WorldWideScience

Sample records for age-associated chaperone capacity

  1. [Age-associated peculiarities of microcirculation system in skeletal muscles and their role in muscle work capacity in human aging (author's transl)].

    Science.gov (United States)

    Korkusko, O V; Sarkisov, K G; Frajfel'd, V E

    1982-01-01

    The muscle blood flow was investigated at rest (MBFR) and after physical load under ischemia conditions (maximal muscle blood flow--MMBF) in 87 practically healthy persons (45 women and 42 men) aged 20--90. The state of muscle blood flow was evaluated by means of the clearance of 133xenon injected into M. tibialis anterior. The data obtained showed a decrease of MBFR and MMBF in older people as compared with younger subjects. In realization of this phenomenon a decrease in muscle capillarisation and a reduction in reactivity of microcirculatory link of vascular system plays an increasingly greater role with aging. The reduction in muscle blood flow forms a circulatory component of the age-associated hypoxia. This fact results in a decrease of muscle blood flow and limits the functional capacity of skeletal muscle under conditions of activity.

  2. The perception and use of chaperones by Nigerian gynecologists.

    Science.gov (United States)

    Nkwo, Peter O; Chigbu, Chibuike O; Ajah, Leonard O; Okoro, Onyemaechi S

    2013-01-01

    To determine how Nigerian gynecologists perceive and use chaperones during intimate gynecologic examinations. A cross-sectional survey of Nigerian gynecologists was conducted with the aid of self-administered, semi-structured questionnaires. Data were analyzed for descriptive and inferential statistics. In all, 97.6% of respondents considered the use of a chaperone necessary during intimate gynecologic examinations and recommended that the Society of Gynaecology and Obstetrics of Nigeria (SOGON) should endorse the routine offer of chaperones for such examinations. However, just 35.9% of male physicians always or often used chaperones, while 76.9% of female physicians used chaperones only under special circumstances. No female physician always or often used a chaperone during pelvic examination. The main obstacles to the use of chaperones were scarcity of personnel to serve in this capacity (87.6%) and patients' refusal to be examined in the presence of a third party (12.4%). Most Nigerian gynecologists use chaperones at least some of the time and also support a policy of routinely offering chaperones during intimate gynecologic examination while respecting patients' right to decline this offer. Scarcity of personnel to serve as chaperones is the greatest challenge to the implementation of this policy. Copyright © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

  3. Chaperone-Mediated Autophagy

    Science.gov (United States)

    Bejarano, Eloy; Cuervo, Ana Maria

    2010-01-01

    Continuous renewal of intracellular components is required to preserve cellular functionality. In fact, failure to timely turnover proteins and organelles leads often to cell death and disease. Different pathways contribute to the degradation of intracellular components in lysosomes or autophagy. In this review, we focus on chaperone-mediated autophagy (CMA), a selective form of autophagy that modulates the turnover of a specific pool of soluble cytosolic proteins. Selectivity in CMA is conferred by the presence of a targeting motif in the cytosolic substrates that, upon recognition by a cytosolic chaperone, determines delivery to the lysosomal surface. Substrate proteins undergo unfolding and translocation across the lysosomal membrane before reaching the lumen, where they are rapidly degraded. Better molecular characterization of the different components of this pathway in recent years, along with the development of transgenic models with modified CMA activity and the identification of CMA dysfunction in different severe human pathologies and in aging, are all behind the recent regained interest in this catabolic pathway. PMID:20160146

  4. Histone chaperone networks shaping chromatin function

    DEFF Research Database (Denmark)

    Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda

    2017-01-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability...... and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....

  5. Chaperone-client complexes: A dynamic liaison

    Science.gov (United States)

    Hiller, Sebastian; Burmann, Björn M.

    2018-04-01

    Living cells contain molecular chaperones that are organized in intricate networks to surveil protein homeostasis by avoiding polypeptide misfolding, aggregation, and the generation of toxic species. In addition, cellular chaperones also fulfill a multitude of alternative functionalities: transport of clients towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver clients towards proteolysis machineries. Until recently, the only available source of atomic resolution information for virtually all chaperones were crystal structures of their client-free, apo-forms. These structures were unable to explain details of the functional mechanisms underlying chaperone-client interactions. The difficulties to crystallize chaperones in complexes with clients arise from their highly dynamic nature, making solution NMR spectroscopy the method of choice for their study. With the advent of advanced solution NMR techniques, in the past few years a substantial number of structural and functional studies on chaperone-client complexes have been resolved, allowing unique insight into the chaperone-client interaction. This review summarizes the recent insights provided by advanced high-resolution NMR-spectroscopy to understand chaperone-client interaction mechanisms at the atomic scale.

  6. Insight into the assembly of chaperones

    Energy Technology Data Exchange (ETDEWEB)

    May, R.P. [Institut Max von Laue - Paul Langevin (ILL), 38 - Grenoble (France); Stegmann, R.; Manakova, E.; Roessle, M.; Hermann, T.; Heumann, H. [Max-Planck-Institut fuer Biochemie, Martinsried (Germany); Axmann, S.; Plueckthun, A. [Zurich Univ. (Switzerland); Wiedenmann, A. [HMI, Berlin (Germany)

    1997-04-01

    Chaperones are proteins that help other proteins (substrate proteins) to acquire a `good` conformation. The folding is a dynamic process and involves repetitive binding and release of the chaperone components and of the substrate protein. Small-angle neutron scattering is used to investigate the structural changes that appear to happen during the folding process. (author). 2 refs.

  7. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  8. Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

    Directory of Open Access Journals (Sweden)

    Lisa Cadavez

    Full Text Available In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP. The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR, perturbing endoplasmic reticulum (ER homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP or protein disulfite isomerase (PDI, and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA or 4-phenylbutyrate (PBA, alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

  9. Molecular chaperones: The modular evolution of cellular networks

    Indian Academy of Sciences (India)

    2007-03-22

    Mar 22, 2007 ... Molecular chaperones play a prominent role in signaling and transcriptional regulatory networks of the cell. Recent advances uncovered that chaperones act as genetic buffers stabilizing the phenotype of various cells and organisms and may serve as potential regulators of evolvability. Chaperones have ...

  10. Conservative fluid management prevents age-associated ventilator induced mortality.

    Science.gov (United States)

    Herbert, Joseph A; Valentine, Michael S; Saravanan, Nivi; Schneck, Matthew B; Pidaparti, Ramana; Fowler, Alpha A; Reynolds, Angela M; Heise, Rebecca L

    2016-08-01

    Approximately 800 thousand patients require mechanical ventilation in the United States annually with an in-hospital mortality rate of over 30%. The majority of patients requiring mechanical ventilation are over the age of 65 and advanced age is known to increase the severity of ventilator-induced lung injury (VILI) and in-hospital mortality rates. However, the mechanisms which predispose aging ventilator patients to increased mortality rates are not fully understood. Ventilation with conservative fluid management decreases mortality rates in acute respiratory distress patients, but to date there has been no investigation of the effect of conservative fluid management on VILI and ventilator associated mortality rates. We hypothesized that age-associated increases in susceptibility and incidence of pulmonary edema strongly promote age-related increases in ventilator associated mortality. 2month old and 20month old male C57BL6 mice were mechanically ventilated with either high tidal volume (HVT) or low tidal volume (LVT) for up to 4h with either liberal or conservative fluid support. During ventilation, lung compliance, total lung capacity, and hysteresis curves were quantified. Following ventilation, bronchoalveolar lavage fluid was analyzed for total protein content and inflammatory cell infiltration. Wet to dry ratios were used to directly measure edema in excised lungs. Lung histology was performed to quantify alveolar barrier damage/destruction. Age matched non-ventilated mice were used as controls. At 4h, both advanced age and HVT ventilation significantly increased markers of inflammation and injury, degraded pulmonary mechanics, and decreased survival rates. Conservative fluid support significantly diminished pulmonary edema and improved pulmonary mechanics by 1h in advanced age HVT subjects. In 4h ventilations, conservative fluid support significantly diminished pulmonary edema, improved lung mechanics, and resulted in significantly lower mortality rates in

  11. Heat shock protein 90: the cancer chaperone

    Indian Academy of Sciences (India)

    2007-04-02

    Apr 2, 2007 ... Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 ...

  12. Age-associated changes in human hematopoietic stem cells.

    Science.gov (United States)

    Pang, Wendy W; Schrier, Stanley L; Weissman, Irving L

    2017-01-01

    Aging has a broad impact on the function of the human hematopoietic system. This review will focus primarily on the effect of aging on the human hematopoietic stem cell (HSC) population. With age, even though human HSCs increase in number, they have decreased self-renewal capacity and reconstitution potential upon transplantation. As a population, human HSCs become more myeloid-biased in their differentiation potential. This is likely due to the human HSC population becoming more clonal with age, selecting for myeloid-biased HSC clones. The HSC clones that come to predominate with age may also contain disease-causing genetic and epigenetic changes that confer an increased risk of developing into an age-associated clonal hematopoietic disease, such as myelodysplastic syndrome, myeloproliferative disorders, or leukemia. The selection of these aged human HSC clones may be in part due to changes in the aging bone marrow microenvironment. While there have been significant advances in the understanding of the effect of aging on mouse hematopoiesis and mouse HSCs, we have comparatively less detailed analyses of the effect of aging on human HSCs. Continued evaluation of human HSCs in the context of aging will be important to determine how applicable the findings in mice and other model organisms are to the human clinical setting. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Molecular chaperones and hypoxic-ischemic encephalopathy

    Directory of Open Access Journals (Sweden)

    Cong Hua

    2017-01-01

    Full Text Available Hypoxic-ischemic encephalopathy (HIE is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological deficits, with a poor prognosis. The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and inflammation. The molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles. The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress-induced pathway. Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic-ischemic preconditioning have proven effective in HIE patients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, regulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In

  14. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  15. Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome.

    Directory of Open Access Journals (Sweden)

    Melody S Clark

    Full Text Available BACKGROUND: The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. METHODOLOGY/PRINCIPAL FINDINGS: The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp. In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS, providing a resource for population and also gene function studies. CONCLUSIONS: This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding

  16. Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome.

    Science.gov (United States)

    Clark, Melody S; Thorne, Michael A S; Toullec, Jean-Yves; Meng, Yan; Guan, Le Luo; Peck, Lloyd S; Moore, Stephen

    2011-01-06

    The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies. This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding marine ectotherms' capacities to cope with environmental change.

  17. Enzyme enhancement therapy through non-competitive pharmacological chaperones

    OpenAIRE

    Aymami Bofarull, Juan; Barril, Xavier; Delgado, Aida; Reves, Marc; Lavilla, Rodolfo; Higaki, Katsumi; García-Collazo, Anan Maria; Rodríguez-Pascau, Laura; Cubero, Elena; Pizcueta, Pilar; Martinell, Marc

    2014-01-01

    Pharmacological chaperones, Chemical chaperones , Enzyme e n- hancement therapy, GM1, Gangliosidosis, Morqui B, Lysosomal Storage Disease, Lysosomal Storage Disorders Most Pharmacological chaperones (PC’s) described until now are substrate analogues which bind to the active site of the target protein. C ons e- quently, such PC’s also inhibit the target protein at higher concentrations thus rendering a narrow therapeutic window and have poor drug-like proper...

  18. Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways.

    Science.gov (United States)

    Douglas, Peter M; Summers, Daniel W; Cyr, Douglas M

    2009-01-01

    The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.

  19. Chaperone-directed ubiquitylation maintains proteostasis at the expense of longevity.

    Science.gov (United States)

    Pokrzywa, Wojciech; Lorenz, Robin; Hoppe, Thorsten

    2017-01-01

    The integrity of the cellular proteome is supported by quality control networks, which govern protein synthesis, folding, and degradation. It is generally accepted that an age-related decline in protein homeostasis (proteostasis) contributes to protein aggregation diseases. However, the mechanistic principles underlying proteostasis imbalance and the impact on life expectancy are not well understood. We recently demonstrated that this interrelation is affected by chaperone-directed ubiquitylation, shifting the amount of the conserved DAF-2/insulin receptor both in Caenorhabditis elegans and Drosophila melanogaster . The ubiquitin ligase CHIP either targets the membrane bound insulin receptor or misfolded proteins for degradation, which depends on the cellular proteostasis status. Increased proteotoxicity triggers chaperone-assisted redirection of CHIP toward protein aggregates, limiting its capacity to degrade the insulin receptor and prevent premature aging. In light of these findings, we discuss a new concept for understanding the impact of proteome imbalance on longevity risk.

  20. Presence of chaperones during pelvic examinations in southeast ...

    African Journals Online (AJOL)

    Objectives: To assess the opinions, attitude, and preferences of Nigerian women to the presence of chaperones during pelvic examinations. Materials and Methods: A cross‑sectional survey of first time gynecology clinic attendees on their opinions, attitudes, and preferences with respect to the presence of chaperones ...

  1. Modulation of human IAPP fibrillation: cosolutes, crowders and chaperones.

    Science.gov (United States)

    Gao, Mimi; Estel, Kathrin; Seeliger, Janine; Friedrich, Ralf P; Dogan, Susanne; Wanker, Erich E; Winter, Roland; Ebbinghaus, Simon

    2015-04-07

    The cellular environment determines the structure and function of proteins. Marginal changes of the environment can severely affect the energy landscape of protein folding. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). By applying biophysical methods such as thioflavin T fluorescence spectroscopy, fluorescence anisotropy, total reflection Fourier-transform infrared spectroscopy, circular dichroism spectroscopy and atomic force microscopy we analyse and compare their inhibition mechanism. We demonstrate that the fibrillation reaction of human IAPP is strongly inhibited by formation of globular, amorphous assemblies by both, the pharmacological and the protein chaperones. We studied the inhibition mechanism under cell-like conditions by using the artificial crowding agents Ficoll 70 and sucrose. Under such conditions the suppressive effect of proline was decreased, whereas the pharmacological chaperone remains active.

  2. Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

    OpenAIRE

    Douglas, Peter M; Summers, Daniel W; Cyr, Douglas M

    2009-01-01

    The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease protei...

  3. Disaggregases, molecular chaperones that resolubilize protein aggregates

    Directory of Open Access Journals (Sweden)

    David Z. Mokry

    2015-08-01

    Full Text Available The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.

  4. Deletion of the Mitochondrial Chaperone TRAP-1 Uncovers Global Reprogramming of Metabolic Networks

    Directory of Open Access Journals (Sweden)

    Sofia Lisanti

    2014-08-01

    Full Text Available Reprogramming of metabolic pathways contributes to human disease, especially cancer, but the regulators of this process are unknown. Here, we have generated a mouse knockout for the mitochondrial chaperone TRAP-1, a regulator of bioenergetics in tumors. TRAP-1−/− mice are viable and showed reduced incidence of age-associated pathologies, including obesity, inflammatory tissue degeneration, dysplasia, and spontaneous tumor formation. This was accompanied by global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing deregulated mitochondrial respiration, oxidative stress, impaired cell proliferation, and a switch to glycolytic metabolism in vivo. These data identify TRAP-1 as a central regulator of mitochondrial bioenergetics, and this pathway could contribute to metabolic rewiring in tumors.

  5. Histological and morphometric studies on the age-associated ...

    African Journals Online (AJOL)

    Histological and morphometric studies on the age-associated changes in the colon of the mouse. MA Motabagani. Abstract. No Abstract. Journal of Experimental and Clinical Anatomy Vol. 5(1) 2006: 48-58. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  6. Aging-associated renal disease in mice is fructokinase dependent.

    Science.gov (United States)

    Roncal-Jimenez, Carlos A; Ishimoto, Takuji; Lanaspa, Miguel A; Milagres, Tamara; Hernando, Ana Andres; Jensen, Thomas; Miyazaki, Makoto; Doke, Tomohito; Hayasaki, Takahiro; Nakagawa, Takahiko; Marumaya, Shoichi; Long, David A; Garcia, Gabriela E; Kuwabara, Masanari; Sánchez-Lozada, Laura G; Kang, Duk-Hee; Johnson, Richard J

    2016-10-01

    Aging-associated kidney disease is usually considered a degenerative process associated with aging. Recently, it has been shown that animals can produce fructose endogenously, and that this can be a mechanism for causing kidney damage in diabetic nephropathy and in association with recurrent dehydration. We therefore hypothesized that low-level metabolism of endogenous fructose might play a role in aging-associated kidney disease. Wild-type and fructokinase knockout mice were fed a normal diet for 2 yr that had minimal (renal injury was amplified by provision of high-salt diet for 3 wk, as noted by the presence of glomerular hypertrophy, mesangial matrix expansion, and alpha smooth muscle actin expression, and with segmental thrombi. Fructokinase knockout mice were protected from renal injury both at baseline and after high salt intake (3 wk) compared with wild-type mice. This was associated with higher levels of active (phosphorylated serine 1177) endothelial nitric oxide synthase in their kidneys. These studies suggest that aging-associated renal disease might be due to activation of specific metabolic pathways that could theoretically be targeted therapeutically, and raise the hypothesis that aging-associated renal injury may represent a disease process as opposed to normal age-related degeneration.

  7. Aging-associated oxidative stress inhibits liver progenitor cell activation in mice.

    Science.gov (United States)

    Cheng, Yiji; Wang, Xue; Wang, Bei; Zhou, Hong; Dang, Shipeng; Shi, Yufang; Hao, Li; Luo, Qingquan; Jin, Min; Zhou, Qianjun; Zhang, Yanyun

    2017-04-29

    Recent studies have discovered aging-associated changes of adult stem cells in various tissues and organs, which potentially contribute to the organismal aging. However, aging-associated changes of liver progenitor cells (LPCs) remain elusive. Employing young (2-month-old) and old (24-month-old) mice, we found diverse novel alterations in LPC activation during aging. LPCs in young mice could be activated and proliferate upon liver injury, whereas the counterparts in old mice failed to respond and proliferate, leading to the impaired liver regeneration. Surprisingly, isolated LPCs from young and old mice did not exhibit significant difference in their clonogenic and proliferative capacity. Later, we uncovered that the decreased activation and proliferation of LPCs were due to excessive reactive oxygen species produced by neutrophils infiltrated into niche, which was resulted from chemokine production from activated hepatic stellate cells during aging. This study demonstrates aging-associated changes in LPC activation and reveals critical roles for the stem cell niche, including neutrophils and hepatic stellate cells, in the negative regulation of LPCs during aging.

  8. The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases

    OpenAIRE

    Rachel E. Lackie; Rachel E. Lackie; Andrzej Maciejewski; Andrzej Maciejewski; Valeriy G. Ostapchenko; Jose Marques-Lopes; Wing-Yiu Choy; Martin L. Duennwald; Vania F. Prado; Vania F. Prado; Vania F. Prado; Vania F. Prado; Marco A. M. Prado; Marco A. M. Prado; Marco A. M. Prado

    2017-01-01

    The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide—either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degrada...

  9. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2012-02-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  10. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2010-05-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  11. Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo

    Directory of Open Access Journals (Sweden)

    Baik L. Seong

    2011-03-01

    Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

  12. Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

    Science.gov (United States)

    Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

    2017-07-04

    Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Age-associated changes in cardiovascular structure and function: a fertile milieu for future disease

    Science.gov (United States)

    Strait, James

    2015-01-01

    Important changes occur in the cardiovascular system with advancing age, even in apparently healthy individuals. Thickening and stiffening of the large arteries develop due to collagen and calcium deposition and loss of elastic fibers in the medial layer. These arterial changes cause systolic blood pressure to rise with age, while diastolic blood pressure generally declines after the sixth decade. In the left ventricle, modest concentric wall thickening occurs due to cellular hypertrophy, but cavity size does not change. Although left ventricular systolic function is preserved across the age span, early diastolic filling rate declines 30–50% between the third and ninth decades. Conversely, an age-associated increase in late diastolic filling due to atrial contraction preserves end-diastolic volume. Aerobic exercise capacity declines approximately 10% per decade in cross-sectional studies; in longitudinal studies, however, this decline is accelerated in the elderly. Reductions in peak heart rate and peripheral oxygen utilization but not stroke volume appear to mediate the age-associated decline in aerobic capacity. Deficits in both cardiac b-adrenergic receptor density and in the efficiency of postsynaptic b-adrenergic signaling contribute significantly to the reduced cardiovascular performance during exercise in older adults. Although these cardiovascular aging changes are considered “normative”, they lower the threshold for the development of cardiovascular disease, which affects the majority of older adults. PMID:21809160

  14. Age-associated changes in cardiovascular structure and function: a fertile milieu for future disease.

    Science.gov (United States)

    Fleg, Jerome L; Strait, James

    2012-09-01

    Important changes occur in the cardiovascular system with advancing age, even in apparently healthy individuals. Thickening and stiffening of the large arteries develop due to collagen and calcium deposition and loss of elastic fibers in the medial layer. These arterial changes cause systolic blood pressure to rise with age, while diastolic blood pressure generally declines after the sixth decade. In the left ventricle, modest concentric wall thickening occurs due to cellular hypertrophy, but cavity size does not change. Although left ventricular systolic function is preserved across the age span, early diastolic filling rate declines 30-50% between the third and ninth decades. Conversely, an age-associated increase in late diastolic filling due to atrial contraction preserves end-diastolic volume. Aerobic exercise capacity declines approximately 10% per decade in cross-sectional studies; in longitudinal studies, however, this decline is accelerated in the elderly. Reductions in peak heart rate and peripheral oxygen utilization but not stroke volume appear to mediate the age-associated decline in aerobic capacity. Deficits in both cardiac β-adrenergic receptor density and in the efficiency of postsynaptic β-adrenergic signaling contribute significantly to the reduced cardiovascular performance during exercise in older adults. Although these cardiovascular aging changes are considered "normative", they lower the threshold for the development of cardiovascular disease, which affects the majority of older adults.

  15. Evaluation of structure, chaperone-like activity and protective ability of peroxynitrite modified human α-Crystallin subunits against copper-mediated ascorbic acid oxidation.

