miR-221-3p was downregulated in OA cartilage tissues, which was significantly correlated with a modified Mankin scale. Through gain-of-function and loss-of-function studies, miR-221-3p was shown to significantly affect matrix synthesis gene expression and chondrocyte proliferation and apoptosis. Using SW1353 and C28I2 cells, SDF1 was identified as a target of miR-221-3p. SDF1 overexpression resulted in increased expression of catabolic genes such as MMP-13 and ADAMTS-5 in response to IL-1β, but these effects were moderated by miR-221-3p. SDF1 treatment antagonized this effect, while knockdown of SDF1 by shSDF1 induced inhibitory effects on the expression of CXCR4 and its main target genes, similar to miR-221-3p. The results indicate that upregulation of miR-221-3p could prevent IL-1β-induced ECM degradation in chondrocytes. Targeting the SDF1/CXCR4 signaling pathway may be used as a therapeutic approach for OA. miR-221-3p is downregulated in human cartilage tissues. miR-221-3p levels are associated with cartilage degeneration grade. miR-221-3p upregulation prevents IL-1β-induced ECM degradation in chondrocytes. Protection of ECM degradation by miR-223-3p occurs via SDF1/CXCR4

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

    Science.gov (United States)

    Nemet, Ina; Monnier, Vincent M

    2011-10-28

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

  12. Maximum flow approach to prioritize potential drug targets of Mycobacterium tuberculosis H37Rv from protein-protein interaction network.

    Science.gov (United States)

    Melak, Tilahun; Gakkhar, Sunita

    2015-12-01

    In spite of the implementations of several strategies, tuberculosis (TB) is overwhelmingly a serious global public health problem causing millions of infections and deaths every year. This is mainly due to the emergence of drug-resistance varieties of TB. The current treatment strategies for the drug-resistance TB are of longer duration, more expensive and have side effects. This highlights the importance of identification and prioritization of targets for new drugs. This study has been carried out to prioritize potential drug targets of Mycobacterium tuberculosis H37Rv based on their flow to resistance genes. The weighted proteome interaction network of the pathogen was constructed using a dataset from STRING database. Only a subset of the dataset with interactions that have a combined score value ≥770 was considered. Maximum flow approach has been used to prioritize potential drug targets. The potential drug targets were obtained through comparative genome and network centrality analysis. The curated set of resistance genes was retrieved from literatures. Detail literature review and additional assessment of the method were also carried out for validation. A list of 537 proteins which are essential to the pathogen and non-homologous with human was obtained from the comparative genome analysis. Through network centrality measures, 131 of them were found within the close neighborhood of the centre of gravity of the proteome network. These proteins were further prioritized based on their maximum flow value to resistance genes and they are proposed as reliable drug targets of the pathogen. Proteins which interact with the host were also identified in order to understand the infection mechanism. Potential drug targets of Mycobacterium tuberculosis H37Rv were successfully prioritized based on their flow to resistance genes of existing drugs which is believed to increase the druggability of the targets since inhibition of a protein that has a maximum flow to

  13. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

    Directory of Open Access Journals (Sweden)

    Annie M Westerlund

    2018-04-01

    Full Text Available Calmodulin (CaM is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

  14. GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

    Science.gov (United States)

    Negahdar, Maria; Aukrust, Ingvild; Molnes, Janne; Solheim, Marie H; Johansson, Bente B; Sagen, Jørn V; Dahl-Jørgensen, Knut; Kulkarni, Rohit N; Søvik, Oddmund; Flatmark, Torgeir; Njølstad, Pål R; Bjørkhaug, Lise

    2014-01-25

    GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

    Science.gov (United States)

    2016-01-01

    Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

  16. Anaerobic digestion of cattle offal: protein and lipid-rich substrate degradation and population dynamics of acidogens and methanogens.

    Science.gov (United States)

    Lee, Joonyeob; Koo, Taewoan; Han, Gyuseong; Shin, Seung Gu; Hwang, Seokhwan

    2015-12-01

    Anaerobic digestion of cattle offal was investigated in batch reactors at 35 °C to determine the feasibility of using cattle offal as a feedstock. The organic content [i.e., volatile solids (VS)] of the cattle offal was mainly composed of protein (33.9%) and lipids (46.1%). Hydrolysis along with acidogenesis was monitored to investigate the substrate degradation and generation of intermediate products (e.g., volatile fatty acids, ammonia). Acetate (2.03 g/L), propionate (0.60 g/L), n-butyrate (0.39 g/L), and iso-valerate (0.37 g/L) were major acidogenesis products (91% of total volatile fatty acid concentration). Overall protein and lipid degradation were 82.9 and 81.8%, respectively. Protein degraded first, and four times faster (0.28 day(-1)) than lipid (0.07 day(-1)). Methane yields were 0.52 L CH4/g VSadded and 0.65 L CH4/g VSremoved, indicating that anaerobic digestion of the offal was feasible. A quantitative QPCR assay was conducted to understand the microbial dynamics. The variation patt erns in the gene concentrations successfully indicated the population dynamics of proteolytic and lipolytic acidogens. A fourth-order Runge-Kutta approximation was used to determine the kinetics of the acidogens. The molecular biotechnology approach was appropriate for the evaluation of the acidogenic biokinetics. The maximum growth rate, μ m, halfsaturation coefficients, K s, microbial yield coefficient, Y, cell mass decay rate coefficient, k d, of the proteolytic acidogens were 9.9 day(-1), 37.8 g protein/L, 1.1 × 10(10) copies/g protein, and 3.8 × 10(-1), respectively. Those for the lipolytic acidogens were 1.2 × 10(-1) day(-1), 8.3 g lipid/L, 1.5 × 10(9) copies/g lipid, and 9.9 × 10(-3) day(-1), respectively.

  17. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    Science.gov (United States)

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PMID:21857022

  18. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    Science.gov (United States)

    Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biological background, 1 PPDT-Module and 22 PCOS potential drug targets were identified, 21 of which were verified by literatures to be associated with the pathogenesis of PCOS. 42 drugs targeting to 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. Evaluated by independent datasets, the whole PPDT-Module and 22 PCOS potential drug targets could not only reveal the drug response, but also distinguish the statuses between normal and disease. Our identified PPDT-Module and PCOS potential drug targets would shed light on the treatment of PCOS. And our approach would provide valuable insights to research on the pathogenesis and drug response of other diseases. PMID:27191267

  19. The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins

    DEFF Research Database (Denmark)

    Madsen, Louise; Kriegenburg, Franziska; Lages Lino Vala, Andrea

    2011-01-01

    is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p......The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded...... protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8...

  20. Indian hedgehog signaling triggers Nkx3.2 protein degradation during chondrocyte maturation

    Science.gov (United States)

    Choi, Seung-Won; Jeong, Da-Un; Kim, Jeong-Ah; Lee, Boyoung; Joeng, Kyu Sang; Long, Fanxin; Kim, Dae-Won

    2015-01-01

    The Indian hedgehog (Ihh) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes, and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in this work, we investigate whether Nkx3.2, an early stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. Here, we show that Ihh signaling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (Wnt co-receptor) and Sfrp (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocyte. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signaling by deletion of either Ihh or Smoothened. Thus, these results suggest that Ihh/Wnt5a signaling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis. PMID:22507129

  1. Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

    Science.gov (United States)

    Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

    2017-08-01

    Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

  2. RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana

    Science.gov (United States)

    Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

    2017-01-01

    RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study als