    Science.gov (United States)

    Ghahramani, Maryam; Yousefi, Reza; Khoshaman, Kazem; Moghadam, Sogand Sasan; Kurganov, Boris I

    2016-06-01

    The copper-catalyzed oxidation of ascorbic acid (ASA) to dehydroascorbate (DHA) and hydrogen peroxide plays a central role in pathology of cataract diseases during ageing and in diabetic patients. In the current study, the structural feature, chaperone-like activity and protective ability of peroxynitrite (PON) modified αA- and αB-Crystallin (Cry) against copper-mediated ASA oxidation were studied using different spectroscopic measurements and gel mobility shift assay. Upon PON modification, additional to protein structural alteration, the contents of nitrotyrosine, nitrotryptophan, dityrosine and carbonyl groups were significantly increased. Moreover, αB-Cry demonstrates significantly larger capacity for PON modification than αA-Cry. Also, based on the extent of PON modification, these proteins may display an improved chaperone-like activity and enhanced protective ability against copper-mediated ASA oxidation. In the presence of copper ions, chaperone-like activity of both native and PON-modified α-Cry subunits were appreciably improved. Additionally, binding of copper ions to native and PON-modified proteins results in the significant reduction of their solvent exposed hydrophobic patches. Overall, the increase in chaperone-like activity/ASA protective ability of PON-modified α-Cry and additional enhancement of its chaperoning action with copper ions appear to be an important defense mechanism offered by this protein. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Patterns of Age-Associated Degeneration Differ in Shoulder Muscles

    Science.gov (United States)

    Raz, Yotam; Henseler, Jan F.; Kolk, Arjen; Riaz, Muhammad; van der Zwaal, Peer; Nagels, Jochem; Nelissen, Rob G. H. H.; Raz, Vered

    2015-01-01

    Shoulder complaints are common in the elderly and hamper daily functioning. These complaints are often caused by tears in the muscle-tendon units of the rotator cuff (RC). The four RC muscles stabilize the shoulder joint. While some RC muscles are frequently torn in shoulder complaints others remain intact. The pathological changes in RC muscles are poorly understood. We investigated changes in RC muscle pathology combining radiological and histological procedures. We measured cross sectional area (CSA) and fatty infiltration from Magnetic Resonance Imaging with Arthrography (MRA) in subjects without (N = 294) and with (N = 109) RC-tears. Normalized muscle CSA of the four RC muscles and the deltoid shoulder muscle were compared and age-associated patterns of muscle atrophy and fatty infiltration were constructed. We identified two distinct age-associated patterns: in the supraspinatus and subscapularis RC muscles CSAs continuously declined throughout adulthood, whereas in the infraspinatus and deltoid reduced CSA was prominent from midlife onwards. In the teres minor, CSA was unchanged with age. Most importantly, age-associated patterns were highly similar between subjects without RC tear and those with RC-tears. This suggests that extensive RC muscle atrophy during aging could contribute to RC pathology. We compared muscle pathology between torn infraspinatus and non-torn teres minor and the deltoid in two patients with a massive RC-tear. In the torn infraspinatus we found pronounced fatty droplets, an increase in extracellular collagen-1, a loss of myosin heavy chain-1 expression in myofibers and an increase in Pax7-positive cells. However, the adjacent intact teres minor and deltoid exhibited healthy muscle features. This suggests that satellite cells and the extracellular matrix may contribute to extensive muscle fibrosis in torn RC. We suggest that torn RC muscles display hallmarks of muscle aging whereas the teres minor could represent an aging

  17. Patterns of age-associated degeneration differ in shoulder muscles

    Directory of Open Access Journals (Sweden)

    Yotam eRaz

    2015-12-01

    Full Text Available Shoulder complaints are common in the elderly and hamper daily functioning. These complaints are often caused by tears in the muscle-tendon units of the rotator cuff (RC. The four RC muscles stabilize the shoulder joint. While some RC muscles are frequently torn in shoulder complaints others remain intact. The pathological changes in RC muscles are poorly understood. We investigated changes in RC muscle pathology combining radiological and histological procedures. We measured cross sectional area (CSA and fatty infiltration from Magnetic Resonance Imaging with Arthrography in subjects without (N=294 and with (N=109 RC-tears. Normalized muscle CSA of the four RC muscles and the deltoid shoulder muscle were compared and age-associated patterns of muscle atrophy and fatty infiltration were constructed. We identified two distinct age-associated patterns: in the supraspinatus and subscapularis RC muscles CSAs continuously declined throughout adulthood, whereas in the infraspinatus and deltoid reduced CSA was prominent from midlife onwards. In the teres minor, CSA was unchanged with age. Most importantly, age-associated patterns were highly similar between subjects without RC tear and those with RC-tears. This suggests that extensive RC muscle atrophy during aging could contribute to RC pathology. We compared muscle pathology between torn infraspinatus and non-torn teres minor and the deltoid in two patients with a massive RC-tear. In the torn infraspinatus we found pronounced fatty droplets, an increase in extracellular collagen-1, a loss of myosin heavy chain-1 expression in myofibers and an increase in Pax7-positive cells. However, the adjacent intact teres minor and deltoid exhibited healthy muscle features. This suggests that satellite cells and the extracellular matrix may contribute to extensive muscle fibrosis in torn RC. We suggest that torn RC muscles display hallmarks of muscle aging whereas the teres minor could represent an aging

  18. Patterns of Age-Associated Degeneration Differ in Shoulder Muscles.

    Science.gov (United States)

    Raz, Yotam; Henseler, Jan F; Kolk, Arjen; Riaz, Muhammad; van der Zwaal, Peer; Nagels, Jochem; Nelissen, Rob G H H; Raz, Vered

    2015-01-01

    Shoulder complaints are common in the elderly and hamper daily functioning. These complaints are often caused by tears in the muscle-tendon units of the rotator cuff (RC). The four RC muscles stabilize the shoulder joint. While some RC muscles are frequently torn in shoulder complaints others remain intact. The pathological changes in RC muscles are poorly understood. We investigated changes in RC muscle pathology combining radiological and histological procedures. We measured cross sectional area (CSA) and fatty infiltration from Magnetic Resonance Imaging with Arthrography (MRA) in subjects without (N = 294) and with (N = 109) RC-tears. Normalized muscle CSA of the four RC muscles and the deltoid shoulder muscle were compared and age-associated patterns of muscle atrophy and fatty infiltration were constructed. We identified two distinct age-associated patterns: in the supraspinatus and subscapularis RC muscles CSAs continuously declined throughout adulthood, whereas in the infraspinatus and deltoid reduced CSA was prominent from midlife onwards. In the teres minor, CSA was unchanged with age. Most importantly, age-associated patterns were highly similar between subjects without RC tear and those with RC-tears. This suggests that extensive RC muscle atrophy during aging could contribute to RC pathology. We compared muscle pathology between torn infraspinatus and non-torn teres minor and the deltoid in two patients with a massive RC-tear. In the torn infraspinatus we found pronounced fatty droplets, an increase in extracellular collagen-1, a loss of myosin heavy chain-1 expression in myofibers and an increase in Pax7-positive cells. However, the adjacent intact teres minor and deltoid exhibited healthy muscle features. This suggests that satellite cells and the extracellular matrix may contribute to extensive muscle fibrosis in torn RC. We suggest that torn RC muscles display hallmarks of muscle aging whereas the teres minor could represent an aging

  19. Inhibitors of the AAA+ Chaperone p97

    Directory of Open Access Journals (Sweden)

    Eli Chapman

    2015-02-01

    Full Text Available It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®, which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+ chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and

  20. Fibroblast screening for chaperone therapy in beta-galactosidosis.

    Science.gov (United States)

    Iwasaki, Hiroyuki; Watanabe, Hiroshi; Iida, Masami; Ogawa, Seiichiro; Tabe, Miho; Higaki, Katsumi; Nanba, Eiji; Suzuki, Yoshiyuki

    2006-09-01

    We performed screening of beta-galactosidase-deficient fibroblasts for possible chemical chaperone therapy using N-octyl-4-epi-beta-valienamine (NOEV) in patients with GM1-gangliosidosis and Morquio B disease (beta-galactosidosis). Fibroblasts were cultured with NOEV for 4 days and beta-galactosidase activity was measured. Mutation analysis was performed simultaneously. Two separate criteria were set for evaluation of the chaperone effect: a relative increase of enzyme activity (more than 3-fold), and an increase up to more than 10% normal enzyme activity. Among the 50 fibroblast strains tested, more than 3-fold increase was achieved in 17 cell strains (34%), and more than 10% normal activity in 10 (20%). Both criteria were satisfied in 6 (12%), and either of them in 21 (42%). Juvenile GM1-gangliosidosis was most responsive, and then infantile GM1-gangliosidosis. This enhancement was mutation-specific. We estimate that the NOEV chaperone therapy will be effective in 20-40% of the patients, mainly in juvenile and infantile GM1-gangliosidosis patients. A molecular design may produce mutation-specific chaperone compounds for the other disease phenotypes. This cellular screening will be useful for identification of human patients with beta-galactosidase deficiency for chaperone therapy to be started in the near future.

  1. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

  2. Bioactive silica nanoparticles reverse age-associated bone loss in mice.

    Science.gov (United States)

    Weitzmann, M Neale; Ha, Shin-Woo; Vikulina, Tatyana; Roser-Page, Susanne; Lee, Jin-Kyu; Beck, George R

    2015-05-01

    We recently reported that in vitro, engineered 50nm spherical silica nanoparticles promote the differentiation and activity of bone building osteoblasts but suppress bone-resorbing osteoclasts. Furthermore, these nanoparticles promote bone accretion in young mice in vivo. We have now investigated the capacity of these nanoparticles to reverse bone loss in aged mice, a model of human senile osteoporosis. Aged mice received nanoparticles weekly and bone mineral density (BMD), bone structure, and bone turnover were quantified. Our data revealed a significant increase in BMD, bone volume, and biochemical markers of bone formation. Biochemical and histological examinations failed to identify any abnormalities caused by nanoparticle administration. Our studies demonstrate that silica nanoparticles effectively blunt and reverse age-associated bone loss in mice by a mechanism involving promotion of bone formation. The data suggest that osteogenic silica nanoparticles may be a safe and effective therapeutic for counteracting age-associated bone loss. Osteoporosis poses a significant problem in the society. Based on their previous in-vitro findings, the authors' group investigated the effects of spherical silica nanoparticles in reversing bone loss in a mouse model of osteoporosis. The results showed that intra-peritoneal injections of silica nanoparticles could increase bone mineral density, with little observed toxic side effects. This novel method may prove important in future therapy for combating osteoporosis. Published by Elsevier Inc.

  3. Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures.

    Directory of Open Access Journals (Sweden)

    Marquiza Sablón-Carrazana

    chaperones are able to protect and recondition the cerebellar granule cells (CGC from the cytotoxicity produced by the hIAPP20-29 fragment or by a low potassium medium, regardless of their capacity for accelerating or inhibiting in vitro formation of fibers. In vivo animal experiments are required to study the impact of chemical chaperones in cognitive and metabolic syndromes.

  4. The Hsp90 chaperone in action: following the ATP cycle of a molecular machine

    NARCIS (Netherlands)

    Karagoz, G.E.

    2011-01-01

    Protein folding in the cell is assisted by molecular chaperones. Hsp90 is the most abundant molecular chaperone in the cytosol. It facilitates the folding and activation of mainly signalling molecules. Its chaperoning of regulatory proteins places Hsp90 in the cross road of several important

  5. The small heat shock proteins family : The long forgotten chaperones

    NARCIS (Netherlands)

    Garrido, C.; Paul, C.; Seigneuric, R.; Kampinga, H. H.

    2012-01-01

    Small heat shock proteins are a rather heterogeneous family of ATP-independent chaperones, some of which have been proven to block protein aggregation and help the cells to survive stressful conditions. Although much less studied than high molecular weight HSPs like HSP70/HSPA or HSP90/HSPC, their

  6. Hsp100/ClpB Chaperone Function and Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Vierling, Elizabeth [Univ. of Massachusetts, Amherst, MA (United States). Dept. of Biochemistry and Molecular Biology

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  7. Mitochondrial Chaperones in the Brain: Safeguarding Brain Health and Metabolism?

    Directory of Open Access Journals (Sweden)

    José Pedro Castro

    2018-04-01

    Full Text Available The brain orchestrates organ function and regulates whole body metabolism by the concerted action of neurons and glia cells in the central nervous system. To do so, the brain has tremendously high energy consumption and relies mainly on glucose utilization and mitochondrial function in order to exert its function. As a consequence of high rate metabolism, mitochondria in the brain accumulate errors over time, such as mitochondrial DNA (mtDNA mutations, reactive oxygen species, and misfolded and aggregated proteins. Thus, mitochondria need to employ specific mechanisms to avoid or ameliorate the rise of damaged proteins that contribute to aberrant mitochondrial function and oxidative stress. To maintain mitochondria homeostasis (mitostasis, cells evolved molecular chaperones that shuttle, refold, or in coordination with proteolytic systems, help to maintain a low steady-state level of misfolded/aggregated proteins. Their importance is exemplified by the occurrence of various brain diseases which exhibit reduced action of chaperones. Chaperone loss (expression and/or function has been observed during aging, metabolic diseases such as type 2 diabetes and in neurodegenerative diseases such as Alzheimer’s (AD, Parkinson’s (PD or even Huntington’s (HD diseases, where the accumulation of damage proteins is evidenced. Within this perspective, we propose that proper brain function is maintained by the joint action of mitochondrial chaperones to ensure and maintain mitostasis contributing to brain health, and that upon failure, alter brain function which can cause metabolic diseases.

  8. Modulation of chaperone-like and membranolytic activities of major ...

    Indian Academy of Sciences (India)

    L-Carnitine is a small polar molecule present in high concentrations inmammalian seminal plasma. The present results demonstrate that L-carnitine binds to HSP-1/2 and increases its thermalstability, enhances cooperativity of its chemical unfolding and decreases both chaperone-like and membranolytic activitiesof this ...

  9. Modulation of chaperone-like and membranolytic activities of major ...

    Indian Academy of Sciences (India)

    C Sudheer Kumar

    2017-06-20

    Jun 20, 2017 ... Modulation of chaperone-like and membranolytic activities of major horse seminal plasma protein HSP-1/2 by L-carnitine. C SUDHEER KUMAR and MUSTI J SWAMY. ,*. School of Chemistry, University of Hyderabad, Hyderabad 500 046, India. *Corresponding author (Email, mjssc@uohyd.ernet.in, ...

  10. Influence of different concentrations of two chemical chaperones on ...

    African Journals Online (AJOL)

    www.nasle-javan.com

    2012-06-21

    Jun 21, 2012 ... The natural osmolytes such as glycerol are thought to enhance correct folding of the mutant proteins. (Bonapace et al., 2004). The aim of this study was to investigate the possible roles of different concentrations of two chemical chaperones named glycerol and spermine on human islet amyloid polypeptide ...

  11. Presence of chaperones during pelvic examinations in southeast ...

    African Journals Online (AJOL)

    2012-12-12

    Dec 12, 2012 ... Results:One hundred and nineteen (51.7%) of the respondents preferred female physicians for pelvic examination, 23 (10%) preferred male ... by a female physician or to be attended by a nurse chaperone if the examining physician is a male. We recommend a .... assistants conducted the interviews.

  12. Challenging muscle homeostasis uncovers novel chaperone interactions in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Anna eFrumkin

    2014-11-01

    Full Text Available Proteome stability is central to cellular function and the lifespan of an organism. This is apparent in muscle cells, where incorrect folding and assembly of the sarcomere contributes to disease and aging. Apart from the myosin-assembly factor UNC-45, the complete network of chaperones involved in assembly and maintenance of muscle tissue is currently unknown. To identify additional factors required for sarcomere quality control, we performed genetic screens based on suppressed or synthetic motility defects in Caenorhabditis elegans. In addition to ethyl methyl sulfonate-based mutagenesis, we employed RNAi-mediated knockdown of candidate chaperones in unc-45 temperature-sensitive mutants and screened for impaired movement at permissive conditions. This approach confirmed the cooperation between UNC-45 and Hsp90. Moreover, the screens identified three novel co-chaperones, CeHop (STI-1, CeAha1 (C01G10.8 and Cep23 (ZC395.10, required for muscle integrity. The specific identification of Hsp90 and Hsp90 co-chaperones highlights the physiological role of Hsp90 in myosin folding. Our work thus provides a clear example of how a combination of mild perturbations to the proteostasis network can uncover specific quality control modules.

  13. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that. FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, ...

  14. Molecular chaperones and proteostasis regulation during redox imbalance☆

    Science.gov (United States)

    Niforou, Katerina; Cheimonidou, Christina; Trougakos, Ioannis P.

    2014-01-01

    Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones. PMID:24563850

  15. Molecular chaperones and proteostasis regulation during redox imbalance.

    Science.gov (United States)

    Niforou, Katerina; Cheimonidou, Christina; Trougakos, Ioannis P

    2014-01-01

    Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones.

  16. Molecular chaperones and proteostasis regulation during redox imbalance

    Directory of Open Access Journals (Sweden)

    Katerina Niforou

    2014-01-01

    Full Text Available Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome network (PN. PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer. In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones.

  17. Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

    OpenAIRE

    Slonim, L N; Pinkner, J S; Brändén, C I; Hultgren, S J

    1992-01-01

    The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pi...

  18. ANTIOXIDANT SUPPLEMENTATION IN THE TREATMENT OF AGING-ASSOCIATED DISEASES

    Directory of Open Access Journals (Sweden)

    Valeria eConti

    2016-02-01

    Full Text Available Oxidative stress is generally considered an imbalance between pro- and antioxidants species, which often results into indiscriminate and global damage at the organismal level. Elderly people are more susceptible to oxidative stress and this depends, almost in part, from a decreased performance of their endogenous antioxidant system. As many studies reported an inverse correlation between systemic levels of antioxidants and several diseases, primarily cardiovascular diseases, but also diabetes and neurological disorders, antioxidant supplementation has been foreseen as an effective preventive and therapeutic intervention for aging-associated pathologies. However, the expectations of this therapeutic approach have often been partially disappointed by clinical trials. The interplay of both endogenous and exogenous antioxidants with the systemic redox system is very complex and represents an issue that is still under debate. In this review a selection of recent clinical studies concerning antioxidants supplementation and the evaluation of their influence in aging-related diseases is analyzed. The controversial outcomes of the antioxidants supplementation therapy that might partially depend, among others, from an underestimation of the patient specific metabolic demand and genetic background, are presented.

  19. Aging-associated excess formaldehyde leads to spatial memory deficits

    Science.gov (United States)

    Tong, Zhiqian; Han, Chanshuai; Luo, Wenhong; Li, Hui; Luo, Hongjun; Qiang, Min; Su, Tao; Wu, Beibei; Liu, Ying; Yang, Xu; Wan, You; Cui, Dehua; He, Rongqiao

    2013-01-01

    Recent studies show that formaldehyde participates in DNA demethylation/methylation cycle. Emerging evidence identifies that neuronal activity induces global DNA demethylation and re-methylation; and DNA methylation is a critical step for memory formation. These data suggest that endogenous formaldehyde may intrinsically link learning-responsive DNA methylation status and memory formation. Here, we report that during spatial memory formation process, spatial training induces an initial global DNA demethylation and subsequent re-methylation associated with hippocampal formaldehyde elevation then decline to baseline level in Sprague Dawley rats. Scavenging this elevated formaldehyde by formaldehyde-degrading enzyme (FDH), or enhancing DNA demethylation by a DNA demethylating agent, both led to spatial memory deficits by blocking DNA re-methylation in rats. Furthermore, we found that the normal adult rats intrahippocampally injected with excess formaldehyde can imitate the aged-related spatial memory deficits and global DNA methylation decline. These findings indicate that aging-associated excess formaldheyde contributes to cognitive decline during aging. PMID:23657727

  20. Pharmaceutical Rejuvenation of Age-Associated Decline in Spatial Memory.

    Science.gov (United States)

    Mendelsohn, Andrew R; Larrick, James W

    2016-12-01

    Spatial memory and cognition decline during aging. Montelukast, an FDA approved drug for the treatment of asthma, can restore spatial memory in old rats to levels similar to those of young animals. Treatment improves three hallmarks of aging in the brain: reducing microglial-mediated neuroinflammation, blood-brain barrier (BBB) permeability, and increasing neurogenesis in the hippocampus although not completely to youthful levels. Other aging-associated parameters, such as reduced synaptic density, are not affected, suggesting that anti-aging therapeutics may be further optimized. Montelukast targets leukotriene receptors GPR17 and CysLTR1 and appears to invert leukotriene signaling, converting an inflammatory signal into an anti-inflammatory signal. This acts as a dominant factor to overcome the dysfunctional effects of aging reportedly mediated, in part, by blood-borne factors such as beta-2 microglobulin that inhibit neurogenesis in the dentate gyrus of the hippocampus. The key mechanism for cognitive improvement by montelukast may be restoration of BBB integrity, which would presumably decrease the amount of deleterious blood-borne factors to enter the brain. Whether or not this hypothesis is true for montelukast, drugs that restore or maintain BBB integrity may be useful in combating age-related loss of cognitive function.

  1. Railway capacity

    OpenAIRE

    Lavrič, Tadeja

    2012-01-01

    In the first part of the thesis, the concepts of railway capacity and capacity consumption are defined. Experts have in the past defined railway capacity in many different ways. This thesis outlines some definitions of railway capacity and capacity consumption, including the definition of railway capacity in accordance with the UIC code 406 (2004), given by the International Union of Railways in order to standardize the definition of railway capacity. Capacity and capacity consumption are dir...

  2. Chaperone use during intimate examinations in primary care: postal survey of family physicians

    Directory of Open Access Journals (Sweden)

    Upshur Ross EG

    2005-12-01

    Full Text Available Abstract Background Physicians have long been advised to have a third party present during certain parts of a physical examination; however, little is known about the frequency of chaperone use for those specific intimate examinations regularly performed in primary care. We aimed to determine the frequency of chaperone use among family physicians across a variety of intimate physical examinations for both male and female patients, and also to identify the factors associated with chaperone use. Methods Questionnaires were mailed to a randomly selected sample of 500 Ontario members of the College of Family Physicians of Canada. Participants were asked about their use of chaperones when performing a variety of intimate examinations, namely female pelvic, breast, and rectal exams and male genital and rectal exams. Results 276 of 500 were returned (56%, of which 257 were useable. Chaperones were more commonly used with female patients than with males (t = 9.09 [df = 249], p Conclusion Clinical practice concerning the use of chaperones during intimate exams continues to be discordant with the recommendations of medical associations and medico-legal societies. Chaperones are used by only a minority of Ontario family physicians. Chaperone use is higher for examinations of female patients than of male patients and is highest for female pelvic exams. The availability of a nurse in the clinic to act as a chaperone is associated with more frequent use of chaperones.

  3. Metal chaperones: a holistic approach to the treatment of AD

    Directory of Open Access Journals (Sweden)

    Paul Anthony Adlard

    2012-03-01

    Full Text Available As the burden of proof for the role of metal ion dysregulation in the pathogenesis of multiple CNS disorders grows, it has become important to more precisely identify and differentiate the biological effects of various pharmacological modulators of metal ion homeostasis. This is particularly evident in disorders such as Alzheimer’s disease, where the use of metal chaperones (that transport metals, as opposed to chelators (which exclude metals from biological interactions, may prove to be the first truly disease modifying approach for this condition. The purpose of this mini-review is to highlight the emerging notion that metal chaperones, such as PBT2 (Prana Biotechnology, modulate a variety of critical pathways affecting key aspects of the AD cascade to provide a more holistic approach to the treatment of this disease.

  4. Structural Characterization of Clusterin-Chaperone Client Protein Complexes*

    OpenAIRE

    Wyatt, Amy R.; Yerbury, Justin J.; Wilson, Mark R.

    2009-01-01

    Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-tr...

  5. Cloning and molecular characterization of a copper chaperone gene ...

    African Journals Online (AJOL)

    The cDNA encoding a copper chaperone, designated as HbCCH1, was isolated from Hevea brasiliensis. HbCC1 was 589 bp long containing a 261 bp open reading frame encoding a putative protein of 86 amino acids, flanked by a 103 bp 5'UTR and a 225 bp 3'UTR. The predicted molecular mass of HbCCH1 was 9.2 kDa, ...

  6. The conformational dynamics of the mitochondrial Hsp70 chaperone.

    Science.gov (United States)

    Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

    2010-04-09

    Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

  7. Transcription elongation factor GreA has functional chaperone activity.

    Science.gov (United States)

    Li, Kun; Jiang, Tianyi; Yu, Bo; Wang, Limin; Gao, Chao; Ma, Cuiqing; Xu, Ping; Ma, Yanhe

    2012-01-01

    Bacterial GreA is an indispensable factor in the RNA polymerase elongation complex. It plays multiple roles in transcriptional elongation, and may be implicated in resistance to various stresses. In this study, we show that Escherichia coli GreA inhibits aggregation of several substrate proteins under heat shock condition. GreA can also effectively promote the refolding of denatured proteins. These facts reveal that GreA has chaperone activity. Distinct from many molecular chaperones, GreA does not form stable complexes with unfolded substrates. GreA overexpression confers the host cells with enhanced resistance to heat shock and oxidative stress. Moreover, GreA expression in the greA/greB double mutant could suppress the temperature-sensitive phenotype, and dramatically alleviate the in vivo protein aggregation. The results suggest that bacterial GreA may act as chaperone in vivo. These results suggest that GreA, in addition to its function as a transcription factor, is involved in protection of cellular proteins against aggregation.

  8. Beta-galactosidase deficiency: an approach to chaperone therapy.

    Science.gov (United States)

    Suzuki, Yoshiyuki

    2006-01-01

    We propose a new molecular therapeutic approach to lysosomal diseases with severe neurological manifestations. Some low-molecular-weight compounds, acting as competitive inhibitors of a lysosomal enzyme in vitro, were found to stabilize and restore catalytic activities of the enzyme molecule as a molecular chaperone. We started this trial first in Fabry disease (generalized vasculopathy) using galactose and 1-deoxygalactonojirimycin, and then in beta-galactosidase deficiency disorders (beta-galactosidosis) with generalized neurosomatic and/or systemic skeletal manifestations (GM(1)-gangliosidosis and Morquio B disease), using a newly developed chemical compound N-octyl-4-epi-beta-valienamine (NOEV). Administration of this chaperone compound resulted in elevation of intracellular enzyme activity in cultured fibroblasts from patients and genetically engineered model mice. In addition, substrate storage was improved after NOEV had been transported into the brain tissue via the blood-brain barrier. We hope this new approach (chemical chaperone therapy) will be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

  9. The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Rachel E. Lackie

    2017-05-01

    Full Text Available The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD. Assembles of beta-amyloid (Aβ peptide—either soluble (oligomers or insoluble (plaques and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a Caenorhabditis elegans model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1, an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrPC. Extracellular STI1 can prevent Aβ toxic signaling by (i interfering with Aβ binding to PrPC and (ii triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in C. elegans can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular

  10. The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases

    Science.gov (United States)

    Lackie, Rachel E.; Maciejewski, Andrzej; Ostapchenko, Valeriy G.; Marques-Lopes, Jose; Choy, Wing-Yiu; Duennwald, Martin L.; Prado, Vania F.; Prado, Marco A. M.

    2017-01-01

    The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide—either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp) family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a Caenorhabditis elegans model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1), an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrPC). Extracellular STI1 can prevent Aβ toxic signaling by (i) interfering with Aβ binding to PrPC and (ii) triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in C. elegans can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular STI1 and the

  11. RNAi-Mediated Reverse Genetic Screen IdentifiedDrosophilaChaperones Regulating Eye and Neuromuscular Junction Morphology.

    Science.gov (United States)

    Raut, Sandeep; Mallik, Bhagaban; Parichha, Arpan; Amrutha, Valsakumar; Sahi, Chandan; Kumar, Vimlesh

    2017-07-05

    Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C -Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development. Copyright © 2017 Raut et al.

  12. RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology

    Directory of Open Access Journals (Sweden)

    Sandeep Raut

    2017-07-01

    Full Text Available Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila. Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development.

  13. Non-equilibrium effects in chaperone-assisted translocation of a stiff polymer

    Science.gov (United States)

    Haji Abdolvahab, Rouhollah

    2018-01-01

    Chaperone-assisted biopolymer translocation is the main model proposed for translocation in vivo. A dynamical Monte Carlo method is used to simulate the translocation of a stiff homopolymer through a nanopore driven by chaperones. Chaperones are proteins that bind to the polymer near the wall and prevent its backsliding through Cis side. The important parameters include binding energy, size and the local concentration of the chaperones. The profile of these local concentrations, build up the chaperones distribution. Here we investigate the effects of binding energy, size and the exponential distribution of chaperones in their equilibration in each step of the polymer translocation needed for stable translocation time. The simulation results show that in case of chaperones with the size of a monomer (λ = 1) and/or positive effective binding energy and/or uniform distribution, the chaperones binding equilibration rate/frequency is less than 5 times per monomer. However, in some special cases in the exponential distribution of chaperones with size λ > 1 and negative effective binding energy the equilibration rate will diverge to more than 20 times per monomer. We show that this non-equilibrium effect results in supper diffusion, seen before. Moreover, we confirm the equilibration process theoretically.

  14. Chaperones and the Proteasome System: Regulating the Construction and Demolition of Striated Muscle

    Directory of Open Access Journals (Sweden)

    Casey Carlisle

    2017-12-01

    Full Text Available Protein folding factors (chaperones are required for many diverse cellular functions. In striated muscle, chaperones are required for contractile protein function, as well as the larger scale assembly of the basic unit of muscle, the sarcomere. The sarcomere is complex and composed of hundreds of proteins and the number of proteins and processes recognized to be regulated by chaperones has increased dramatically over the past decade. Research in the past ten years has begun to discover and characterize the chaperones involved in the assembly of the sarcomere at a rapid rate. Because of the dynamic nature of muscle, wear and tear damage is inevitable. Several systems, including chaperones and the ubiquitin proteasome system (UPS, have evolved to regulate protein turnover. Much of our knowledge of muscle development focuses on the formation of the sarcomere but recent work has begun to elucidate the requirement and role of chaperones and the UPS in sarcomere maintenance and disease. This review will cover the roles of chaperones in sarcomere assembly, the importance of chaperone homeostasis and the cooperation of chaperones and the UPS in sarcomere integrity and disease.

  15. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  16. A thermodynamic assay to test pharmacological chaperones for Fabry disease.

    Science.gov (United States)

    Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria

    2014-03-01

    The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Catapult mechanism renders the chaperone action of Hsp70 unidirectional.

    Science.gov (United States)

    Gisler, S M; Pierpaoli, E V; Christen, P

    1998-06-19

    Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins. The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis. We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions. (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1). (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide. For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1. The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2). Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached. This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding. By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated. Copyright 1998 Academic Press Limited.

  18. Translational autocontrol of the Escherichia coli hfq RNA chaperone gene.

    Science.gov (United States)

    Vecerek, Branislav; Moll, Isabella; Bläsi, Udo

    2005-06-01

    The conserved bacterial RNA chaperone Hfq has been shown to play an important role in post-transcriptional regulation. Here, we demonstrate that Hfq synthesis is autoregulated at the translational level. We have mapped two Hfq binding sites in the 5'-untranslated region of hfq mRNA and show that Hfq binding inhibits formation of the translation initiation complex. In vitro translation and in vivo studies further revealed that Hfq binding to both sites is required for efficient translational repression of hfq mRNA.

  19. Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

    NARCIS (Netherlands)

    Eerde, André van; Hamiaux, Cyril; Pérez, Javier; Parsot, Claude; Dijkstra, Bauke W.

    2004-01-01

    Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15

  20. 25 CFR 39.704 - Are schools eligible to receive chaperone expenses to transport residential students?

    Science.gov (United States)

    2010-04-01

    ....704 Are schools eligible to receive chaperone expenses to transport residential students? Yes. Schools... 25 Indians 1 2010-04-01 2010-04-01 false Are schools eligible to receive chaperone expenses to transport residential students? 39.704 Section 39.704 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE...

  1. Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1

    NARCIS (Netherlands)

    Nollen, E A; Kabakov, A E; Brunsting, J F; Kanon, B; Höhfeld, J; Kampinga, H H

    2001-01-01

    The chaperone activity of Hsp70 is influenced by the activities of both positive and negative regulatory proteins. In this study, we provide first time evidence for the stimulating effect of the Hsp70-interacting protein Hip on the chaperone activity in the mammalian cytosol. Overexpressing Hip

  2. Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

    OpenAIRE

    Colvin, Teresa A; Gabai, Vladimir L; Sherman, Michael Y

    2014-01-01

    Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e....

  3. Small intestinal mucosa expression of putative chaperone fls485

    Directory of Open Access Journals (Sweden)

    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  4. A passive physical model for DnaK chaperoning

    Science.gov (United States)

    Uhl, Lionel; Dumont, Audrey; Dukan, Sam

    2018-03-01

    Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

  5. Degradation of AF1Q by chaperone-mediated autophagy

    International Nuclear Information System (INIS)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru; Li, Huanjie; Cui, Taixing; Li Wang, Xing; Tang, Dongqi; Ji, Chunyan

    2014-01-01

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components

  6. Humanin is an endogenous activator of chaperone-mediated autophagy.

    Science.gov (United States)

    Gong, Zhenwei; Tasset, Inmaculada; Diaz, Antonio; Anguiano, Jaime; Tas, Emir; Cui, Lingguang; Kuliawat, Regina; Liu, Honghai; Kühn, Bernhard; Cuervo, Ana Maria; Muzumdar, Radhika

    2018-02-05

    Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects. © 2018 Gong et al.

  7. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  8. UBL/BAG-domain co-chaperones cause cellular stress upon overexpression through constitutive activation of Hsf1

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl; Kampmeyer, Caroline; Kriegenburg, Franziska

    2017-01-01

    of molecular chaperones and other stress-relieving proteins. Here, we show that the fission yeast Schizosaccharomyces pombe orthologues of human BAG-1, Bag101, and Bag102, are Hsp70 co-chaperones that associate with 26S proteasomes. Only a subgroup of Hsp70-type chaperones, including Ssa1, Ssa2, and Sks2...

  9. Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70.

    Science.gov (United States)

    Colvin, Teresa A; Gabai, Vladimir L; Sherman, Michael Y

    2014-01-01

    Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

  10. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    Directory of Open Access Journals (Sweden)

    Murat Cetinbaş

    Full Text Available Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics.

  11. Sylvie Chaperon, Les origines de la sexologie (1850-1900)

    OpenAIRE

    Anne-Claire Rebreyend

    2008-01-01

    Le mot sexologie apparaît seulement au début des années 1910 en France. Mais dès la seconde moitié du XIXe siècle s’affirme un nouveau savoir médical sur la sexualité, une protosexologie dont Sylvie Chaperon retrace l’histoire par le biais de sources médicales et policières, de romans érotiques, de mémoires. « Premier panorama synthétique » (p. 11) d’une histoire de la sexologie encore lacunaire en France, Les origines de la sexologie pointe la lente émergence d’une nouvelle discipline sur la...

  12. Sylvie Chaperon, Les origines de la sexologie (1850-1900

    Directory of Open Access Journals (Sweden)

    Anne-Claire Rebreyend

    2008-07-01

    Full Text Available Le mot sexologie apparaît seulement au début des années 1910 en France. Mais dès la seconde moitié du XIXe siècle s’affirme un nouveau savoir médical sur la sexualité, une protosexologie dont Sylvie Chaperon retrace l’histoire par le biais de sources médicales et policières, de romans érotiques, de mémoires. « Premier panorama synthétique » (p. 11 d’une histoire de la sexologie encore lacunaire en France, Les origines de la sexologie pointe la lente émergence d’une nouvelle discipline sur la...

  13. Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

    Science.gov (United States)

    Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Sinha Ray, Sougata; Biswas, Ashis

    2015-01-01

    Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

  14. Structural Characterization of Clusterin-Chaperone Client Protein Complexes*

    Science.gov (United States)

    Wyatt, Amy R.; Yerbury, Justin J.; Wilson, Mark R.

    2009-01-01

    Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU ≥ 4 × 107 Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57 ± 18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06 ± 6.87 nm and 52.61 ± 7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins. PMID:19535339

  15. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  16. The FKBP51 Glucocorticoid Receptor Co-Chaperone: Regulation, Function, and Implications in Health and Disease.

    Science.gov (United States)

    Fries, Gabriel R; Gassen, Nils C; Rein, Theo

    2017-12-05

    Among the chaperones and co-chaperones regulating the glucocorticoid receptor (GR), FK506 binding protein (FKBP) 51 is the most intensely investigated across different disciplines. This review provides an update on the role of the different co-chaperones of Hsp70 and Hsp90 in the regulation of GR function. The development leading to the focus on FKBP51 is outlined. Further, a survey of the vast literature on the mechanism and function of FKBP51 is provided. This includes its structure and biochemical function, its regulation on different levels-transcription, post-transcription, and post-translation-and its function in signaling pathways. The evidence portraying FKBP51 as a scaffolding protein organizing protein complexes rather than a chaperone contributing to the folding of individual proteins is collated. Finally, FKBP51's involvement in physiology and disease is outlined, and the promising efforts in developing drugs targeting FKBP51 are discussed.

  17. The Role of the Co-Chaperone, CHIP, in Androgen Independent Prostate Cancer

    National Research Council Canada - National Science Library

    Hassen, Waleed A

    2008-01-01

    Expression of Chip, a Co-Chaperone Which Interacts with the Androgen Receptor, Results in Loss of AR Expression and Growth Inhibition of Prostate Cancer Cells Waleed Hassen, Xiaoyoung Zheng, Antonio...

  18. The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease

    Directory of Open Access Journals (Sweden)

    Erica L. Gorenberg

    2017-05-01

    Full Text Available Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs: Cysteine String Protein alpha (CSPα; DNAJC5, auxilin (DNAJC6, and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13. These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70, enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110, which interacts with Hsc70, DNAJAs, and

  19. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Science.gov (United States)

    Dixit, Anshuman; Verkhivker, Gennady M

    2012-01-01

    Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be

  20. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  1. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

    Energy Technology Data Exchange (ETDEWEB)

    Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo; Miller Jenkins, Lisa M.; Wlodawer, Alexander; Chattoraj, Dhruba; Dunny, Gary M.

    2017-04-18

    Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that

  2. A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation1[OPEN

    Science.gov (United States)

    Pareek, Ashwani; Singla-Pareek, Sneh Lata

    2016-01-01

    Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants. PMID:27342307

  3. The heat-shock protein/chaperone network and multiple stress resistance.

    Science.gov (United States)

    Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

    2017-04-01

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Structural Analysis of Protein Folding by the Long-Chain Archaeal Chaperone FKBP26

    Energy Technology Data Exchange (ETDEWEB)

    E Martinez-Hackert; W Hendrickson

    2011-12-31

    In the cell, protein folding is mediated by folding catalysts and chaperones. The two functions are often linked, especially when the catalytic module forms part of a multidomain protein, as in Methanococcus jannaschii peptidyl-prolyl cis/trans isomerase FKBP26. Here, we show that FKBP26 chaperone activity requires both a 50-residue insertion in the catalytic FKBP domain, also called 'Insert-in-Flap' or IF domain, and an 80-residue C-terminal domain. We determined FKBP26 structures from four crystal forms and analyzed chaperone domains in light of their ability to mediate protein-protein interactions. FKBP26 is a crescent-shaped homodimer. We reason that folding proteins are bound inside the large crescent cleft, thus enabling their access to inward-facing peptidyl-prolyl cis/trans isomerase catalytic sites and ipsilateral chaperone domain surfaces. As these chaperone surfaces participate extensively in crystal lattice contacts, we speculate that the observed lattice contacts reflect a proclivity for protein associations and represent substrate interactions by FKBP26 chaperone domains. Finally, we find that FKBP26 is an exceptionally flexible molecule, suggesting a mechanism for nonspecific substrate recognition.

  5. The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

    Directory of Open Access Journals (Sweden)

    Kris E Spaeth

    2009-09-01

    Full Text Available In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone. Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion". The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

  6. Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones.

    Science.gov (United States)

    Narayan, Priyanka; Meehan, Sarah; Carver, John A; Wilson, Mark R; Dobson, Christopher M; Klenerman, David

    2012-11-20

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer's disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, and amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells, while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes, thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment.

  7. IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle.

    Science.gov (United States)

    Dagdeviren, Sezin; Jung, Dae Young; Friedline, Randall H; Noh, Hye Lim; Kim, Jong Hun; Patel, Payal R; Tsitsilianos, Nicholas; Inashima, Kunikazu; Tran, Duy A; Hu, Xiaodi; Loubato, Marilia M; Craige, Siobhan M; Kwon, Jung Yeon; Lee, Ki Won; Kim, Jason K

    2017-02-01

    Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging-associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18-mo-old transgenic mice with muscle-specific overexpression of IL-10 (M IL10 ) and in wild-type mice during hyperinsulinemic-euglycemic clamping. Despite similar fat mass and energy balance, M IL10 mice were protected from aging-associated insulin resistance with significant increases in glucose infusion rates, whole-body glucose turnover, and skeletal muscle glucose uptake (∼60%; P Friedline, R. H., Noh, H. L., Kim, J. H., Patel, P. R., Tsitsilianos, N., Inashima, K., Tran, D. A., Hu, X., Loubato, M. M., Craige, S. M., Kwon, J. Y., Lee, K. W., Kim, J. K. IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle. © FASEB.

  8. Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs.

    Science.gov (United States)

    Young, Brent; Wertman, Jaime; Dupré, Denis J

    2015-01-01

    G protein-coupled receptors (GPCRs) make up a superfamily of integral membrane proteins that respond to a wide variety of extracellular stimuli, giving them an important role in cell function and survival. They have also proven to be valuable targets in the fight against various diseases. As such, GPCR signal regulation has received considerable attention over the last few decades. With the amplitude of signaling being determined in large part by receptor density at the plasma membrane, several endogenous mechanisms for modulating GPCR expression at the cell surface have come to light. It has been shown that cell surface expression is determined by both exocytic and endocytic processes. However, the body of knowledge surrounding GPCR trafficking from the endoplasmic reticulum to the plasma membrane, commonly known as anterograde trafficking, has considerable room for growth. We focus here on the current paradigms of anterograde GPCR trafficking. We will discuss the regulatory role of both the general and "nonclassical private" chaperone systems in GPCR trafficking as well as conserved motifs that serve as modulators of GPCR export from the endoplasmic reticulum and Golgi apparatus. Together, these topics summarize some of the known mechanisms by which the cell regulates anterograde GPCR trafficking. © 2015 Elsevier Inc. All rights reserved.

  9. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    Science.gov (United States)

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  10. An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP

    Science.gov (United States)

    Siegenthaler, Kevin D; Pareja, Kristeen A; Wang, Jie; Sevier, Carolyn S

    2017-01-01

    Unfavorable redox conditions in the endoplasmic reticulum (ER) can decrease the capacity for protein secretion, altering vital cell functions. While systems to manage reductive stress are well-established, how cells cope with an overly oxidizing ER remains largely undefined. In previous work (Wang et al., 2014), we demonstrated that the chaperone BiP is a sensor of overly oxidizing ER conditions. We showed that modification of a conserved BiP cysteine during stress beneficially alters BiP chaperone activity to cope with suboptimal folding conditions. How this cysteine is reduced to reestablish 'normal' BiP activity post-oxidative stress has remained unknown. Here we demonstrate that BiP's nucleotide exchange factor – Sil1 – can reverse BiP cysteine oxidation. This previously unexpected reductant capacity for yeast Sil1 has potential implications for the human ataxia Marinesco-Sjögren syndrome, where it is interesting to speculate that a disruption in ER redox-signaling (due to genetic defects in SIL1) may influence disease pathology. DOI: http://dx.doi.org/10.7554/eLife.24141.001 PMID:28257000

  11. Probing the Inhibitor versus Chaperone Properties of sp2-Iminosugars towards Human β-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease

    Directory of Open Access Journals (Sweden)

    Teresa Mena-Barragán

    2018-04-01

    Full Text Available A series of sp2-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido, the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 β-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N′-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase. The 1-deoxynojirimycin (DNJ-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM. At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

  12. The suppression of ghrelin signaling mitigates age-associated thermogenic impairment

    Science.gov (United States)

    Aging is associated with severe thermogenic impairment, which contributes to obesity and diabetes in aging. We previously reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), attenuates age-associated obesity and insulin resistance. Ghrelin and obestatin are ...

  13. Therapeutic Potential and Recent Advances of Curcumin in the Treatment of Aging-Associated Diseases

    Directory of Open Access Journals (Sweden)

    Sathish Sundar Dhilip Kumar

    2018-04-01

    Full Text Available Curcumin, a low molecular weight, lipophilic, major yellow natural polyphenolic, and the most well-known plant-derived compound, is extracted from the rhizomes of the turmeric (Curcuma longa plant. Curcumin has been demonstrated as an effective therapeutic agent in traditional medicine for the treatment and prevention of different diseases. It has also shown a wide range of biological and pharmacological effects in drug delivery, and has actively been used for the treatment of aging-associated diseases, including cardiovascular diseases, atherosclerosis, neurodegenerative diseases, cancer, rheumatoid arthritis, ocular diseases, osteoporosis, diabetes, hypertension, chronic kidney diseases, chronic inflammation and infection. The functional application and therapeutic potential of curcumin in the treatment of aging-associated diseases is well documented in the literature. This review article focuses mainly on the potential role of plant-derived natural compounds such as curcumin, their mechanism of action and recent advances in the treatment of aging-associated diseases. Moreover, the review briefly recaps on the recent progress made in the preparation of nanocurcumins and their therapeutic potential in clinical research for the treatment of aging-associated diseases.

  14. Senescence of the adaptive immune system in health and aging-associated autoimmune disease

    NARCIS (Netherlands)

    van der Geest, Kornelis Stephan Mario

    2015-01-01

    Aging of the immune system may contribute to the development of aging-associated autoimmune diseases, such as giant cell arteritis, polymyalgia rheumatica and rheumatoid arthritis. The aim of this thesis was to identify aging-dependent changes of the adaptive immune system that promote autoimmunity

  15. Identification of novel type III secretion chaperone-substrate complexes of Chlamydia trachomatis.

    Science.gov (United States)

    Pais, Sara V; Milho, Catarina; Almeida, Filipe; Mota, Luís Jaime

    2013-01-01

    Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipitation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.

  16. Aging-associated DNA methylation changes in middle-aged individuals: the Young Finns study.

    Science.gov (United States)

    Kananen, L; Marttila, S; Nevalainen, T; Jylhävä, J; Mononen, N; Kähönen, M; Raitakari, O T; Lehtimäki, T; Hurme, M

    2016-02-09

    Chronological aging-associated changes in the human DNA methylome have been studied by multiple epigenome-wide association studies (EWASs). Certain CpG sites have been identified as aging-associated in multiple studies, and the majority of the sites identified in various studies show common features regarding location and direction of the methylation change. However, as a whole, the sets of aging-associated CpGs identified in different studies, even with similar tissues and age ranges, show only limited overlap. In this study, we further explore and characterize CpG sites that show close relationship between their DNA methylation level and chronological age during adulthood and which bear the relationship regardless of blood cell type heterogeneity. In this study, with a multivariable regression model adjusted for cell type heterogeneity, we identified 1202 aging-associated CpG sites (a-CpGs, FDR age range (40 - 49 years). Repeatedly reported a-CpGs located in genes ELOVL2, FHL2, PENK and KLF14 were also identified. Regions with aging-associated hypermethylation were enriched regarding several gene ontology (GO) terms (especially in the cluster of developmental processes), whereas hypomethylated sites showed no enrichment. The genes with higher numbers of a-CpG hits were more often hypermethylated with advancing age. The comparison analysis revealed that of the 1202 a-CpGs identified in the present study, 987 were identified as differentially methylated also between nonagenarians and young adults in a previous study (The Vitality 90+ study), and importantly, the directions of changes were identical in the previous and in the present study. Here we report that aging-associated DNA methylation features can be identified in a middle-aged population with an age range of only 9 years. A great majority of these sites have been previously reported as aging-associated in a population aged 19 to 90 years. Aging is associated with different types of changes in DNA

  17. Carrying Capacity

    DEFF Research Database (Denmark)

    Schroll, Henning; Andersen, Jan; Kjærgård, Bente

    2012-01-01

    A spatial planning act was introduced inIndonesia 1992 and renewed in 2008. It emphasised the planning role of decentralised authorities. The spatial planning act covers both spatial and environmental issues. It defines the concept of carrying capacity and includes definitions of supportive....../cities. Four different sectors (water, food production, waste, and forests) were selected as core areas for decentralised spatial planning. Indicators for SCC and ACC were identified and assessed with regard to relevance and quantifiability. For each of the indicators selected, a legal threshold or guiding...... was introduced inIndonesia 1992 and renewed in 2008. It emphasised the planning role of decentralised authorities. The spatial planning act covers both spatial and environmental issues. It defines the concept of carrying capacity and includes definitions of supportive carrying capacity (SCC) and assimilative...

  18. The role of chaperone-mediated autophagy in huntingtin degradation.

    Directory of Open Access Journals (Sweden)

    Lin Qi

    Full Text Available Huntington Disease (HD is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt. HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA components, heat shock protein cognate 70 (Hsc70 and lysosome-associated protein 2A (LAMP-2A in degradation of Htt fragment 1-552aa (Htt-552. A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.

  19. Biologic activities of molecular chaperones and pharmacologic chaperone imidazole-containing dipeptide-based compounds: natural skin care help and the ultimate challenge: implication for adaptive responses in the skin.

    Science.gov (United States)

    Babizhayev, Mark A; Nikolayev, Gennady M; Nikolayeva, Juliana G; Yegorov, Yegor E

    2012-03-01

    Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of photoaged skin and pathogenesis of human cutaneous disease. Growing evidence demonstrates the ability of molecular chaperone proteins and of pharmacologic chaperones to decrease the environmental stress and ameliorate the oxidation stress-related and glycation disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for skin diseases and aging. In this review, we examine the evidence suggesting a role for molecular chaperone proteins in the skin and their inducer and protecting agents: pharmacologic chaperone imidazole dipeptide-based agents (carcinine and related compounds) in cosmetics and dermatology. Furthermore, we discuss the use of chaperone therapy for the treatment of skin photoaging diseases and other skin pathologies that have a component of increased glycation and/or free radical-induced oxidation in their genesis. We examine biologic activities of molecular and pharmacologic chaperones, including strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human skin disease. This allows the protein to function and traffic to the appropriate location in the skin, thereby increasing protein activity and cellular function and reducing stress on skin cells. The benefits of imidazole dipeptide antioxidants with transglycating activity (such as carcinine) in skin care are that they help protect and repair cell membrane damage and help retain youthful, younger-looking skin. All skin types will benefit from daily, topical application of pharmacologic chaperone antioxidants, anti-irritants, in combination with water-binding protein agents that work to mimic the structure and function of healthy skin. General strategies are presented addressing ground techniques to improve absorption of usually active chaperone proteins and dipeptide compounds, include

  20. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    Science.gov (United States)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  1. Chaperone activity of human small heat shock protein-GST fusion proteins.

    Science.gov (United States)

    Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

    2017-07-01

    Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

  2. Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.

    Science.gov (United States)

    Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M; Onikubo, Takashi; Cahill, Sean; Brenowitz, Michael; Cowburn, David; Girvin, Mark; Shechter, David

    2017-12-20

    Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

  3. Engineering and evolution of molecular chaperones and protein disaggregases with enhanced activity

    Directory of Open Access Journals (Sweden)

    Korrie eMack

    2016-03-01

    Full Text Available Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson’s disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector.

  4. Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima

    2018-01-01

    The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

    Directory of Open Access Journals (Sweden)

    Thomas eSimmen

    2014-10-01

    Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

  6. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  7. Molecular Chaperones of Leishmania: Central Players in Many Stress-Related and -Unrelated Physiological Processes

    Directory of Open Access Journals (Sweden)

    Jose M. Requena

    2015-01-01

    Full Text Available Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Folding of nascent polypeptides is supported by molecular chaperones, which avoid the formation of aggregates by preventing nonspecific interactions and aid, when necessary, the translocation of proteins to their correct intracellular localization. Furthermore, when proteins are damaged, molecular chaperones may also facilitate their refolding or, in the case of irreparable proteins, their removal by the protein degradation machinery of the cell. During their digenetic lifestyle, Leishmania parasites encounter and adapt to harsh environmental conditions, such as nutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature; all these factors are potential triggers of cellular stress. We summarize here our current knowledge on the main types of molecular chaperones in Leishmania and their functions. Among them, heat shock proteins play important roles in adaptation and survival of this parasite against temperature changes associated with its passage from the poikilothermic insect vector to the warm-blooded vertebrate host. The study of structural features and the function of chaperones in Leishmania biology is providing opportunities (and challenges for drug discovery and improving of current treatments against leishmaniasis.

  8. Station Capacity

    DEFF Research Database (Denmark)

    Landex, Alex

    2011-01-01

    the probability of conflicts and the minimum headway times into account. The last method analyzes how optimal platform tracks are used by examining the arrival and departure pattern of the trains. The developed methods can either be used separately to analyze specific characteristics of the capacity of a station...

  9. Mnemonics usage and cognitive decline in age-associated memory impairment.

    Science.gov (United States)

    Small, G W; La Rue, A; Komo, S; Kaplan, A; Mandelkern, M A

    1997-03-01

    To determine predictors of cognitive deterioration, the authors performed baseline and 1- to 5-year follow-up (mean +/- SD = 2.5 +/- 1.2 years) neuropsychological assessments on 36 persons (mean age +/- SD = 62.1 +/- 8.0; range = 50 to 81 years) with age-associated memory impairment. Subjects were recruited from a larger group of volunteers, had minimal medical comorbidity, and 25 of them had a family history of Alzheimer's disease. Baseline age and a subjective memory measure indicating reported frequency of mnemonics usage were significant decline predictors. Subjects reporting more frequent mnemonics use at baseline were more likely to show objective cognitive decline at follow-up. Baseline full-scale IQ, educational level, and family history of Alzheimer's disease failed to predict decline. These findings suggest that although age is the strongest decline predictor in some people with age-associated memory impairment, self-perception of memory function may also predict subsequent cognitive loss.

  10. Side effects of chaperone gene co-expression in recombinant protein production

    Directory of Open Access Journals (Sweden)

    Martínez-Alonso Mónica

    2010-09-01

    Full Text Available Abstract Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.

  11. On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60

    Science.gov (United States)

    Deocaris, Custer C.; Kaul, Sunil C.; Wadhwa, Renu

    2006-01-01

    The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular “brotherhood,” their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging. PMID:16817317

  12. Identification of Aging-Associated Gene Expression Signatures That Precede Intestinal Tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Yoshihisa Okuchi

    Full Text Available Aging-associated alterations of cellular functions have been implicated in various disorders including cancers. Due to difficulties in identifying aging cells in living tissues, most studies have focused on aging-associated changes in whole tissues or certain cell pools. Thus, it remains unclear what kinds of alterations accumulate in each cell during aging. While analyzing several mouse lines expressing fluorescent proteins (FPs, we found that expression of FPs is gradually silenced in the intestinal epithelium during aging in units of single crypt composed of clonal stem cell progeny. The cells with low FP expression retained the wild-type Apc allele and the tissues composed of them did not exhibit any histological abnormality. Notably, the silencing of FPs was also observed in intestinal adenomas and the surrounding normal mucosae of Apc-mutant mice, and mediated by DNA methylation of the upstream promoter. Our genome-wide analysis then showed that the silencing of FPs reflects specific gene expression alterations during aging, and that these alterations occur in not only mouse adenomas but also human sporadic and hereditary (familial adenomatous polyposis adenomas. Importantly, pharmacological inhibition of DNA methylation, which suppresses adenoma development in Apc-mutant mice, reverted the aging-associated silencing of FPs and gene expression alterations. These results identify aging-associated gene expression signatures that are heterogeneously induced by DNA methylation and precede intestinal tumorigenesis triggered by Apc inactivation, and suggest that pharmacological inhibition of the signature genes could be a novel strategy for the prevention and treatment of intestinal tumors.

  13. IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle

    OpenAIRE

    Dagdeviren, Sezin; Jung, Dae Young; Friedline, Randall H.; Noh, Hye Lim; Kim, Jong Hun; Patel, Payal R.; Tsitsilianos, Nicholas; Inashima, Kunikazu; Tran, Duy A.; Hu, Xiaodi; Loubato, Marilia M.; Craige, Siobhan M.; Kwon, Jung Yeon; Lee, Ki Won; Kim, Jason K.

    2016-01-01

    Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging-associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18-mo-old transgenic mice with muscle-specific overexpression of IL-10 (MIL10) and in wild-type mice during hyperinsulinemic–euglycemic clamping. Despite similar fat mass and energy balance, MIL10 mice were ...

  14. Exerting Capacity.

    Science.gov (United States)

    Leger, J Michael; Phillips, Carolyn A

    2017-05-01

    Patient safety has been at the forefront of nursing research since the release of the Institute of Medicine's report estimating the number of preventable adverse events in hospital settings; yet no research to date has incorporated the perspectives of bedside nurses using classical grounded theory (CGT) methodology. This CGT study explored the perceptions of bedside registered nurses regarding patient safety in adult acute care hospitals. Data analysis used three techniques unique to CGT-the constant comparative method, coding, and memoing-to explore the values, realities, and beliefs of bedside nurses about patient safety. The analysis resulted in a substantive theory, Exerting Capacity, which explained how bedside nurses balance the demands of keeping their patients safe. Exerting Capacity has implications for health care organization leaders, nursing leaders, and bedside nurses; it also has indications for future research into the concept of patient safety.

  15. Oxidative Stress andSalvia miltiorrhizain Aging-Associated Cardiovascular Diseases.

    Science.gov (United States)

    Chang, Cheng-Chieh; Chang, Yu-Chun; Hu, Wen-Long; Hung, Yu-Chiang

    2016-01-01

    Aging-associated cardiovascular diseases (CVDs) have some risk factors that are closely related to oxidative stress. Salvia miltiorrhiza (SM) has been used commonly to treat CVDs for hundreds of years in the Chinese community. We aimed to explore the effects of SM on oxidative stress in aging-associated CVDs. Through literature searches using Medicine, PubMed, EMBASE, Cochrane library, CINAHL, and Scopus databases, we found that SM not only possesses antioxidant, antiapoptotic, and anti-inflammatory effects but also exerts angiogenic and cardioprotective activities. SM may reduce the production of reactive oxygen species by inhibiting oxidases, reducing the production of superoxide, inhibiting the oxidative modification of low-density lipoproteins, and ameliorating mitochondrial oxidative stress. SM also increases the activities of catalase, manganese superoxide dismutase, glutathione peroxidase, and coupled endothelial nitric oxide synthase. In addition, SM reduces the impact of ischemia/reperfusion injury, prevents cardiac fibrosis after myocardial infarction, preserves cardiac function in coronary disease, maintains the integrity of the blood-brain barrier, and promotes self-renewal and proliferation of neural stem/progenitor cells in stroke. However, future clinical well-designed and randomized control trials will be necessary to confirm the efficacy of SM in aging-associated CVDs.

  16. Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

    Directory of Open Access Journals (Sweden)

    Sergio Cepeda

    2018-01-01

    Full Text Available Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs, such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

  17. Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Graham P. Holloway

    2018-03-01

    Full Text Available Summary: It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress. : Holloway et al. show that an inability of ADP to decrease mitochondrial reactive oxygen species emission contributes to redox stress in skeletal muscle tissue of older individuals and that this process is not recovered following prolonged resistance-type exercise training, despite the general benefits of resistance training for muscle health. Keywords: mitochondria, aging, muscle, ROS, H2O2, ADP, respiration, bioenergetics, exercise, resistance training

  18. Age-associated differences in motor output variability and coordination during the simultaneous dorsiflexion of both feet.

    Science.gov (United States)

    Marchini, Amanda; Pereira, Rafael; Pedroso, Wellington; Christou, Evangelos; Neto, Osmar Pinto

    2017-06-01

    Older adults are more variable than young adults on tasks that demand the simultaneous control of more than one effector, and the difference between age groups may be related to their different capacity of coordinating the force output of the involved effectors. The goal of this study was to determine whether age-associated differences in motor output variability during tasks involving the simultaneous dorsiflexion of two feet can be partially explained by differences in coordination and possibly attenuated by physical training. Ten young and 22 old adults (10 trained and 12 untrained old adults) volunteered to participate in the study. Trained older adults had experience in a high-intensity mixed modality training (MMT) regime for a minimum of 1 year. Volunteers performed successive trials of a constant force task and a goal-directed task, with and without visual feedback. Within- and between-trial variability were calculated and coordination was quantified using the uncontrolled manifold (UCM) approach (i.e., co-variation of the force outputs of both feet were used to quantify a motor synergy index). Older adults exhibited greater variability and lower synergy (p motor output variability in tasks involving the simultaneous dorsiflexion of both feet possibly due to a lack of coordination between the feet.

  19. 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...

  20. Possible Function of Molecular Chaperones in Diseases Caused by Propagating Amyloid Aggregates

    Directory of Open Access Journals (Sweden)

    Vladimir F. Lazarev

    2017-05-01

    Full Text Available The vast majority of neurodegenerative pathologies stem from the formation of toxic oligomers and aggregates composed of wrongly folded proteins. These protein complexes can be released from pathogenic cells and enthralled by other cells, causing the formation of new aggregates in a prion-like manner. By this mechanism, migrating complexes can transmit a disorder to distant regions of the brain and promote gradually transmitting degenerative processes. Molecular chaperones can counteract the toxicity of misfolded proteins. In this review, we discuss recent data on the possible cytoprotective functions of chaperones in horizontally transmitting neurological disorders.

  1. Iminosugars and isoiminosugars as pharmacological chaperones for Krabbe disease - a SAR study

    DEFF Research Database (Denmark)

    Viuff, Agnete

    2015-01-01

    The overall theme of this thesis is the use of iminosugars and isoiminosugars as inhibitors or chaperones for glycosidases. Glycosidases are a group of enzymes with a large variety of biological roles, and the ability to modulate their activity is, therefore, relevant for the development of new......-imino-D-lyxitol (10) and iso-galacto-fagomine lactam (43)) have been synthesised and tested for their ability to act as pharmacological chaperones for galactocerebrosidase. X-ray crystal structures and thermal shift assays as well as K i experiments have shown that the ability to stabilise galactocerebrosidase...

  2. Both Hsp70 chaperone and Clp protease plastidial systems are required for protection against oxidative stress.

    Science.gov (United States)

    Pulido, Pablo; Llamas, Ernesto; Rodriguez-Concepcion, Manuel

    2017-03-04

    Environmental stress conditions such as high light, extreme temperatures, salinity or drought trigger oxidative stress and eventually protein misfolding in plants. In chloroplasts, chaperone systems refold proteins after stress, while proteases degrade misfolded and aggregated proteins that cannot be refolded. We observed that reduced activity of chloroplast Hsp70 chaperone or Clp protease systems both prevented growth of Arabidopsis thaliana seedlings after treatment with the oxidative agent methyl viologen. Besides showing a role for these particular protein quality control components on the protection against oxidative stress, we provide evidence supporting the existence of a yet undiscovered pathway for Clp-mediated degradation of the damaged proteins.

  3. Effects of HSP27 chaperone on THP-1 tumor cell apoptosis.

    Science.gov (United States)

    Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Maroshkina, A N; Belkina, M V

    2012-11-01

    The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.

  4. Capacity Building

    International Nuclear Information System (INIS)

    Molloy, Brian; Mallick, Shahid

    2014-01-01

    Outcomes & Recommendations: • Significant increase needed in the nuclear workforce both to replace soon-to-retire current generation and to staff large numbers of new units planned • Key message, was the importance of an integrated approach to workforce development. • IAEA and other International Organisations were asked to continue to work on Knowledge Management, Networks and E&T activities • IAEA requested to conduct Global Survey of HR needs – survey initiated but only 50% of operating countries (30% of capacity) took part, so results inconclusive

  5. Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body.

    Directory of Open Access Journals (Sweden)

    Katrin B Gehring

    Full Text Available In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals.

  6. Improvement of aging-associated cardiovascular dysfunction by the orally administered copper(II)-aspirinate complex.

    Science.gov (United States)

    Radovits, Tamás; Gerö, Domokos; Lin, Li-ni; Loganathan, Sivakkanan; Hoppe-Tichy, Torsten; Szabó, Csaba; Karck, Matthias; Sakurai, Hiromu; Szabó, Gábor

    2008-10-01

    Aging-associated nitro-oxidative stress causes tissue injury and activates proinflammatory pathways that play an important role in the pathogenesis of aging-associated cardiovascular dysfunction. It has been recently reported, that the copper(II)-aspirinate complex (CuAsp) exerts not only the well-known anti-inflammatory and platelet antiaggregating effects of aspirin, but, due to its superoxide dismutase mimetic activity, it acts as a potent antioxidant as well. In this study we investigated the effects of CuAsp on aging-associated myocardial and endothelial dysfunction. Aging and young rats were treated for 3 weeks with vehicle, or with CuAsp (200 mg/kg per day per os). Left ventricular pressure-volume relations were measured by using a microtip pressure-volume conductance catheter, and indexes of contractility (e.g., slope of end-systolic pressure-volume relationships [ESPVR] [E(es)], and dP/dt(max) - end-diastolic volume [EDV]) were calculated. In organ bath experiments for isometric tension with isolated aortic rings, endothelium-dependent and -independent vasorelaxation were investigated by using acetylcholine and sodium nitroprusside. When compared to the young controls, aging rats showed impaired left ventricular contractility (E(es), 0.51 +/- 0.04 vs. 2.16 +/- 0.28 mmHg/microL; dP/dt(max) - EDV, 10.71 +/- 2.02 vs. 37.23 +/- 4.18 mmHg/sec per microL; p aging animals (94.83 +/- 0.73 vs. 66.66 +/- 1.30%; p cardiovascular functions of young rats. Our results demonstrate that oxidative stress and inflammatory pathways contribute to the pathogenesis of cardiovascular dysfunction in the aging organism, which can be reversed by CuAsp.

  7. Capacity and Capacity Utilization in Fishing Industries

    OpenAIRE

    Kirkley, James E; Squires, Dale

    1999-01-01

    Excess capacity of fishing fleets is one of the most pressing problems facing the world's fisheries and the sustainable harvesting of resource stocks. Considerable confusion persists over the definition and measurement of capacity and capacity utilization in fishing. Fishing capacity and capacity utilization, rather than capital (or effort) utilization, provide the appropriate framework. This paper provides both technological-economic and economic definitions of capacity and excess capacity i...

  8. Spearmint Extract Improves Working Memory in Men and Women with Age-Associated Memory Impairment

    OpenAIRE

    Herrlinger, Kelli A.; Nieman, Kristin M.; Sanoshy, Kristen D.; Fonseca, Brenda A.; Lasrado, Joanne A.; Schild, Arianne L.; Maki, Kevin C.; Wesnes, Keith A.; Ceddia, Michael A.

    2018-01-01

    Abstract Objective: The purpose of this study was to investigate the effects of supplementation with a spearmint (Mentha spicata L.) extract, high in polyphenols including rosmarinic acid, on cognitive performance, sleep, and mood in individuals with age-associated memory impairment (AAMI). Design: Subjects with AAMI (N = 90; 67% female; age = 59.4 ± 0.6 years) were randomly assigned (n = 30/group) to consume 900, 600, or 0 mg/day (two capsules, once daily) spearmint extract for 90 days, in t...

  9. Chaperone network composition in Solanum lycopersicum explored by transcriptome profiling and microarray meta-analysis.

    Science.gov (United States)

    Fragkostefanakis, Sotirios; Simm, Stefan; Paul, Puneet; Bublak, Daniela; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-04-01

    Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato. © 2014 John Wiley & Sons Ltd.

  10. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

    Science.gov (United States)

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-01-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

  11. Investigation of original multivalent iminosugars as pharmacological chaperones for the treatment of Gaucher disease

    NARCIS (Netherlands)

    Laigre, Eugénie; Hazelard, Damien; Casas, Josefina; Serra-Vinardell, Jenny; Michelakakis, Helen; Mavridou, Irene; Aerts, Johannes M. F. G.; Delgado, Antonio; Compain, Philippe

    2016-01-01

    Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a

  12. Patients attitudes to vaginal examination and use of chaperones at a ...

    African Journals Online (AJOL)

    Conclusion: In an SA public hospital, women are more likely to regard VE as the most intimate examination. Women are equivocal on the use of a chaperone and if it was necessary; nurses are their preferred choice except for teenagers, who preferred their mothers. Key words: Intimate examinations, patient-physician ...

  13. The Attitudes and Practices of General Practitioners about the Use of Chaperones in Melbourne, Australia

    Directory of Open Access Journals (Sweden)

    Oliver van Hecke

    2012-01-01

    Full Text Available Introduction. To consider the use of medical chaperones during certain clinical examinations is important whether one practises as a specialist, nurse, medical student, or generalist. Chaperones have been used by doctors conducting intimate examinations for many years but their true extent remains largely unknown. Until recently, there was no national guidance in Australia. Aim. To explore the attitudes and practices of general practitioners (GP regarding their use of chaperones in urban Melbourne, Australia. Method. Qualitative two focus groups involving seventeen GPs from two locations. Discussions were audio-taped, transcribed verbatim and analysed. Results. Common themes and subthemes emerged which were grouped into three main areas: (a practitioner-related, (b patient-related and (c practice related. Discussion. This is the first study from an Australian primary care perspective to gauge the attitudes and experiences of GPs on their use of chaperones. It will provide vital information to inform the next step of extending this research to a national GP audience. From an international perspective, this study provides an excellent template for other primary care clinicians to conduct research in this important field of doctor-patient relationship.

  14. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  15. Analytical background and discussion of the chaperone model of prion diseases.

    Science.gov (United States)

    Liautard, J P

    1999-01-01

    It is generally accepted that prion infection is due solely to a protein i.e. the protein-only hypothesis. The essential constituent of infectious prions is the scrapie prion protein (PrPSc) which is chemically indistinguishable from the normal, cellular protein (PrPC) but exhibits distinct secondary and tertiary structure. This very unusual feature seems to be in contradiction with a major paradigm of present structural biology stated by Anfinsen: a protein folds to the most stable conformation, this means only one structure. In order to reconcile the results obtained on prions with the biophysics of protein folding, a model is proposed. It is based on the hypothesis that a thermodynamically irreversible step is involved in protein folding. The model is then extended to chaperone-assisted protein folding. It is shown that, under certain conditions, the transitory secondary structure formed during the earlier step of folding could interact with chaperone. Analysis shows that chaperone may help the protein to find correct conformation. On the other hand, analysis reveals the possibility that more than one structure may form from a single polypeptide chain. Under these conditions, the behaviour of chaperones resembles the characteristics of prion diseases.

  16. Hsp90-Tau complex reveals molecular basis for specificity in chaperone action

    NARCIS (Netherlands)

    Karagöz, G. Elif; Duarte, Afonso M.S.; Akoury, Elias; Ippel, Hans|info:eu-repo/dai/nl/115343415; Biernat, Jacek; Morán Luengo, Tania|info:eu-repo/dai/nl/369406036; Radli, Martina|info:eu-repo/dai/nl/375810420; Didenko, Tatiana|info:eu-repo/dai/nl/304829455; Nordhues, Bryce A.; Veprintsev, Dmitry B.; Dickey, Chad A.; Mandelkow, Eckhard; Zweckstetter, Markus; Boelens, Rolf|info:eu-repo/dai/nl/070151407; Madl, Tobias; Rüdiger, Stefan G.D.|info:eu-repo/dai/nl/314076662

    2014-01-01

    Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is

  17. Promiscuous histone mis-assembly is actively prevented by chaperones | Center for Cancer Research

    Science.gov (United States)

    About the Cover Chaperone HJURP drives the proper loading of protein CENP-A to the centromere of a chromosome. The effect of HJURP on CENP-A's structural dynamics are observed and explained using dual-resolution in silico simulations, while in vivo experiments demonstrate how CENP-A mutations influence its specific localization in human cells. Abstract

  18. Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes

    DEFF Research Database (Denmark)

    Kudirkiene, Egle; Bager, Ragnhild Jørgensen; Johnson, Timothy J.

    2014-01-01

    encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low...

  19. Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

    NARCIS (Netherlands)

    Y.M. Moshkin (Yuri); C.M. Doyen (Cécile); T.W. Kan (Tsung Wai); G.E. Chalkley (Gillian); K. Sap (Karen); K. Bezstarosti (Karel); J.A.A. Demmers (Jeroen); Z. Özgür (Zeliha); W.F.J. van IJcken (Wilfred); C.P. Verrijzer (Peter)

    2013-01-01

    textabstractChromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed

  20. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  1. Hypochlorite-induced structural modifications enhance the chaperone activity of human α2-macroglobulin.

    Science.gov (United States)

    Wyatt, Amy R; Kumita, Janet R; Mifsud, Richard W; Gooden, Cherrie A; Wilson, Mark R; Dobson, Christopher M

    2014-05-20

    Hypochlorite, an oxidant generated in vivo by the innate immune system, kills invading pathogens largely by inducing the misfolding of microbial proteins. Concomitantly, the nonspecific activity of hypochlorite also damages host proteins, and the accumulation of damaged (misfolded) proteins is implicated in the pathology of a variety of debilitating human disorders (e.g., Alzheimer's disease, atherosclerosis, and arthritis). It is well-known that cells respond to oxidative stress by up-regulating proteostasis machinery, but the direct activation of mammalian chaperones by hypochlorite has not, to our knowledge, been previously reported. In this study, we show that hypochlorite-induced modifications of human α2-macroglobulin (α2M) markedly increase its chaperone activity by generating species, particularly dimers formed by dissociation of the native tetramer, which have enhanced surface hydrophobicity. Moreover, dimeric α2M is generated in whole-blood plasma in the presence of physiologically relevant amounts of hypochlorite. The chaperone activity of hypochlorite-modified α2M involves the formation of stable soluble complexes with misfolded client proteins, including heat-denatured enzymes, oxidized fibrinogen, oxidized LDL, and native or oxidized amyloid β-peptide (Aβ1-42). Here, we show that hypochlorite-modified α2M delivers its misfolded cargo to lipoprotein receptors on macrophages and reduces Aβ1-42 neurotoxicity. Our results support the conclusion that α2M is a specialized chaperone that prevents the extracellular accumulation of misfolded and potentially pathogenic proteins, particularly during innate immune system activity.

  2. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may

  3. The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo.

    Science.gov (United States)

    Park, Kyung-Won; Eun Kim, Gyoung; Morales, Rodrigo; Moda, Fabio; Moreno-Gonzalez, Ines; Concha-Marambio, Luis; Lee, Amy S; Hetz, Claudio; Soto, Claudio

    2017-03-23

    Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo. In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrP Sc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrP Sc in vitro. Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins.

  4. Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins*

    Science.gov (United States)

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-01-01

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development. PMID:20385547

  5. Mitochondrial matrix chaperone and c-myc inhibition causes enhanced lethality in glioblastoma.

    Science.gov (United States)

    Ishida, Chiaki Tsuge; Shu, Chang; Halatsch, Marc-Eric; Westhoff, Mike-Andrew; Altieri, Dario C; Karpel-Massler, Georg; Siegelin, Markus David

    2017-06-06

    Malignant gliomas display high levels of the transcription factor c-myc and organize a tumor specific chaperone network within mitochondria. Here, we show that c-myc along with mitochondrial chaperone inhibition displays massive tumor cell death. Inhibition of mitochondrial matrix chaperones and c-myc was established by utilizing genetic as well as pharmacological approaches. Bromodomain and extraterminal (BET) family protein inhibitors, JQ1 and OTX015, were used for c-myc inhibition. Gamitrinib was applied to interfere with mitochondrial matrix chaperones. A xenograft model was used to determine the in vivo efficacy. Combined inhibition of c-myc and mitochondrial matrix chaperones led to a synergistic reduction of cellular proliferation (CI values less than 1) in established glioblastoma, patient-derived xenograft and stem cell-like glioma cultures. The combinatorial treatment of BET inhibitors and Gamitrinib elicited massive apoptosis induction with dissipation of mitochondrial membrane potential and activation of caspases. Mechanistically, BET-inhibitors and Gamitrinib mediated a pronounced integrated stress response with a PERK-dependent up regulation of ATF4 and subsequent modulation of Bcl-2 family of proteins with down-regulation of Mcl-1 and its interacting partner, Usp9X, and an increase in pro-apoptotic Noxa. Blocking ATF4 by siRNA attenuated Gamitrinib/BET inhibitor mediated increase of Noxa. Knockdown of Noxa and Bak protected from the combinatorial treatment. Finally, the combination treatment of Gamitrinib and OTX015 led to a significantly stronger reduction of tumor growth as compared to single treatments in a xenograft model of human glioma without induction of toxicity. Thus, Gamitrinib in combination with BET-inhibitors should be considered for the development for clinical application.

  6. Oxidative stress induces monocyte necrosis with enrichment of cell-bound albumin and overexpression of endoplasmic reticulum and mitochondrial chaperones.

    Directory of Open Access Journals (Sweden)

    Haiping Tang

    Full Text Available In the present study, monocytes were treated with 5-azacytidine (azacytidine, gossypol or hydrogen peroxide to induce cell death through oxidative stress. A shift from apoptotic to necrotic cell death occurred when monocytes were treated with 100 µM azacytidine for more than 12 hours. Necrotic monocytes exhibited characteristics, including enrichment of cell-bound albumin and up-regulation of endoplasmic reticulum (ER- and mitochondrial-specific chaperones to protect mitochondrial integrity, which were not observed in other necrotic cells, including HUH-7, A2780, A549 and HOC1a. Our results show that the cell-bound albumin originates in the culture medium rather than from monocyte-derived hepatocytes, and that HSP60 is a potential binding partner of the cell-bound albumin. Proteomic analysis shows that HSP60 and protein disulfide isomerase are the most abundant up-regulated mitochondrial and ER-chaperones, and that both HSP60 and calreticulin are ubiquitinated in necrotic monocytes. In contrast, expression levels of the cytosolic chaperones HSP90 and HSP71 were down-regulated in the azacytidine-treated monocytes, concomitant with an increase in the levels of these chaperones in the cell culture medium. Collectively, our results demonstrates that chaperones from different organelles behave differently in necrotic monocytes, ER- and mitochondrial chaperones being retained and cytosolic and nuclear chaperones being released into the cell culture medium through the ruptured cell membrane. HSP60 may serve as a new target for development of myeloid leukemia treatment.

  7. Rapamycin Attenuates Age-associated Changes in Tibialis Anterior Tendon Viscoelastic Properties

    Science.gov (United States)

    Zaseck, Lauren Wood; Miller, Richard A.

    2016-01-01

    Rapamycin extends mouse life span, but the extent to which rapamycin prevents aging-associated changes in specific tissues remains unclear. Stiffness increases and collagen turnover decreases in mouse tendon with aging; thus, our aim was to determine the effect of long-term rapamycin treatment on the mechanical and structural properties of tendons from old mice. Tendons were harvested from female UM-HET3 mice maintained on a standard chow diet for 4 (adult) or 22 (old) months or fed chow containing polymer-encapsulated rapamycin (eRAPA) from 9 to 22 months of age (old RAPA). Stiffness was twofold higher for tendons of old compared with adult mice, but in old RAPA mice, tendon stiffness was maintained at a value not different from that of adults. Additionally, expression of collagen decreased, expression of matrix metalloproteinase-8 increased, and total hydroxyproline content trended toward decreased levels in tendons of old eRAPA-fed mice compared with controls. Finally, age-associated calcification of Achilles tendons and accompanying elevations in expression of chondrocyte and osteoblast markers were all lower in old eRAPA-fed mice. These results suggest that long-term administration of rapamycin alters the molecular pathways responsible for aging of tendon extracellular matrix, resulting in tissue that is structurally and mechanically similar to tendons in adult mice. PMID:26809496

  8. Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

    Directory of Open Access Journals (Sweden)

    Hicham Mahboubi

    2015-12-01

    Full Text Available Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i the physiological state of the cell and (ii the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

  9. Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate

    DEFF Research Database (Denmark)

    Wahlestedt, Martin; Erlandsson, Eva; Kristiansen, Trine

    2017-01-01

    Ageing associates with significant alterations in somatic/adult stem cells and therapies to counteract these might have profound benefits for health. In the blood, haematopoietic stem cell (HSC) ageing is linked to several functional shortcomings. However, besides the recent realization...... with the generation of induced pluripotent stem (iPS) cells. This allows us to specifically focus on aged HSCs presenting with a pronounced lineage skewing, a hallmark of HSC ageing. Functional and molecular evaluations reveal haematopoiesis from these iPS clones to be indistinguishable from that associating...... that individual HSCs might be preset differentially already from young age, HSCs might also age asynchronously. Evaluating the prospects for HSC rejuvenation therefore ultimately requires approaching those HSCs that are functionally affected by age. Here we combine genetic barcoding of aged murine HSCs...

  10. Age-associated epigenetic upregulation of the FKBP5 gene selectively impairs stress resiliency.

    Directory of Open Access Journals (Sweden)

    Jonathan J Sabbagh

    Full Text Available Single nucleotide polymorphisms (SNPs in the FK506 binding protein 5 (FKBP5 gene combine with traumatic events to increase risk for post-traumatic stress and major depressive disorders (PTSD and MDD. These SNPs increase FKBP51 protein expression through a mechanism involving demethylation of the gene and altered glucocorticoid signaling. Aged animals also display elevated FKBP51 levels, which contribute to impaired resiliency to depressive-like behaviors through impaired glucocorticoid signaling, a phenotype that is abrogated in FKBP5-/- mice. But the age of onset and progressive stability of these phenotypes remain unknown. Moreover, it is unclear how FKBP5 deletion affects other glucocorticoid-dependent processes or if age-associated increases in FKBP51 expression are mediated through a similar epigenetic process caused by SNPs in the FKBP5 gene. Here, we show that FKBP51-mediated impairment in stress resiliency and glucocorticoid signaling occurs by 10 months of age and this increased over their lifespan. Surprisingly, despite these progressive changes in glucocorticoid responsiveness, FKBP5-/- mice displayed normal longevity, glucose tolerance, blood composition and cytokine profiles across lifespan, phenotypes normally associated with glucocorticoid signaling. We also found that methylation of Fkbp5 decreased with age in mice, a process that likely explains the age-associated increases in FKBP51 levels. Thus, epigenetic upregulation of FKBP51 with age can selectively impair psychological stress-resiliency, but does not affect other glucocorticoid-mediated physiological processes. This makes FKBP51 a unique and attractive therapeutic target to treat PTSD and MDD. In addition, aged wild-type mice may be a useful model for investigating the mechanisms of FKBP5 SNPs associated with these disorders.

  11. Involvement of NF-κBIZ and related cytokines in age-associated renal fibrosis.

    Science.gov (United States)

    Chung, Ki Wung; Jeong, Hyeong Oh; Lee, Bonggi; Park, Daeui; Kim, Dae Hyun; Choi, Yeun Ja; Lee, Eun Kyeong; Kim, Kyung Mok; Park, June Whoun; Yu, Byung Pal; Chung, Hae Young

    2017-01-31

    Chronic inflammation is a major contributor to age-related nephropathic changes, including renal fibrosis. In this study, various experimental paradigms were designed to delineate the role played by NF-κBIZ (also known as IκBζ) in age-associated renal fibrosis. Analyses based on RNA-sequencing findings obtained by next generation sequencing (NGS) revealed the upregulations of NF-κBIZ and of IL-6 and MCP-1 (both known to be regulated by NF-κBIZ) during aging. The up-regulation of NF-κBIZ in aged rat kidneys coincided with increased macrophage infiltration. In LPS-treated macrophages, oxidative stress was found to play a pivotal role in NF-κBIZ expression, suggesting age-related oxidative stress is associated with NF-κBIZ activation. Furthermore, these in vitro findings were confirmed in LPS-treated old rats, which showed higher levels of oxidative stress and NF-κBIZ in kidneys than LPS-treated young rats. Additional in vitro experiments using macrophages and kidney fibroblasts demonstrated NF-κBIZ and related cytokines participate in fibrosis. In particular, increased levels of NF-κBIZ-associated cytokines in macrophages significantly up-regulated TGF-β induced kidney fibroblast activation. Moreover, experiments with NF-κBIZ knocked down macrophages showed reduced TGF-β-induced kidney fibroblast activation. The findings of the present study provide evidence regarding an involvement of NF-κBIZ in age-associated progressive renal fibrosis and provides potential targets for its prevention.

  12. Structural and Functional Consequences of Chaperone Site Deletion in αA-Crystallin

    Science.gov (United States)

    Santhoshkumar, Puttur; Karmakar, Srabani; Sharma, Krishna K.

    2016-01-01

    The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70–88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70–76 and αAΔ70–88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70–88 was completely inactive as a chaperone, while αAΔ70–76 demonstrated a 40–50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70–88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45 °C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70–88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53 °C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70–88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70–88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins. PMID:27524665

  13. Age-Associated Methylation Suppresses SPRY1, Leading to a Failure of Re-quiescence and Loss of the Reserve Stem Cell Pool in Elderly Muscle

    Directory of Open Access Journals (Sweden)

    Anne Bigot

    2015-11-01

    Full Text Available The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.

  14. Interaction of the Disordered Yersinia Effector Protein YopE with Its Cognate Chaperone SycE

    Science.gov (United States)

    2009-01-01

    of the disordered effector protein to its cognate chaperone in the type III secretion system ( T3SS ). Starting from de novo models, we generated...interactions and devising strategies for interfering with T3SS transport. The type III secretion system ( T3SS ) utilized by many Gram- negative bacteria plays an...of T3SS effector/chaperone complexes have been identified, only a few have been structurally deter- mined so far (5). Here, we explore computational

  15. Heterologous Expression of Chaperones from Hyperthermophilic Archaea Inhibits Aminoglycoside-Induced Protein Misfolding in Escherichia coli.

    Science.gov (United States)

    Peng, S; Chu, Z; Lu, J; Li, D; Wang, Y; Yang, S; Zhang, Y

    2017-10-01

    Aminoglycoside antibiotics affect protein translation fidelity and lead to protein aggregation and an increase in intracellular oxidative stress level as well. The overexpression of the chaperonin GroEL/GroES system promotes short-term tolerance to aminoglycosides in Escherichia coli. Here, we demonstrated that the coexpression of prefoldin or Hsp60 originating from the hyperthermophilic archaeon Pyrococcus furiosus in E. coli cells can rescue cell growth and inhibit protein aggregation induced by streptomycin exposure. The results of our study show that hyperthermophilic chaperones endow E. coli with a higher tolerance to streptomycin than the GroEL/GroES system, and that they exert better effects on the reduction of intracellular protein misfolding, indicating that these chaperones have unique features and functions.

  16. The Role of System-Specific Molecular Chaperones in the Maturation of Molybdoenzymes in Bacteria

    Directory of Open Access Journals (Sweden)

    Meina Neumann

    2011-01-01

    Full Text Available Biogenesis of prokaryotic molybdoenzymes is a complex process with the final step representing the insertion of a matured molybdenum cofactor (Moco into a folded apoenzyme. Usually, specific chaperones of the XdhC family are required for the maturation of molybdoenzymes of the xanthine oxidase family in bacteria. Enzymes of the xanthine oxidase family are characterized to contain an equatorial sulfur ligand at the molybdenum center of Moco. This sulfur ligand is inserted into Moco while bound to the XdhC-like protein and before its insertion into the target enzyme. In addition, enzymes of the xanthine oxidase family bind either the molybdopterin (Mo-MPT form of Moco or the modified molybdopterin cytosine dinucleotide cofactor (MCD. In both cases, only the matured cofactor is inserted by a proofreading process of XdhC. The roles of these specific XdhC-like chaperones during the biogenesis of enzymes of the xanthine oxidase family in bacteria are described.

  17. The heat shock protein/chaperone network and multiple stress resistance

    KAUST Repository

    Jacob, Pierre

    2016-11-15

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

  18. Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

    Science.gov (United States)

    Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

    2012-07-01

    Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. Copyright © 2012 Elsevier GmbH. All rights reserved.

  19. PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Anat Florentin

    2017-11-01

    Full Text Available Summary: The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex. : Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells. Keywords: malaria, Plasmodium, apicoplast, IPP, Clp, chaperone, caseinolytic protease

  20. The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.

    Science.gov (United States)

    Tang, Fenfen; Xia, Hongjie; Wang, Peipei; Yang, Jie; Zhao, Tianyong; Zhang, Qi; Hu, Yuanyang; Zhou, Xi

    2014-09-01

    Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1.

    Science.gov (United States)

    Cockburn, Ingrid L; Pesce, Eva-Rachele; Pryzborski, Jude M; Davies-Coleman, Michael T; Clark, Peter G K; Keyzers, Robert A; Stephens, Linda L; Blatch, Gregory L

    2011-05-01

    Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

  2. Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones.

    Directory of Open Access Journals (Sweden)

    Xun Cai

    Full Text Available Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc. facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup[1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit and CupB6 (the putative adhesin using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5.

  3. Chaperone heat shock protein 90 mobilization and hydralazine cytoprotection against acrolein-induced carbonyl stress.

    Science.gov (United States)

    Burcham, Philip C; Raso, Albert; Kaminskas, Lisa M

    2012-11-01

    Toxic carbonyls such as acrolein participate in many degenerative diseases. Although the nucleophilic vasodilatory drug hydralazine readily traps such species under "test-tube" conditions, whether these reactions adequately explain its efficacy in animal models of carbonyl-mediated disease is uncertain. We have previously shown that hydralazine attacks carbonyl-adducted proteins in an "adduct-trapping" reaction that appears to take precedence over direct "carbonyl-sequestering" reactions, but how this reaction conferred cytoprotection was unclear. This study explored the possibility that by increasing the bulkiness of acrolein-adducted proteins, adduct-trapping might alter the redistribution of chaperones to damaged cytoskeletal proteins that are known targets for acrolein. Using A549 lung adenocarcinoma cells, the levels of chaperones heat shock protein (Hsp) 40, Hsp70, Hsp90, and Hsp110 were measured in intermediate filament extracts prepared after a 3-h exposure to acrolein. Exposure to acrolein alone modestly increased the levels of all four chaperones. Coexposure to hydralazine (10-100 μM) strongly suppressed cell ATP loss while producing strong adduct-trapping in intermediate filaments. Most strikingly, hydralazine selectively boosted the levels of cytoskeletal-associated Hsp90, including a high-mass species that was sensitive to the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin. Biochemical fractionation of acrolein- and hydralazine-treated cells revealed that hydralazine likely promoted Hsp90 migration from cytosol into other subcellular compartments. A role for Hsp90 mobilization in cytoprotection was confirmed by the finding that brief heat shock treatment suppressed acute acrolein toxicity in A549 cells. Taken together, these findings suggest that by increasing the steric bulk of carbonyl-adducted proteins, adduct-trapping drugs trigger the intracellular mobilization of the key molecular chaperone Hsp90.

  4. The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

    Czech Academy of Sciences Publication Activity Database

    Trčka, F.; Durech, M.; Hernychová, L.; Man, Petr; Müller, P.; Vojtěšek, B.

    2014-01-01

    Roč. 289, č. 14 (2014), s. 9887-9901 ISSN 0021-9258 R&D Projects: GA ČR(CZ) P301/11/1678; GA MZd(CZ) 00209805 Grant - others:Regional Center for Applied Molecular Oncology(CZ) CZ.1.05/2.1.00/03.0101 Institutional support: RVO:61388971 Keywords : HSP90 * chaperone * protein assembly Subject RIV: EE - Microbiology, Virology Impact factor: 4.573, year: 2014

  5. Rescue of a pathogenic mutant human glucagon receptor by pharmacological chaperones.

    Science.gov (United States)

    Yu, Run; Chen, Chun-Rong; Liu, Xiaohong; Kodra, János T

    2012-10-01

    We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.

  6. Conversion of scFv peptide-binding specificity for crystal chaperone development

    Energy Technology Data Exchange (ETDEWEB)

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A. (GIT); (UMM); (Texas)

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  7. Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo.

    Directory of Open Access Journals (Sweden)

    Christine R Langlois

    2016-11-01

    Full Text Available Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.

  8. Molecular transformers in the cell: lessons learned from the DegP protease-chaperone.

    Science.gov (United States)

    Sawa, Justyna; Heuck, Alexander; Ehrmann, Michael; Clausen, Tim

    2010-04-01

    Structure-function analysis of DegP revealed a novel mechanism for protease and chaperone regulation. Binding of unfolded proteins induces the oligomer reassembly from the resting hexamer (DegP6) into the functional protease-chaperone DegP12/24. The newly formed cage exhibits the characteristics of a proteolytic folding chamber, shredding those proteins that are severely misfolded while stabilizing and protecting proteins present in their native state. Isolation of native DegP complexes with folded outer membrane proteins (OMPs) highlights the importance of DegP in OMP biogenesis. The encapsulated OMP beta-barrel is significantly stabilized in the hydrophobic chamber of DegP12/24 and thus DegP seems to employ a reciprocal mechanism to those chaperones assisting the folding of water soluble proteins via polar interactions. In addition, we discuss in this review similarities to other complex proteolytic machines that, like DegP, are under control of a substrate-induced or stress-induced oligomer conversion.

  9. N. meningitidis 1681 is a member of the FinO family of RNA chaperones.

    Energy Technology Data Exchange (ETDEWEB)

    Chaulk, S.; Lu, J.; Tan, K.; Arthur, D.; Edwards, R.; Frost, L.; Joachimiak, A.; Glover, J. (Biosciences Division); (Univ. of Alberta)

    2010-11-01

    The conjugative transfer of F-like plasmids between bacteria is regulated by the plasmid-encoded RNA chaperone, FinO, which facilitates sense - antisense RNA interactions to regulate plasmid gene expression. FinO was thought to adopt a unique structure, however many putative homologs have been identified in microbial genomes and are considered members of the FinO-conjugation-repressor superfamily. We were interested in determining whether other members were also able to bind RNA and promote duplex formation, suggesting that this motif does indeed identify a putative RNA chaperone. We determined the crystal structure of the N. meningitidis MC58 protein NMB1681. It revealed striking similarity to FinO, with a conserved fold and a large, positively charged surface that could function in RNA interactions. Using assays developed to study FinO-FinP sRNA interactions, NMB1681, like FinO, bound tightly to FinP RNA stem-loops with short 5-foot and 3-foot single-stranded tails but not to ssRNA. It also was able to catalyze strand exchange between an RNA duplex and a complementary single-strand, and facilitated duplexing between complementary RNA hairpins. Finally, NMB1681 was able to rescue a finO deficiency and repress F plasmid conjugation. This study strongly suggests that NMB1681 is a FinO-like RNA chaperone that likely regulates gene expression through RNA-based mechanisms in N. meningitidis.

  10. A novel protease activity assay using a protease-responsive chaperone protein

    Energy Technology Data Exchange (ETDEWEB)

    Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  11. Maf-dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export.

    Science.gov (United States)

    Parker, Jennifer L; Lowry, Rebecca C; Couto, Narciso A S; Wright, Phillip C; Stafford, Graham P; Shaw, Jonathan G

    2014-04-01

    Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  12. Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

    Science.gov (United States)

    Boczek, Edgar E.; Reefschläger, Lasse G.; Dehling, Marco; Struller, Tobias J.; Häusler, Elisabeth; Seidl, Andreas; Kaila, Ville R. I.; Buchner, Johannes

    2015-01-01

    Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated. PMID:26056257

  13. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    Science.gov (United States)

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  14. TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon

    Directory of Open Access Journals (Sweden)

    Zijing Chen

    2015-10-01

    Full Text Available TRP channels and G protein-coupled receptors (GPCRs play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1 and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which, in turn, associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells.

  15. Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones.

    Science.gov (United States)

    Dollins, D Eric; Warren, Joshua J; Immormino, Robert M; Gewirth, Daniel T

    2007-10-12

    GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 A crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a "twisted V" conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.

  16. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides.

    Science.gov (United States)

    Gillis, Judith; Schipper-Krom, Sabine; Juenemann, Katrin; Gruber, Anna; Coolen, Silvia; van den Nieuwendijk, Rian; van Veen, Henk; Overkleeft, Hermen; Goedhart, Joachim; Kampinga, Harm H; Reits, Eric A

    2013-06-14

    Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

  17. Hsp90 chaperone inhibitor 17-AAG attenuates Aβ-induced synaptic toxicity and memory impairment.

    Science.gov (United States)

    Chen, Yaomin; Wang, Bin; Liu, Dan; Li, Jing Jing; Xue, Yueqiang; Sakata, Kazuko; Zhu, Ling-qiang; Heldt, Scott A; Xu, Huaxi; Liao, Francesca-Fang

    2014-02-12

    The excessive accumulation of soluble amyloid peptides (Aβ) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aβ. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aβ significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aβ and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.

  18. Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

    Science.gov (United States)

    Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

    Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

  19. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    International Nuclear Information System (INIS)

    Marcianò, G.; Huang, D. T.

    2016-01-01

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding

  20. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  1. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  2. Schizosaccharomyces pombe disaggregation machinery chaperones support Saccharomyces cerevisiae growth and prion propagation.

    Science.gov (United States)

    Reidy, Michael; Sharma, Ruchika; Masison, Daniel C

    2013-05-01

    Hsp100 chaperones protect microorganisms and plants from environmental stress by cooperating with Hsp70 and its nucleotide exchange factor (NEF) and Hsp40 cochaperones to resolubilize proteins from aggregates. The Saccharomyces cerevisiae Hsp104 (Sc-Hsp104)-based disaggregation machinery also is essential for replication of amyloid-based prions. Escherichia coli ClpB can substitute for Hsp104 to propagate [PSI(+)] prions in yeast, but only if E. coli DnaK and GrpE (Hsp70 and NEF) are coexpressed. Here, we tested if the reported inability of Schizosaccharomyces pombe Hsp104 (Sp-Hsp104) to support [PSI(+)] propagation was due to similar species-specific chaperone requirements and find that Sp-Hsp104 alone supported propagation of three different yeast prions. Sp-Hsp70 and Sp-Fes1p (NEF) likewise functioned in place of their Sa. cerevisiae counterparts. Thus, chaperones of these long-diverged species possess conserved activities that function in processes essential for both cell growth and prion propagation, suggesting Sc. pombe can propagate its own prions. We show that curing by Hsp104 overexpression and inactivation can be distinguished and confirm the observation that, unlike Sc-Hsp104, Sp-Hsp104 cannot cure yeast of [PSI(+)] when it is overexpressed. These results are consistent with a view that mechanisms underlying prion replication and elimination are distinct.

  3. Failure of delayed nonsynaptic neuronal plasticity underlies age-associated long-term associative memory impairment

    Directory of Open Access Journals (Sweden)

    Watson Shawn N

    2012-08-01

    Full Text Available Abstract Background Cognitive impairment associated with subtle changes in neuron and neuronal network function rather than widespread neuron death is a feature of the normal aging process in humans and animals. Despite its broad evolutionary conservation, the etiology of this aging process is not well understood. However, recent evidence suggests the existence of a link between oxidative stress in the form of progressive membrane lipid peroxidation, declining neuronal electrical excitability and functional decline of the normal aging brain. The current study applies a combination of behavioural and electrophysiological techniques and pharmacological interventions to explore this hypothesis in a gastropod model (Lymnaea stagnalis feeding system that allows pinpointing the molecular and neurobiological foundations of age-associated long-term memory (LTM failure at the level of individual identified neurons and synapses. Results Classical appetitive reward-conditioning induced robust LTM in mature animals in the first quartile of their lifespan but failed to do so in animals in the last quartile of their lifespan. LTM failure correlated with reduced electrical excitability of two identified serotonergic modulatory interneurons (CGCs critical in chemosensory integration by the neural network controlling feeding behaviour. Moreover, while behavioural conditioning induced delayed-onset persistent depolarization of the CGCs known to underlie appetitive LTM formation in this model in the younger animals, it failed to do so in LTM-deficient senescent animals. Dietary supplementation of the lipophilic anti-oxidant α-tocopherol reversed the effect of age on CGCs electrophysiological characteristics but failed to restore appetitive LTM function. Treatment with the SSRI fluoxetine reversed both the neurophysiological and behavioural effects of age in senior animals. Conclusions The results identify the CGCs as cellular loci of age-associated appetitive

  4. The suppression of ghrelin signaling mitigates age-associated thermogenic impairment

    Science.gov (United States)

    Bongmba, Odelia Y. N.; Ma, Xiaojun; Zhu, Xiongwei; Sheikh-Hamad, David; Sun, Yuxiang

    2014-01-01

    Aging is associated with severe thermogenic impairment, which contributes to obesity and diabetes in aging. We previously reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), attenuates age-associated obesity and insulin resistance. Ghrelin and obestatin are derived from the same preproghrelin gene. Here we showed that in brown adipocytes, ghrelin decreases the expression of thermogenic regulator but obestatin increases it, thus showing the opposite effects. We also found that during aging, plasma ghrelin and GHS-R expression in brown adipose tissue (BAT) are increased, but plasma obestatin is unchanged. Increased plasma ghrelin and unchanged obestatin during aging may lead to an imbalance of thermogenic regulation, which may in turn exacerbate thermogenic impairment in aging. Moreover, we found that GHS-R ablation activates thermogenic signaling, enhances insulin activation, increases mitochondrial biogenesis, and improves mitochondrial dynamics of BAT. In addition, we detected increased norepinephrine in the circulation, and observed that GHS-R knockdown in brown adipocytes directly stimulates thermogenic activity, suggesting that GHS-R regulates thermogenesis via both central and peripheral mechanisms. Collectively, our studies demonstrate that ghrelin signaling is an important thermogenic regulator in aging. Antagonists of GHS-R may serve as unique anti-obesity agents, combating obesity by activating thermogenesis. PMID:25543537

  5. Aging associated changes in the motor control of ankle movements in the brain.

    Science.gov (United States)

    Linortner, Patricia; Jehna, Margit; Johansen-Berg, Heidi; Matthews, Paul; Schmidt, Reinhold; Fazekas, Franz; Enzinger, Christian

    2014-10-01

    Although age-related gait changes have been well characterized, little is known regarding potential functional changes in central motor control of distal lower limb movements with age. We hypothesized that there are age-related changes in brain activity associated with the control of repetitive ankle movements, an element of gait feasible for study with functional magnetic resonance imaging. We analyzed standardized functional magnetic resonance imaging data from 102 right-foot dominant healthy participants aged 20-83 years for age-associated effects using FSL and a meta-analysis using coordinate-based activation likelihood estimation. For the first time, we have confirmed age-related changes in brain activity with this gait-related movement of the lower limb in a large population. Increasing age correlated strongly with increased movement-associated activity in the cerebellum and precuneus. Given that task performance did not vary with age, we interpret these changes as potentially compensatory for other age-related changes in the sensorimotor network responsible for control of limb function. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Age-associated chromatin relaxation is enhanced in Huntington's disease mice

    Science.gov (United States)

    Jeon, Kyuheum; Cho, Sunwha; Park, Jung Sun; Kim, Jisun; Jeon, Jeha; Song, Jinhoi; Kim, Seokho; Jeong, Sangkyun; Seo, Hyemyung; Kang, Yong-Kook

    2017-01-01

    Expansion of polyglutamine stretch in the huntingtin (HTT) protein is a major cause of Huntington's disease (HD). The polyglutamine part in HTT interacts with various proteins implicated in epigenetic regulation of genes, suggesting that mutant HTT may disturb the integrity of the epigenetic system. Here, we used a PCRseq-based method to examine expression profile of 395 exonic segments from 260 “epi-driver” genes in splenic T lymphocytes from aged HD mice. We identified 67 exonic segments differentially expressed between young and aged HD mice, most of them upregulated in the aged. Polycomb-repressive complex (PRC)-regulated genes (PRGs) were markedly upregulated in aged HD mice, consistent with downregulation of PRC genes. Epi-driver gene categories of lysine-methylation, lysine-demethylation, arginine-methylation, and PRG showed differential age-associated changes between HD and control. Analyzing the pattern of change in epi-driver gene expressions hinted at an enhanced shift in HD chromatin to a more accessible state with age, which was experimentally demonstrated by DNase-I-hypersensitivity sequencing showing increased chromatin accessibility in HD cells compared to control. We suggest the global change can potentially relieve chromatin-induced repression of many genes, and the unintended expressions of some detrimental proteins could alter T cell function to a greater degree in aged HD mice. PMID:28288000

  7. L-Lactate Protects Skin Fibroblasts against Aging-Associated Mitochondrial Dysfunction via Mitohormesis

    Directory of Open Access Journals (Sweden)

    Jaroslav Zelenka

    2015-01-01

    Full Text Available A moderate elevation of reactive oxygen species (ROS production and a mild inhibition of mitochondrial respiratory chain have been associated with a health promotion and a lifespan extension in several animal models of aging. Here, we tested whether this phenomenon called mitohormesis could be mediated by L-lactate. The treatment with 5 mM L-lactate significantly increased H2O2 production and slightly inhibited the respiration in cultured skin fibroblasts and in isolated mitochondria. The L-lactate exposure was associated with oxidation of intracellular glutathione, phosphorylation of 5′AMP-activated protein kinase (AMPK, and induction of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α transcription. A replicative aging of fibroblasts (L0 with a constant (LC, or intermittent 5 mM L-lactate (LI in media showed that the high-passage LI fibroblasts have higher respiration, lower H2O2 release, and lower secretion of L-lactate compared to L0 and LC. This protection against mitochondrial dysfunction in LI cells was associated with lower activity of mechanistic target of rapamycin complex 1 (mTORC1, less signs of cellular senescence, and increased autophagy compared to L0 and LC. In conclusion, we demonstrated that intermittent but not constant exposure to L-lactate triggers mitohormesis, prevents aging-associated mitochondrial dysfunction, and improves other markers of aging.

  8. Age-associated changes in rich-club organisation in autistic and neurotypical human brains.

    Science.gov (United States)

    Watanabe, Takamitsu; Rees, Geraint

    2015-11-05

    Macroscopic structural networks in the human brain have a rich-club architecture comprising both highly inter-connected central regions and sparsely connected peripheral regions. Recent studies show that disruption of this functionally efficient organisation is associated with several psychiatric disorders. However, despite increasing attention to this network property, whether age-associated changes in rich-club organisation occur during human adolescence remains unclear. Here, analysing a publicly shared diffusion tensor imaging dataset, we found that, during adolescence, brains of typically developing (TD) individuals showed increases in rich-club organisation and inferred network functionality, whereas individuals with autism spectrum disorders (ASD) did not. These differences between TD and ASD groups were statistically significant for both structural and functional properties. Moreover, this typical age-related changes in rich-club organisation were characterised by progressive involvement of the right anterior insula. In contrast, in ASD individuals, did not show typical increases in grey matter volume, and this relative anatomical immaturity was correlated with the severity of ASD social symptoms. These results provide evidence that rich-club architecture is one of the bases of functionally efficient brain networks underpinning complex cognitive functions in adult human brains. Furthermore, our findings suggest that immature rich-club organisation might be associated with some neurodevelopmental disorders.

  9. Aging-associated changes in hematopoiesis and leukemogenesis: what's the connection?

    Science.gov (United States)

    Henry, Curtis J; Marusyk, Andriy; DeGregori, James

    2011-06-01

    Aging is associated with a marked increase in a number of diseases, including many types of cancer. Due to the complex and multi-factorial nature of both aging and cancer, accurate deciphering of causative links between aging and cancer remains a major challenge. It is generally accepted that initiation and progression of cancers are driven by a process of clonal evolution. In principle, this somatic evolution should follow the same Darwinian logic as evolutionary processes in populations in nature: diverse heritable types arising as a result of mutations are subjected to selection, resulting in expansion of the fittest clones. However, prevalent paradigms focus primarily on mutational aspects in linking aging and cancer. In this review, we will argue that age-related changes in selective pressures are likely to be equally important. We will focus on aging-related changes in the hematopoietic system, where age-associated alterations are relatively well studied, and discuss the impact of these changes on the development of leukemias and other malignancies.

  10. C-terminal Domain Modulates the Nucleic Acid Chaperone Activity of Human T-cell Leukemia Virus Type 1 Nucleocapsid Protein via an Electrostatic Mechanism*

    OpenAIRE

    Qualley, Dominic F.; Stewart-Maynard, Kristen M.; Wang, Fei; Mitra, Mithun; Gorelick, Robert J.; Rouzina, Ioulia; Williams, Mark C.; Musier-Forsyth, Karin

    2009-01-01

    Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retrovir...

  11. Food restriction prevents an age-associated increase in rat liver beta-adrenergic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Dax, E.M.; Ingram, D.K.; Partilla, J.S.; Gregerman, R.I.

    1989-05-01

    In male Wistar rats fed ad libitum (24% protein, 4.5 Kcal/gm), the (/sup 125/I)iodopindolol binding capacity of the beta-adrenergic receptors in liver of 24-month-old animals is 3-4 times greater than that of 6-month-old counterparts. In rats fed the same diet, on alternate days from weaning, the receptor capacity did not increase significantly between 6 and 24 months (10.20 +/- 0.55 vs 9.20 +/- 0.72 fmol/mg) or between 24 and 30 months. This was not due to acute dietary deprivation, as rats food-restricted for only 2 weeks, at 23.5 months of age, also showed elevated receptor capacities compared to 6-month-old ad libitum fed animals. Moreover, intermittent feeding produced no significant effects among 6-month-old animals, whether restricted since weaning or for two weeks prior to sacrifice. Many biochemical parameters that decrease with aging in rats fed ad libitum are prevented by dietary restriction. Our results demonstrate that a reproducible biochemical process that increases with aging is also prevented with dietary restriction. The age-related, liver beta-receptor increase may be a potentially reliable marker for studying biochemical perturbations that modify life span.

  12. The role of the molecular chaperone heat shock protein A2 (HSPA2 in regulating human sperm-egg recognition

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    Brett Nixon

    2015-01-01

    Full Text Available One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI, recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2, as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  13. Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization.

    Science.gov (United States)

    McCullough, Laura L; Connell, Zaily; Xin, Hua; Studitsky, Vasily M; Feofanov, Alexey V; Valieva, Maria E; Formosa, Tim

    2018-03-07

    The essential histone chaperone FAcilitates Chromatin Transcription (FACT) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo. Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Age-associated B cells (ABC) inhibit B lymphopoiesis and alter antibody repertoires in old age.

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    Riley, Richard L; Khomtchouk, Kelly; Blomberg, Bonnie B

    2017-11-01

    With old age (∼2y old), mice show substantial differences in B cell composition within the lymphoid tissues. In particular, a novel subset of IgM + CD21/35 lo/- CD23 - mature B cells, the age-associated B cells or ABC, increases numerically and proportionately. This occurs at the expense of other B cell subsets, including B2 follicular B cells in spleen and recirculating primary B cells in bone marrow. Our studies suggest that ABC have a distinctive antibody repertoire, as evidenced by relatively high reactivity to the self-antigens phosphorylcholine (PC) and malondialdehyde (MDA). While PC and MDA are found on apoptotic cells and oxidized lipoproteins, antibodies to these antigens are also cross-reactive with epitopes on bacterial species. In old mice, ABC express TNFα and are pro-inflammatory. ABC can inhibit growth and/or survival in pro-B cells as well as common lymphoid progenitors (CLP). In particular, ABC cause apoptosis in pro-B cells with relatively high levels of the surrogate light chain (SLC) and, consequently, promote an "SLC low" pathway of B cell differentiation in old mice. SLC together with μ heavy chain comprises the pre-B cell receptor (preBCR) critical for pre-B cell expansion and selection of the μ heavy chain Vh repertoire. The low level of SLC likely impairs normal preBCR driven proliferation and alters μ heavy chain Vh selection thereby affecting the antibody specificities of new B cells. In this manner, ABC may contribute to both qualitative and quantitative disruptions of normal B lymphopoiesis in old age. Copyright © 2017. Published by Elsevier Inc.

  15. Spearmint Extract Improves Working Memory in Men and Women with Age-Associated Memory Impairment.

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    Herrlinger, Kelli A; Nieman, Kristin M; Sanoshy, Kristen D; Fonseca, Brenda A; Lasrado, Joanne A; Schild, Arianne L; Maki, Kevin C; Wesnes, Keith A; Ceddia, Michael A

    2018-01-01

    The purpose of this study was to investigate the effects of supplementation with a spearmint (Mentha spicata L.) extract, high in polyphenols including rosmarinic acid, on cognitive performance, sleep, and mood in individuals with age-associated memory impairment (AAMI). Subjects with AAMI (N = 90; 67% female; age = 59.4 ± 0.6 years) were randomly assigned (n = 30/group) to consume 900, 600, or 0 mg/day (two capsules, once daily) spearmint extract for 90 days, in this double-blind, placebo-controlled trial. Assessments were completed for cognition (days 0, 45, and 90), sleep (days 0 and 90), and mood (days 0 and 90) by using the Cognitive Drug Research (CDR) System ™ , Leeds Sleep Evaluation Questionnaire (LSEQ), and Profile of Mood States (POMS ™ ), respectively. Quality of working memory and spatial working memory accuracy improved after supplementation with 900 mg/day spearmint extract by 15% (p = 0.0469) and 9% (p = 0.0456), respectively, versus placebo. Subjects consuming 900 mg/day spearmint extract reported improvement in their ability to fall asleep, relative to subjects consuming placebo (p = 0.0046). Overall treatment effects were evident for vigor-activity (p = 0.0399), total mood disturbance (p = 0.0374), and alertness and behavior following wakefulness (p = 0.0415), with trends observed for improvements after spearmint supplementation relative to placebo. These results suggest that the distinct spearmint extract may be a beneficial nutritional intervention for cognitive health in older subjects with AAMI.

  16. Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

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    Jenkins, Timothy G; Aston, Kenneth I; Pflueger, Christian; Cairns, Bradley R; Carrell, Douglas T

    2014-07-01

    Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

  17. Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

    Directory of Open Access Journals (Sweden)

    Timothy G Jenkins

    2014-07-01

    Full Text Available Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc., trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc. and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body. Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

  18. [Validation of a screening test for age associated cognitive impairment, in Chile].

    Science.gov (United States)

    Quiroga, Pilar; Albala, Cecilia; Klaasen, Gonzalo

    2004-04-01

    The real prevalence of dementia in a given population must be determined through prevalence studies, using validated screening tests. To validate and determine cutoff points for a cognitive impairment screening test composed by the Folstein Mini Mental State Examination (MMSE) and Pfeffer Functional Activities Questionnaire (PFAQ). Validation of the diagnostic test in a sample of 100 subjects over 65 years old (85 from the project "Age associated dementias" and 15 with a confirmed diagnosis of dementia). All were subjected to a complete neuropsychological test by a trained neurologist, that constituted the "gold standard" for the diagnosis of dementia. An independent interviewer applied the MMSE to the subjects and the PFAQ to a next of kin informer. Cutoff points were calculated using ROC curves. The points with the better equilibrium between sensitivity and specificity were selected, considering differences in results between groups with low and high educational level. The cutoff point for MMSE was 21/22, with a sensitivity of 93.6% (95% CI 70.6-99.7%) and a specificity of 46.1% (95% CI 34.7-57.8%). The figure for PFAQ was 5/6, with a sensitivity of 89.2% (95% CI 70.6-99.7%) and a specificity of 70.7% (95% CI 58.9-80.3%). The combination of both instruments gave a sensitivity of 94.4% (95% CI 58.9-80.3%) and a specificity of 83.3% (95% CI 72.3-90.7%). This screening test, using MMSE and PFAQ, has a good sensitivity and specificity for the diagnosis of dementia in Chile. Being simple and of low cost, it can be applied in primary health care.

  19. Intermittent bout exercise training down-regulates age-associated inflammation in skeletal muscles.

    Science.gov (United States)

    Kim, Jeong-Seok; Yi, Ho-Keun

    2015-12-01

    Aging is characterized by the progressive decline in mass and function of the skeletal muscle along with increased susceptibility to inflammation, oxidative stress, and atrophy. In this study, we investigate the effect of intermittent bout and single bout exercise training on inflammatory molecules in young (3 months) and old (22 months) male Sprague-Dawley rats. The rats were divided into 6 groups. Young and old rats were randomly assigned for control and two exercise training groups, single bout (S type): 30 min/day, 5 days/week for 6 weeks and intermittent bout (I type): three times for 10 min/day, 5 days/week for 6 weeks respectively. The exercise training was carried out by a treadmill at a speed of 15m/min (young) or 10 m/min (old) with a slope of 5°. After 48 h of the final exercise bout, muscle samples were collected for biochemical assay. I type exercise training reduced the serum levels of inflammatory molecules such as interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and malondialdehyde (MDA) in old rats. By contrast, interleukin-4 (IL-4) and superoxide dismutase (SOD) were elevated. Consequently in skeletal muscles, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were decreased significantly in the old group of I type. However, the matrix metalloproteinase-2 (MMP-2) level had no positive effects. Also, phosphorylation of mammalian target of rapamycin (p-mTOR) and myogenic differentiation (MyoD) were increased markedly in S and I types of old rats. These results suggest that I type exercise training appears more effective to reduce age-associated inflammatory molecules, and may recommend in regulating against chronic complicated disease induced by aging. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. The SseC translocon component in Salmonella enterica serovar Typhimurium is chaperoned by SscA.

    Science.gov (United States)

    Cooper, Colin A; Mulder, David T; Allison, Sarah E; Pilar, Ana Victoria C; Coombes, Brian K

    2013-10-04

    Salmonella enterica is a causative agent of foodborne gastroenteritis and the systemic disease known as typhoid fever. This bacterium uses two type three secretion systems (T3SSs) to translocate protein effectors into host cells to manipulate cellular function. Salmonella pathogenicity island (SPI)-2 encodes a T3SS required for intracellular survival of the pathogen. Genes in SPI-2 include apparatus components, secreted effectors and chaperones that bind to secreted cargo to coordinate their release from the bacterial cell. Although the effector repertoire secreted by the SPI-2 T3SS is large, only three virulence-associated chaperones have been characterized. Here we report that SscA is the chaperone for the SseC translocon component. We show that SscA and SseC interact in bacterial cells and that deletion of sscA results in a loss of SseC secretion, which compromises intracellular replication and leads to a loss of competitive fitness in mice. This work completes the characterization of the chaperone complement within SPI-2 and identifies SscA as the chaperone for the SseC translocon.

  1. Aging-associated changes in oxidative stress, cell proliferation, and apoptosis are prevented in the prostate of transgenic rats overexpressing regucalcin.

    Science.gov (United States)

    Vaz, Cátia V; Marques, Ricardo; Maia, Cláudio J; Socorro, Sílvia

    2015-12-01

    Regucalcin (RGN) is a calcium (Ca(2+))-binding protein that displays a characteristic downregulated expression with aging in several tissues. Besides its role in regulating intracellular Ca(2+) homeostasis, RGN has been associated with the control of oxidative stress, cell proliferation, and apoptosis. Thus, the diminished expression of RGN with aging may contribute to the age-associated deterioration of cell function. In the present study, we hypothesized that the maintenance of high expression levels of RGN may prevent age-related alterations in the processes mentioned previously. First, we confirmed that RGN expression is significantly diminished in the prostate of 8-, 9-, 12-, and 24-months wild-type rats. Then, the effect of aging on lipid peroxidation, antioxidant defenses, cell proliferation, and apoptosis in the prostate of wild-type controls and transgenic rats overexpressing RGN (Tg-RGN) was investigated. The activity of glutathione and the antioxidant capacity were increased in Tg-RGN rats in response to the age-associated increase in thiobarbituric acid reactive substances levels, an effect not seen in wild type. Overexpression of RGN also counteracted the effect of aging increasing prostate cell proliferation. In contrast to wild-type animals, the prostate weight of Tg-RGN did not change with aging and was underpinned by the diminished expression of stem cell factor and c-kit, and increased expression of p53. In addition, aged Tg-RGN animals displayed increased expression (activity) of apoptosis regulators, therefore not showing the age-induced resistance to apoptosis observed in wild type. Altogether, these findings indicate the protective role of RGN against the development of age-related pathologies, such as, for example, prostate cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

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    Cláudia Caldeira

    2017-08-01

    increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

  3. The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

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    Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.; Thanassi, David G.; Li, Huilin; Christie, P. J.

    2016-06-27

    ABSTRACT

    The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function.

    IMPORTANCEPili are critical virulence factors for many bacterial pathogens. UropathogenicE. colirelies on P and type 1 pili assembled by the chaperone/usher pathway to

  4. Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

    Science.gov (United States)

    Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

    2017-10-01

    By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

  5. Spinal Muscular Atrophy: From Defective Chaperoning of snRNP Assembly to Neuromuscular Dysfunction

    Directory of Open Access Journals (Sweden)

    Maia Lanfranco

    2017-06-01

    Full Text Available Spinal Muscular Atrophy (SMA is a neuromuscular disorder that results from decreased levels of the survival motor neuron (SMN protein. SMN is part of a multiprotein complex that also includes Gemins 2–8 and Unrip. The SMN-Gemins complex cooperates with the protein arginine methyltransferase 5 (PRMT5 complex, whose constituents include WD45, PRMT5 and pICln. Both complexes function as molecular chaperones, interacting with and assisting in the assembly of an Sm protein core onto small nuclear RNAs (snRNAs to generate small nuclear ribonucleoproteins (snRNPs, which are the operating components of the spliceosome. Molecular and structural studies have refined our knowledge of the key events taking place within the crowded environment of cells and the numerous precautions undertaken to ensure the faithful assembly of snRNPs. Nonetheless, it remains unclear whether a loss of chaperoning in snRNP assembly, considered as a “housekeeping” activity, is responsible for the selective neuromuscular phenotype in SMA. This review thus shines light on in vivo studies that point toward disturbances in snRNP assembly and the consequential transcriptome abnormalities as the primary drivers of the progressive neuromuscular degeneration underpinning the disease. Disruption of U1 snRNP or snRNP assembly factors other than SMN induces phenotypes that mirror aspects of SMN deficiency, and splicing defects, described in numerous SMA models, can lead to a DNA damage and stress response that compromises the survival of the motor system. Restoring the correct chaperoning of snRNP assembly is therefore predicted to enhance the benefit of SMA therapeutic modalities based on augmenting SMN expression.

  6. Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor.

    Science.gov (United States)

    Fujimoto, Michiko; Hayashi, Teruo; Urfer, Roman; Mita, Shiro; Su, Tsung-Ping

    2012-07-01

    The sigma-1 receptor (Sig-1R) is a novel endoplasmic reticulum (ER) molecular chaperone that regulates protein folding and degradation. The Sig-1R activation by agonists is known to improve memory, promote cell survival, and exert an antidepressant-like action in animals. Cutamesine (SA4503), a selective Sig-1R ligand, was shown to increase BDNF in the hippocampus of rats. How exactly the intracellular chaperone Sig-1R or associated ligand causes the increase of BDNF or any other neurotrophins is unknown. We examined here whether the action of Sig-1Rs may relate to the post-translational processing and release of BDNF in neuroblastoma cell lines. We used in vitro assays and confirmed that cutamesine possesses the bona fide Sig-1R agonist property by causing the dissociation of BiP from Sig-1Rs. The C-terminus of Sig-1Rs exerted robust chaperone activity by completely blocking the aggregation of BDNF and GDNF in vitro. Chronic treatment with cutamesine in rat B104 neuroblastoma caused a time- and dose-dependent potentiation of the secretion of BDNF without affecting the mRNA level of BDNF. Cutamesine decreased the intracellular level of pro-BDNF and mature BDNF whereas increased the extracellular level of mature BDNF. The pulse-chase experiment indicated that the knockdown of Sig-1Rs decreased the secreted mature BDNF in B104 cells without affecting the synthesis of BDNF. Our findings indicate that, in contrast to clinically used antidepressants that promote the transcriptional upregulation of BDNF, the Sig-1R agonist cutamesine potentiates the post-translational processing of neurotrophins. This unique pharmacological profile may provide a novel therapeutic opportunity for the treatment of neuropsychiatric disorders. Copyright © 2012 Wiley Periodicals, Inc.

  7. The histone chaperone HJURP is a new independent prognostic marker for luminal A breast carcinoma.

    Science.gov (United States)

    Montes de Oca, Rocío; Gurard-Levin, Zachary A; Berger, Frédérique; Rehman, Haniya; Martel, Elise; Corpet, Armelle; de Koning, Leanne; Vassias, Isabelle; Wilson, Laurence O W; Meseure, Didier; Reyal, Fabien; Savignoni, Alexia; Asselain, Bernard; Sastre-Garau, Xavier; Almouzni, Geneviève

    2015-03-01

    Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high-risk patients from good prognosis patients. This is particularly difficult in the low-grade, ER-positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions. Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker. Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly-expressed in the proliferative subtypes (basal-like, HER2-positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF-1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP-A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients. The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low-risk luminal A breast carcinoma patients. Copyright © 2014. Published by Elsevier B.V.

  8. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

    Directory of Open Access Journals (Sweden)

    Alexandre Chlenski

    Full Text Available Secreted Protein Acidic and Rich in Cysteine (SPARC is one of the major non-structural proteins of the extracellular matrix (ECM in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+ concentrations are low, high extracellular concentrations of Ca(2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide

  9. The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    El Bakkouri, Majida; Pow, Andre; Mulichak, Anne; Cheung, Kevin L Y; Artz, Jennifer D; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F; Goodman, C Dean; McFadden, Geoffrey I; Ortega, Joaquin; Hui, Raymond; Houry, Walid A

    2010-12-03

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System

    Directory of Open Access Journals (Sweden)

    Stephen T. Hallett

    2017-10-01

    Full Text Available Summary: Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions. : Hallett et al. reconstitute CDK4/6 client kinase handover from Cdc37-Hsp90 to CDK regulatory partners and propose a model for the assembly factor activity of CIP/KIP CDK inhibitors. They find that CDK4/6 inhibitors in clinical use can displace G1 CDKs from the Cdc37-Hsp90 chaperone system at submicromolar concentrations. Keywords: Cdc37, CDK, chaperone, CIP/KIP, cyclin D, Hsp90, INK, kinase, palbociclib, ribociclib

  11. Interactions of Escherichia coli molecular chaperone HtpG with DnaA replication initiator DNA

    OpenAIRE

    Grudniak, Anna M.; Markowska, Katarzyna; Wolska, Krystyna I.

    2015-01-01

    The bacterial chaperone high-temperature protein G (HtpG), a member of the Hsp90 protein family, is involved in the protection of cells against a variety of environmental stresses. The ability of HtpG to form complexes with other bacterial proteins, especially those involved in fundamental functions, is indicative of its cellular role. An interaction between HtpG and DnaA, the main initiator of DNA replication, was studied both in vivo, using a bacterial two-hybrid system, and in vitro with a...

  12. Lack of the RNA chaperone Hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Jakobsen, Henrik; Struve, Carsten

    2012-01-01

    as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq...... is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host.......Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans...

  13. Breast cancer metastasis suppressor 1 (BRMS1) is stabilized by the Hsp90 chaperone #

    OpenAIRE

    Hurst, Douglas R.; Mehta, Alka; Moore, Blake P.; Phadke, Pushkar A.; Meehan, William J.; Accavitti, Mary Ann; Shevde, Lalita A.; Hopper, James E.; Xie, Yi; Welch, Danny R.; Samant, Rajeev S.

    2006-01-01

    Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins. In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (M...

  14. Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

    DEFF Research Database (Denmark)

    Gunnarsen, Kristin S; Kristinsson, Solveig G; Justesen, Sune

    2013-01-01

    We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random...... mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCR(mut) variants selected were produced in high yields and isolated...

  15. Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease

    DEFF Research Database (Denmark)

    Hughes, Derralynn A.; Nicholls, Kathleen; Shankar, Suma P.

    2017-01-01

    Background Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, resulting in α-galactosidase (α-Gal) deficiency and accumulation of lysosomal substrates. Migalastat, an oral pharmacological chaperone being developed as an alternative to intravenous enzyme replacement......, to receive 18 months open-label migalastat or remain on ERT. Four patients had nonamenable mutant forms of α-Gal based on the validated cell-based assay conducted after treatment initiation and were excluded from primary efficacy analyses only. Migalastat and ERT had similar effects on renal function. Left...

  16. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    Energy Technology Data Exchange (ETDEWEB)

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.; (Harvard-Med); (Brandeis)

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  17. Pineal Calcification, Melatonin Production, Aging, Associated Health Consequences and Rejuvenation of the Pineal Gland.

    Science.gov (United States)

    Tan, Dun Xian; Xu, Bing; Zhou, Xinjia; Reiter, Russel J

    2018-01-31

    The pineal gland is a unique organ that synthesizes melatonin as the signaling molecule of natural photoperiodic environment and as a potent neuronal protective antioxidant. An intact and functional pineal gland is necessary for preserving optimal human health. Unfortunately, this gland has the highest calcification rate among all organs and tissues of the human body. Pineal calcification jeopardizes melatonin's synthetic capacity and is associated with a variety of neuronal diseases. In the current review, we summarized the potential mechanisms of how this process may occur under pathological conditions or during aging. We hypothesized that pineal calcification is an active process and resembles in some respects of bone formation. The mesenchymal stem cells and melatonin participate in this process. Finally, we suggest that preservation of pineal health can be achieved by retarding its premature calcification or even rejuvenating the calcified gland.

  18. Pineal Calcification, Melatonin Production, Aging, Associated Health Consequences and Rejuvenation of the Pineal Gland

    Directory of Open Access Journals (Sweden)

    Dun Xian Tan

    2018-01-01

    Full Text Available The pineal gland is a unique organ that synthesizes melatonin as the signaling molecule of natural photoperiodic environment and as a potent neuronal protective antioxidant. An intact and functional pineal gland is necessary for preserving optimal human health. Unfortunately, this gland has the highest calcification rate among all organs and tissues of the human body. Pineal calcification jeopardizes melatonin’s synthetic capacity and is associated with a variety of neuronal diseases. In the current review, we summarized the potential mechanisms of how this process may occur under pathological conditions or during aging. We hypothesized that pineal calcification is an active process and resembles in some respects of bone formation. The mesenchymal stem cells and melatonin participate in this process. Finally, we suggest that preservation of pineal health can be achieved by retarding its premature calcification or even rejuvenating the calcified gland.

  19. Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

    Science.gov (United States)

    Mares, Rosa E; Meléndez-López, Samuel G; Ramos, Marco A

    2011-01-01

    Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI

  20. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Science.gov (United States)

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  1. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

    Directory of Open Access Journals (Sweden)

    Hongjie Xia

    2015-07-01

    Full Text Available RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71, which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16, another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings

  2. Distinct roles of molecular chaperones HSP90α and HSP90β in the biogenesis of KCNQ4 channels.

    Directory of Open Access Journals (Sweden)

    Yanhong Gao

    Full Text Available Loss-of-function mutations in the KCNQ4 channel cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss. Previous studies have demonstrated that the majority of the pathogenic KCNQ4 mutations lead to trafficking deficiency and loss of KCNQ4 currents. Over the last two decades, various strategies have been developed to rescue trafficking deficiency of pathogenic mutants; the most exciting advances have been made by manipulating activities of molecular chaperones involved in the biogenesis and quality control of the target protein. However, such strategies have not been established for KCNQ4 mutants and little is known about the molecular chaperones governing the KCNQ4 biogenesis. To identify KCNQ4-associated molecular chaperones, a proteomic approach was used in this study. As a result, two major molecular chaperones, HSP70 and HSP90, were identified and then confirmed by reciprocal co-immunoprecipitation assays, suggesting that the HSP90 chaperone pathway might be involved in the KCNQ4 biogenesis. Manipulating chaperone expression further revealed that two different isoforms of HSP90, the inducible HSP90α and the constitutive HSP90β, had opposite effects on the cellular level of the KCNQ4 channel; that HSP40, HSP70, and HOP, three key components of the HSP90 chaperone pathway, were crucial in facilitating KCNQ4 biogenesis. In contrast, CHIP, a major E3 ubiquitin ligase, had an opposite effect. Collectively, our data suggest that HSP90α and HSP90β play key roles in controlling KCNQ4 homeostasis via the HSP40-HSP70-HOP-HSP90 chaperone pathway and the ubiquitin-proteasome pathway. Most importantly, we found that over-expression of HSP90β significantly improved cell surface expression of the trafficking-deficient, pathogenic KCNQ4 mutants L274H and W276S. KCNQ4 surface expression was restored by HSP90β in cells mimicking heterozygous conditions of the DFNA2 patients

  3. Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

    Directory of Open Access Journals (Sweden)

    Michael Reidy

    2014-10-01

    Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

  4. Streptococcus mutans copper chaperone, CopZ, is critical for biofilm formation and competitiveness.

    Science.gov (United States)

    Garcia, S S; Du, Q; Wu, H

    2016-12-01

    The oral cavity is a dynamic environment characterized by hundreds of bacterial species, saliva, and an influx of nutrients and metal ions such as copper. Although there is a physiologic level of copper in the saliva, the oral cavity is often challenged with an influx of copper ions. At high concentrations copper is toxic and must therefore be strictly regulated by pathogens for them to persist and cause disease. The cariogenic pathogen Streptococcus mutans manages excess copper using the copYAZ operon that encodes a negative DNA-binding repressor (CopY), the P1-ATPase copper exporter (CopA), and the copper chaperone (CopZ). These hypothetical roles of the copYAZ operon in regulation and copper transport to receptors led us to investigate their contribution to S. mutans virulence. Mutants defective in the copper chaperone CopZ, but not CopY or CopA, were impaired in biofilm formation and competitiveness against commensal streptococci. Characterization of the CopZ mutant biofilm revealed a decreased secretion of glucosyltransferases and reduced expression of mutacin genes. These data suggest that the function of copZ on biofilm and competitiveness is independent of copper resistance and CopZ is a global regulator for biofilm and other virulence factors. Further characterization of CopZ may lead to the identification of new biofilm pathways. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. High dose of antibiotic colistin induces oligomerization of molecular chaperone HSP90.

    Science.gov (United States)

    Togashi, Shuntaro; Takahashi, Kyosuke; Tamura, Arisa; Toyota, Ikumi; Hatakeyama, Shiori; Komatsuda, Atsushi; Kudo, Ikuru; Sasaki Kudoh, Erina; Okamoto, Tomoya; Haga, Asami; Miyamoto, Asuka; Grave, Ewa; Sugawara, Taku; Shimizu, Hiroaki; Itoh, Hideaki

    2017-07-01

    Colistin is an antimicrobial cationic peptide that belongs to the polymyxin family. Colistin was clinically used for the treatment of gram-negative infections but fell out of favour because of its significant side effects including neurotoxicity and nephrotoxicity. More recently, colistin has been regarded as one of the important options for nosocomial infections caused by multidrug resistant bacteria. Mechanisms of both the side effect onset of the drug and the side effect reduction are yet to be elucidated. In this study, we identified the specific binding protein of colistin using an affinity column chromatography. Colistin binds to the molecular chaperone HSP90. Although colistin slightly suppressed the chaperone activity of HSP90, there are no effects on the ATPase activity for a low concentration of colistin. Interestingly, colistin-induced aggregation of HSP90 via the N-domain. As for the cell viability of the SHSY5Y cell, the cell viability decreased to approximately 80% by the colistin 300 μM. However, the cell viability recovered to approximately 100% by adding ATP dosage. The same result was obtained by dot blot assay using anti-HSP90 antibody. Our results may help to understand the side effect mechanism of colistin. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes.

    Science.gov (United States)

    Pfab, Alexander; Breindl, Matthias; Grasser, Klaus D

    2018-03-01

    The histone chaperone FACT is involved in the expression of genes encoding anthocyanin biosynthetic enzymes also upon induction by moderate high-light and therefore contributes to the stress-induced plant pigmentation. The histone chaperone FACT consists of the SSRP1 and SPT16 proteins and associates with transcribing RNAPII (RNAPII) along the transcribed region of genes. FACT can promote transcriptional elongation by destabilising nucleosomes in the path of RNA polymerase II, thereby facilitating efficient transcription of chromatin templates. Transcript profiling of Arabidopsis plants depleted in SSRP1 or SPT16 demonstrates that only a small subset of genes is differentially expressed relative to wild type. The majority of these genes is either up- or down-regulated in both the ssrp1 and spt16 plants. Among the down-regulated genes, those encoding enzymes of the biosynthetic pathway of the plant secondary metabolites termed anthocyanins (but not regulators of the pathway) are overrepresented. Upon exposure to moderate high-light stress several of these genes are up-regulated to a lesser extent in ssrp1/spt16 compared to wild type plants, and accordingly the mutant plants accumulate lower amounts of anthocyanin pigments. Moreover, the expression of SSRP1 and SPT16 is induced under these conditions. Therefore, our findings indicate that FACT is a novel factor required for the accumulation of anthocyanins in response to light-induction.

  7. Characterization of the Grp94/OS-9 chaperone-lectin complex.

    Science.gov (United States)

    Seidler, Paul M; Shinsky, Stephen A; Hong, Feng; Li, Zihai; Cosgrove, Michael S; Gewirth, Daniel T

    2014-10-23

    Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains "mammalian-specific insets" that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Intracellular dynamics of the Hsp90 co-chaperone p23 is dictated by Hsp90

    International Nuclear Information System (INIS)

    Picard, Didier

    2006-01-01

    p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90-substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the 'fluorescence recovery after photobleaching' (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions

  9. Phosphorylation-mediated control of histone chaperone ASF1 levels by Tousled-like kinases.

    Directory of Open Access Journals (Sweden)

    Maxim Pilyugin

    Full Text Available Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.

  10. A Clp/Hsp100 chaperone functions in Myxococcus xanthus sporulation and self-organization.

    Science.gov (United States)

    Yan, Jinyuan; Garza, Anthony G; Bradley, Michael D; Welch, Roy D

    2012-04-01

    The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.

  11. Chemical chaperone therapy for brain pathology in G(M1)-gangliosidosis.

    Science.gov (United States)

    Matsuda, Junichiro; Suzuki, Osamu; Oshima, Akihiro; Yamamoto, Yoshie; Noguchi, Akira; Takimoto, Kazuhiro; Itoh, Masayuki; Matsuzaki, Yuji; Yasuda, Yosuke; Ogawa, Seiichiro; Sakata, Yuko; Nanba, Eiji; Higaki, Katsumi; Ogawa, Yoshimi; Tominaga, Lika; Ohno, Kousaku; Iwasaki, Hiroyuki; Watanabe, Hiroshi; Brady, Roscoe O; Suzuki, Yoshiyuki

    2003-12-23

    We synthesized a galactose derivative, N-octyl-4-epi-beta-valienamine (NOEV), for a molecular therapy (chemical chaperone therapy) of a human neurogenetic disease, beta-galactosidosis (GM1-gangliosidosis and Morquio B disease). It is a potent inhibitor of lysosomal beta-galactosidase in vitro. Addition of NOEV in the culture medium restored mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage. Short-term oral administration of NOEV to a model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C, resulted in significant enhancement of the enzyme activity in the brain and other tissues. Immunohistochemical stain revealed a decrease in the amount of GM1 and GA1 in neuronal cells in the fronto-temporal cerebral cortex and brainstem. However, mass biochemical analysis did not show the substrate reduction observed histochemically in these limited areas in the brain probably because of the brief duration of this investigation. Chemical chaperone therapy may be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

  12. Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

    Directory of Open Access Journals (Sweden)

    Zi Yang

    Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

  13. Impaired folding of the mitochondrial small TIM chaperones induces clearance by the i-AAA protease.

    Science.gov (United States)

    Baker, Michael J; Mooga, Ved P; Guiard, Bernard; Langer, Thomas; Ryan, Michael T; Stojanovski, Diana

    2012-12-14

    The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1). Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

    Science.gov (United States)

    Charnaud, Sarah C; Dixon, Matthew W A; Nie, Catherine Q; Chappell, Lia; Sanders, Paul R; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J; Beeson, James G; Rayner, Julian C; Przyborski, Jude M; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2017-01-01

    Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

  15. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

    Directory of Open Access Journals (Sweden)

    Sarah C Charnaud

    Full Text Available Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

  16. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

  17. The Citrus Flavanone Naringenin Protects Myocardial Cells against Age-Associated Damage

    Directory of Open Access Journals (Sweden)

    Eleonora Da Pozzo

    2017-01-01

    Full Text Available In recent years, the health-promoting effects of the citrus flavanone naringenin have been examined. The results have provided evidence for the modulation of some key mechanisms involved in cellular damage by this compound. In particular, naringenin has been revealed to have protective properties such as an antioxidant effect in cardiometabolic disorders. Very recently, beneficial effects of naringenin have been demonstrated in old rats. Because aging has been demonstrated to be directly related to the occurrence of cardiac disorders, in the present study, the ability of naringenin to prevent cardiac cell senescence was investigated. For this purpose, a cellular model of senescent myocardial cells was set up and evaluated using colorimetric, fluorimetric, and immunometric techniques. Relevant cellular senescence markers, such as X-gal staining, cell cycle regulator levels, and the percentage of cell cycle-arrested cells, were found to be reduced in the presence of naringenin. In addition, cardiac markers of aging-induced damage, including radical oxidative species levels, mitochondrial metabolic activity, mitochondrial calcium buffer capacity, and estrogenic signaling functions, were also modulated by the compound. These results suggested that naringenin has antiaging effects on myocardial cells.

  18. Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

    DEFF Research Database (Denmark)

    Bunprajun, Tipwadee; Henriksen, Tora Ida; Scheele, Camilla

    2013-01-01

    Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes......, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact...... chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma...

  19. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

    DEFF Research Database (Denmark)

    Bacos, Karl; Gillberg, Linn; Volkov, Petr

    2016-01-01

    Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA...... methylation genome-wide in islets from 87 non-diabetic donors, aged 26-74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes...... identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we...

  20. Role of PGC-1α in exercise training- and resveratrol-induced prevention of age-associated inflammation

    DEFF Research Database (Denmark)

    Olesen, Jesper; Jørgensen, Stine Ringholm; Nielsen, Maja Munk

    2013-01-01

    Age-related metabolic diseases are often associated with low-grade inflammation. The aim of the present study was to investigate the role of the transcriptional co-activator PGC-1α in the potential beneficial effects of exercise training and/or resveratrol in the prevention of age-associated low......-grade inflammation. To address this, a long-term voluntary exercise training and resveratrol supplementation study was conducted....

  1. The TWEAK–Fn14 dyad is involved in age-associated pathological changes in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Tajrishi, Marjan M.; Sato, Shuichi; Shin, Jonghyun [Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202 (United States); Zheng, Timothy S.; Burkly, Linda C. [Department of Immunology, Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142 (United States); Kumar, Ashok, E-mail: ashok.kumar@louisville.edu [Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202 (United States)

    2014-04-18

    Highlights: • The levels of TWEAK receptor Fn14 are increased in skeletal muscle during aging. • Deletion of Fn14 attenuates age-associated skeletal muscle fiber atrophy. • Deletion of Fn14 inhibits proteolysis in skeletal muscle during aging. • TWEAK–Fn14 signaling activates transcription factor NF-κB in aging skeletal muscle. • TWEAK–Fn14 dyad is involved in age-associated fibrosis in skeletal muscle. - Abstract: Progressive loss of skeletal muscle mass and strength (sarcopenia) is a major clinical problem in the elderly. Recently, proinflammatory cytokine TWEAK and its receptor Fn14 were identified as key mediators of muscle wasting in various catabolic states. However, the role of the TWEAK–Fn14 pathway in pathological changes in skeletal muscle during aging remains unknown. In this study, we demonstrate that the levels of Fn14 are increased in skeletal muscle of 18-month old (aged) mice compared with adult mice. Genetic ablation of Fn14 significantly increased the levels of specific muscle proteins and blunted the age-associated fiber atrophy in mice. While gene expression of two prominent muscle-specific E3 ubiquitin ligases MAFBx and MuRF1 remained comparable, levels of ubiquitinated proteins and the expression of autophagy-related molecule Atg12 were significantly reduced in Fn14-knockout (KO) mice compared with wild-type mice during aging. Ablation of Fn14 significantly diminished the DNA-binding activity of transcription factor nuclear factor-kappa B (NF-κB), gene expression of various inflammatory molecules, and interstitial fibrosis in skeletal muscle of aged mice. Collectively, our study suggests that the TWEAK–Fn14 signaling axis contributes to age-associated muscle atrophy and fibrosis potentially through its local activation of proteolytic systems and inflammatory pathways.

  2. Cadmium exposure and age-associated DNA methylation changes in non-smoking women from northern Thailand.

    Science.gov (United States)

    Demanelis, Kathryn; Virani, Shama; Colacino, Justin A; Basu, Niladri; Nishijo, Muneko; Ruangyuttikarn, Werawan; Swaddiwudhipong, Witaya; Nambunmee, Kowit; Rozek, Laura S

    2017-05-01

    DNA methylation changes with age, and may serve as a biomarker of aging. Cadmium (Cd) modifies cellular processes that promote aging and disrupts methylation globally. Whether Cd modifies aging processes by influencing establishment of age-associated methylation marks is currently unknown. In this pilot study, we characterized methylation profiles in > 450 000 CpG sites in 40 non-smoking women (age 40-80) differentially exposed to environmental Cd from Thailand. Based on specific gravity adjusted urinary Cd, we classified them as high (HE) and low (LE) exposed and age-matched within 5 years. Urinary Cd was defined as below 2 µg/l in the LE group. We predicted epigenetic age (DNAm-age) using two published methods by Horvath and Hannum and examined the difference between epigenetic age and chronologic age (Δage). We assessed differences by Cd exposure using linear mixed models adjusted for estimated white blood cell proportions, BMI, and urinary creatinine. We identified 213 age-associated CpG sites in our population ( P LE (Hannum: 3.6 vs. 7.6 years, P = 0.0093; Horvath: 2.4 vs. 4.5 years, P = 0.1308). The Cd exposed group was associated with changes in methylation ( P < 0.05) at 12, 8, and 20 age-associated sites identified in our population, Hannum, and Horvath. From the results of this pilot study, elevated Cd exposure is associated with methylation changes at age-associated sites and smaller differences between DNAm-age and chronologic age, in contrast to expected age-accelerating effects. Cd may modify epigenetic aging, and biomarkers of aging warrant further investigation when examining Cd and its relationship with chronic disease and mortality.

  3. The TWEAK–Fn14 dyad is involved in age-associated pathological changes in skeletal muscle

    International Nuclear Information System (INIS)

    Tajrishi, Marjan M.; Sato, Shuichi; Shin, Jonghyun; Zheng, Timothy S.; Burkly, Linda C.; Kumar, Ashok

    2014-01-01

    Highlights: • The levels of TWEAK receptor Fn14 are increased in skeletal muscle during aging. • Deletion of Fn14 attenuates age-associated skeletal muscle fiber atrophy. • Deletion of Fn14 inhibits proteolysis in skeletal muscle during aging. • TWEAK–Fn14 signaling activates transcription factor NF-κB in aging skeletal muscle. • TWEAK–Fn14 dyad is involved in age-associated fibrosis in skeletal muscle. - Abstract: Progressive loss of skeletal muscle mass and strength (sarcopenia) is a major clinical problem in the elderly. Recently, proinflammatory cytokine TWEAK and its receptor Fn14 were identified as key mediators of muscle wasting in various catabolic states. However, the role of the TWEAK–Fn14 pathway in pathological changes in skeletal muscle during aging remains unknown. In this study, we demonstrate that the levels of Fn14 are increased in skeletal muscle of 18-month old (aged) mice compared with adult mice. Genetic ablation of Fn14 significantly increased the levels of specific muscle proteins and blunted the age-associated fiber atrophy in mice. While gene expression of two prominent muscle-specific E3 ubiquitin ligases MAFBx and MuRF1 remained comparable, levels of ubiquitinated proteins and the expression of autophagy-related molecule Atg12 were significantly reduced in Fn14-knockout (KO) mice compared with wild-type mice during aging. Ablation of Fn14 significantly diminished the DNA-binding activity of transcription factor nuclear factor-kappa B (NF-κB), gene expression of various inflammatory molecules, and interstitial fibrosis in skeletal muscle of aged mice. Collectively, our study suggests that the TWEAK–Fn14 signaling axis contributes to age-associated muscle atrophy and fibrosis potentially through its local activation of proteolytic systems and inflammatory pathways

  4. Effect of procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3.

    Science.gov (United States)

    Deevska, Gergana; Sunkara, Manjula; Karakashian, Claudia; Peppers, Benjamin; Morris, Andrew J; Nikolova-Karakashian, Mariana N

    2014-10-01

    In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.

  5. Proteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts

    Science.gov (United States)

    Waldera-Lupa, Daniel M.; Kalfalah, Faiza; Florea, Ana-Maria; Sass, Steffen; Kruse, Fabian; Rieder, Vera; Tigges, Julia; Fritsche, Ellen; Krutmann, Jean; Busch, Hauke; Boerries, Melanie; Meyer, Helmut E.; Boege, Fritz; Theis, Fabian

    2014-01-01

    We analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts’ aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77% of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging. PMID:25411231

  6. Sarcopenic obesity or obese sarcopenia: A cross talk between age-associated adipose tissue and skeletal muscle inflammation as a main mechanism of the pathogenesis.

    Science.gov (United States)

    Kalinkovich, Alexander; Livshits, Gregory

    2017-05-01

    Sarcopenia, an age-associated decline in skeletal muscle mass coupled with functional deterioration, may be exacerbated by obesity leading to higher disability, frailty, morbidity and mortality rates. In the combination of sarcopenia and obesity, the state called sarcopenic obesity (SOB), some key age- and obesity-mediated factors and pathways may aggravate sarcopenia. This review will analyze the mechanisms underlying the pathogenesis of SOB. In obese adipose tissue (AT), adipocytes undergo hypertrophy, hyperplasia and activation resulted in accumulation of pro-inflammatory macrophages and other immune cells as well as dysregulated production of various adipokines that together with senescent cells and the immune cell-released cytokines and chemokines create a local pro-inflammatory status. In addition, obese AT is characterized by excessive production and disturbed capacity to store lipids, which accumulate ectopically in skeletal muscle. These intramuscular lipids and their derivatives induce mitochondrial dysfunction characterized by impaired β-oxidation capacity and increased reactive oxygen species formation providing lipotoxic environment and insulin resistance as well as enhanced secretion of some pro-inflammatory myokines capable of inducing muscle dysfunction by auto/paracrine manner. In turn, by endocrine manner, these myokines may exacerbate AT inflammation and also support chronic low grade systemic inflammation (inflammaging), overall establishing a detrimental vicious circle maintaining AT and skeletal muscle inflammation, thus triggering and supporting SOB development. Under these circumstances, we believe that AT inflammation dominates over skeletal muscle inflammation. Thus, in essence, it redirects the vector of processes from "sarcopenia→obesity" to "obesity→sarcopenia". We therefore propose that this condition be defined as "obese sarcopenia", to reflect the direction of the pathological pathway. Copyright © 2016 Elsevier B.V. All rights

  7. Age-associated power decline from running, jumping, and throwing male masters world records.

    Science.gov (United States)

    Gava, Paolo; Kern, Helmut; Carraro, Ugo

    2015-01-01

    BACKGROUND/STUDY CONTEXT: The capacity to perform everyday tasks is directly related to the muscular power the body can develop (see Appendix). The age-related loss of power is a fact, but the characterization or the rate of muscle power loss remains an open issue. Data useful to study the decline of the skeletal muscles power are largely available from sources other than medical tests, e.g., from track and field competitions of Masters athletes. The aim of our study is to identify the age-related decline trend of the power developed by the athletes in carrying out the track and field events. Absolute male world records of 16 events were collected along with world records of male Masters categories. Performance was normalized with respect to the absolute record; the performance of various age groups is consequently represented by a number ranging from 1 (world absolute records) to 0 (null performance). The performance of a jumping event is transformed into a parameter proportional to the power developed by the athletes: the displacement of the center of gravity of the athlete. Throwing events are further normalized for the decreasing weight of the implements with the increasing age of the Masters athletes. Most track and field events show a linear decline to 70 years. The annual rate of power decline for all the events (running, throwing, and jumping), using a simplified synthesis, is 1.25% per year. The events that involve mostly upper limbs (shot put, javelin throw) show a higher rate of decline (1.4% per year) compared to those where the lower limbs are mostly involved (long jump 1.1%, track events 0.6-0.7% per year). This analysis of muscle power decline is only partially in line with the results of works based on clinical tests. A clarification of the reasons for such discrepancy may provide clinically significant information. Human power decline in Masters athletes was analyzed, adopting a coherent approach based on an extended database. Skeletal muscle power

  8. c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Diana M. Dunn

    2015-08-01

    Full Text Available The ability of Heat Shock Protein 90 (Hsp90 to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1, promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

  9. Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

    CSIR Research Space (South Africa)

    Makhoba, XH

    2015-09-01

    Full Text Available . coli host. However, some proteins have not been produced as pure, soluble or active proteins. For this reason this has led to some researchers to suggest the use of engineered host systems that do not produce endogenous molecular chaperones such as Hsp...

  10. Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites

    Czech Academy of Sciences Publication Activity Database

    Ettrich, R.; Brandt, W.; Kopecký ml., V.; Baumruk, V.; Hofbauerová, Kateřina; Pavlíček, Z.

    2002-01-01

    Roč. 383, č. 10 (2002), s. 1667-1676 ISSN 1431-6730 Grant - others:GA UK(CZ) 220/2000/B-CH; Volkswagen Foundation(DE) I/74679 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100001 Keywords : chaperone * haptoglobin * molecular modeling Subject RIV: BO - Biophysics Impact factor: 2.548, year: 2002

  11. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  12. Responsibility and Capacities

    DEFF Research Database (Denmark)

    Ryberg, Jesper

    2014-01-01

    That responsible moral agency presupposes certain mental capacities, constitutes a widely accepted view among theorists. Moreover, it is often assumed that degrees in the development of the relevant capacities co-vary with degrees of responsibility. In this article it is argued that, the move from...... the view that responsibility requires certain mental capacities to the position that degrees of responsibility co-vary with degrees of the development of the mental capacities, is premature....

  13. CDMA systems capacity engineering

    CERN Document Server

    Kim, Kiseon

    2004-01-01

    This new hands-on resource tackles capacity planning and engineering issues that are crucial to optimizing wireless communication systems performance. Going beyond the system physical level and investigating CDMA system capacity at the service level, this volume is the single-source for engineering and analyzing systems capacity and resources.

  14. The Mitochondrial-Derived Peptides, HumaninS14G and Small Humanin-like Peptide 2, Exhibit Chaperone-like Activity.

    Science.gov (United States)

    Okada, Alan K; Teranishi, Kazuki; Lobo, Fleur; Isas, J Mario; Xiao, Jialin; Yen, Kelvin; Cohen, Pinchas; Langen, Ralf

    2017-08-10

    Mitochondrial-derived peptides (MDPs) and their analogs have emerged as wide-spectrum, stress response factors protective in amyloid disease models. MDP cytoprotective functions are generally attributed to anti-apoptotic activity, however, little is known about their capacity to facilitate the cell's unfolded protein response via direct interactions with amyloidogenic proteins. Here, we explored the effects of the MDP-analog, humaninS14G (HNG), and the MDP, small humanin-like peptide 2 (SHLP2), on the misfolding of islet amyloid polypeptide (IAPP), a critical pathogenic step in type 2 diabetes mellitus (T2DM). Our thioflavin T fluorescence studies show that HNG inhibits IAPP misfolding at highly substoichiometric concentrations. Seeded fluorescence and co-sedimentation studies demonstrate MDPs block amyloid seeding and directly bind misfolded, seeding-capable IAPP species. Furthermore, our electron paramagnetic resonance spectroscopy and circular dichroism data indicate MDPs do not act by binding IAPP monomers. Taken together our results reveal a novel chaperone-like activity wherein these MDPs specifically target misfolded amyloid seeds to inhibit IAPP misfolding which, along with direct anti-apoptotic activity and beneficial metabolic effects, make HNG and SHLP2 exciting prospects as T2DM therapeutics. These data also suggest that other mitochondrial stress response factors within the MDP family may be amenable to development into therapeutics for protein-misfolding diseases.

  15. Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip.

    Science.gov (United States)

    Nelson, Gregory M; Huffman, Holly; Smith, David F

    2003-01-01

    Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function.

  16. Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

    Directory of Open Access Journals (Sweden)

    Marta del Alamo

    2011-07-01

    Full Text Available Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP, which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC, whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

  17. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

    Science.gov (United States)

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid.

  18. The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

    DEFF Research Database (Denmark)

    Mollerup, Jens; Berchtold, Martin Werner

    2005-01-01

    The heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFa-receptor activation as well as staurosporine...... treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase-3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18 kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis....... Furthermore, we found that the anti-aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis....

  19. Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

    Science.gov (United States)

    Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

    2015-10-01

    Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

  20. The Role of Bacterial Chaperones in the Circulative Transmission of Plant Viruses by Insect Vectors

    Directory of Open Access Journals (Sweden)

    Murad Ghanim

    2013-06-01

    Full Text Available Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.

  1. Christine Bard, Sylvie Chaperon (dirs), Dictionnaire des féministes. France xviiie-

    OpenAIRE

    Rouch, Marine

    2017-01-01

    Dix années auront été nécessaires pour la réalisation de cet ouvrage, véritable événement éditorial de l’année 2017 pour celles et ceux qui s’intéressent de près ou de loin au féminisme, à ses figures, à ses groupes. Comme l’indiquent Christine Bard et Sylvie Chaperon, les directrices de publication, il est désormais le premier du genre à alimenter l’« espace mémoriel immatériel du féminisme » (p. IX) et à inviter au « voyage dans le temps et l’espace du féminisme » (p. X). Il faut souligner ...

  2. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    Directory of Open Access Journals (Sweden)

    Angela Lavado-Roldán

    2016-07-01

    Full Text Available One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2 acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.

  3. The RNA chaperone Hfq enables the environmental stress tolerance super-phenotype of Pseudomonas putida.

    Science.gov (United States)

    Arce-Rodríguez, Alejandro; Calles, Belén; Nikel, Pablo I; de Lorenzo, Víctor

    2016-10-01

    The natural physiological regime of the soil bacterium Pseudomonas putida involves incessant exposure to endogenous metabolic conflicts and environmental physicochemical insults. Yet, the role of assisted small RNA-mRNA pairing in the stress tolerance super-phenotype that is the trademark of this bacterium has not been accredited. We have thoroughly explored the physiological consequences -in particular those related to exogenous stress - of deleting the hfq gene of P. putida, which encodes the major RNA chaperone that promotes sRNA-target mRNA interactions. While the overall trend was a general weakening of every robustness descriptor of the Δhfq strain, growth parameters and production of central metabolic enzymes were comparatively less affected than other qualities that depend directly on energy status (e.g. motility, DNA repair). The overall catalytic vigour of the mutant decreased to putida. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex.

    Science.gov (United States)

    Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J A

    2018-01-01

    Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking. © 2017 Federation of European Biochemical Societies.

  5. Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

    Science.gov (United States)

    Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C

    2009-04-15

    Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

  6. The mitochondrial chaperone protein TRAP1 mitigates α-Synuclein toxicity.

    Directory of Open Access Journals (Sweden)

    Erin K Butler

    2012-02-01

    Full Text Available Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1 was found to enhance age-dependent loss of fly head dopamine (DA and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein-induced sensitivity to rotenone treatment. In human (nonneuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein-induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein.

  7. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

    Science.gov (United States)

    Kim, Jong Hun; Lee, Eunjung; Friedline, Randall H; Suk, Sujin; Jung, Dae Young; Dagdeviren, Sezin; Hu, Xiaodi; Inashima, Kunikazu; Noh, Hye Lim; Kwon, Jung Yeon; Nambu, Aya; Huh, Jun R; Han, Myoung Sook; Davis, Roger J; Lee, Amy S; Lee, Ki Won; Kim, Jason K

    2018-04-01

    Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78 -/- ) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78 -/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78 -/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78 -/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

  8. Structural Analysis of Mycobacterium tuberculosis Homologues of the Eukaryotic Proteasome Assembly Chaperone 2 (PAC2).

    Science.gov (United States)

    Bai, Lin; Jastrab, Jordan B; Isasa, Marta; Hu, Kuan; Yu, Hongjun; Gygi, Steven P; Darwin, K Heran; Li, Huilin

    2017-05-01

    A previous bioinformatics analysis identified the Mycobacterium tuberculosis proteins Rv2125 and Rv2714 as orthologs of the eukaryotic proteasome assembly chaperone 2 (PAC2). We set out to investigate whether Rv2125 or Rv2714 can function in proteasome assembly. We solved the crystal structure of Rv2125 at a resolution of 3.0 Å, which showed an overall fold similar to that of the PAC2 family proteins that include the archaeal PbaB and the yeast Pba1. However, Rv2125 and Rv2714 formed trimers, whereas PbaB forms tetramers and Pba1 dimerizes with Pba2. We also found that purified Rv2125 and Rv2714 could not bind to M. tuberculosis 20S core particles. Finally, proteomic analysis showed that the levels of known proteasome components and substrate proteins were not affected by disruption of Rv2125 in M. tuberculosis Our work suggests that Rv2125 does not participate in bacterial proteasome assembly or function. IMPORTANCE Although many bacteria do not encode proteasomes, M. tuberculosis not only uses proteasomes but also has evolved a posttranslational modification system called pupylation to deliver proteins to the proteasome. Proteasomes are essential for M. tuberculosis to cause lethal infections in animals; thus, determining how proteasomes are assembled may help identify new ways to combat tuberculosis. We solved the structure of a predicted proteasome assembly factor, Rv2125, and isolated a genetic Rv2125 mutant of M. tuberculosis Our structural, biochemical, and genetic studies indicate that Rv2125 and Rv2714 do not function as proteasome assembly chaperones and are unlikely to have roles in proteasome biology in mycobacteria. Copyright © 2017 American Society for Microbiology.

  9. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    International Nuclear Information System (INIS)

    Qualley, Dominic F.; Sokolove, Victoria L.; Ross, James L.

    2015-01-01

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC

  10. Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression.

    Science.gov (United States)

    Bunprajun, Tipwadee; Henriksen, Tora Ida; Scheele, Camilla; Pedersen, Bente Klarlund; Green, Charlotte Jane

    2013-01-01

    Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

  11. Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell Fate.

    Science.gov (United States)

    Matsumoto, Collin; Jiang, Yan; Emathinger, Jacqueline; Quijada, Pearl; Nguyen, Nathalie; De La Torre, Andrea; Moshref, Maryam; Nguyen, Jonathan; Levinson, Aimee B; Shin, Minyoung; Sussman, Mark A; Hariharan, Nirmala

    2018-02-14

    Aging severely limits myocardial repair and regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesized that short telomeres activate p53 and induce autophagy to elicit the age-associated change in CPC fate. We isolated CPCs and compared mouse strains with different telomere lengths for phenotypic characteristics of aging. Wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres exhibits early cardiac aging with cardiac dysfunction, hypertrophy, fibrosis, and senescence, as compared with common lab strains FVB and C57 bearing longer telomeres. CAST CPCs with short telomeres demonstrate altered cell fate as characterized by cell cycle arrest, senescence, basal commitment, and loss of quiescence. Elongation of telomeres using a modified mRNA for telomerase restores youthful properties to CAST CPCs. Short telomeres induce autophagy in CPCs, a catabolic protein degradation process, as evidenced by reduced p62 and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation reverses the cell fate to a more youthful phenotype. Mechanistically, cell fate changes induced by short telomeres are partially p53 dependent, as p53 inhibition rescues senescence and commitment observed in CAST CPCs, coincident with attenuation of autophagy. In conclusion, short telomeres activate p53 and autophagy to tip the equilibrium away from quiescence and proliferation toward differentiation and senescence, leading to exhaustion of CPCs. This study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to prevent senescence of CPCs. Stem Cells 2018. © AlphaMed Press 2018.

  12. Capacity Statement for Railways

    DEFF Research Database (Denmark)

    Landex, Alex

    2007-01-01

    The subject “Railway capacity” is a combination of the capacity consumption and how the capacity is utilized. The capacity utilization of railways can be divided into 4 core elements: The number of trains; the average speed; the heterogeneity of the operation; and the stability. This article desc...... trains. This is due to network effects in the railway system and due to the fact that more trains results in lower punctuality....

  13. The French capacity mechanism

    International Nuclear Information System (INIS)

    2014-01-01

    The French capacity mechanism has been design to ensure security of supply in the context of the energy transition. This energy transition challenges the electricity market design with several features: peak load growth, the development of renewables, demand response,... To ensure security of supply in this context, a capacity mechanism is being implemented in France. It is a market wide capacity obligation on electricity suppliers, based on market principles. Suppliers are responsible for forecasting their obligation, which corresponds to their contribution to winter peak load, and must procure enough capacity certificates to meet their obligations. Capacity certificates are granted to capacities through a certification process, which assesses their contribution to security of supply on the basis of availability commitments. This certification process is technology neutral and performance based, associated with controls and penalties in case of non compliance. Demand Side is fully integrated in the market, either through the reduction of suppliers' capacity obligation or direct participation after certification. In addition to the expected benefits in terms of security of supply, the French capacity market will foster the development of demand response. The participation of foreign capacities will require adaptations which are scheduled in a road-map, and could pave the way for further European integration of energy policies. (authors)

  14. PAI-1–regulated miR-21 defines a novel age-associated fibrogenic pathway in muscular dystrophy

    Science.gov (United States)

    Ardite, Esther; Perdiguero, Eusebio; Vidal, Berta; Gutarra, Susana; Serrano, Antonio L.

    2012-01-01

    Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)–21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming growth factor (TGF)–β in muscle fibroblasts and the activation of miR-21 expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-β with a remarkable cell proliferation–promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1−/− mdx mice, could be reversed by miR-21 or uPA-selective interference, whereas forced miR-21 overexpression aggravated disease severity. The PAI-1–miR-21 fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals. PMID:22213800

  15. Effect of lifelong resveratrol supplementation and exercise training on skeletal muscle oxidative capacity in aging mice

    DEFF Research Database (Denmark)

    Ringholm, Stine; Olesen, Jesper; Pedersen, Jesper Thorhauge

    2013-01-01

    The present study tested the hypothesis that lifelong resveratrol (RSV) supplementation counteracts an age-associated decrease in skeletal muscle oxidative capacity through peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and that RSV combined with lifelong exercise training (ET...

  16. Heat Capacity Analysis Report

    International Nuclear Information System (INIS)

    Findikakis, A.

    2004-01-01

    The purpose of this report is to provide heat capacity values for the host and surrounding rock layers for the waste repository at Yucca Mountain. The heat capacity representations provided by this analysis are used in unsaturated zone (UZ) flow, transport, and coupled processes numerical modeling activities, and in thermal analyses as part of the design of the repository to support the license application. Among the reports that use the heat capacity values estimated in this report are the ''Multiscale Thermohydrologic Model'' report, the ''Drift Degradation Analysis'' report, the ''Ventilation Model and Analysis Report, the Igneous Intrusion Impacts on Waste Packages and Waste Forms'' report, the ''Dike/Drift Interactions report, the Drift-Scale Coupled Processes (DST and TH Seepage) Models'' report, and the ''In-Drift Natural Convection and Condensation'' report. The specific objective of this study is to determine the rock-grain and rock-mass heat capacities for the geologic stratigraphy identified in the ''Mineralogic Model (MM3.0) Report'' (BSC 2004 [DIRS 170031], Table 1-1). This report provides estimates of the heat capacity for all stratigraphic layers except the Paleozoic, for which the mineralogic abundance data required to estimate the heat capacity are not available. The temperature range of interest in this analysis is 25 C to 325 C. This interval is broken into three separate temperature sub-intervals: 25 C to 95 C, 95 C to 114 C, and 114 C to 325 C, which correspond to the preboiling, trans-boiling, and postboiling regimes. Heat capacity is defined as the amount of energy required to raise the temperature of a unit mass of material by one degree (Nimick and Connolly 1991 [DIRS 100690], p. 5). The rock-grain heat capacity is defined as the heat capacity of the rock solids (minerals), and does not include the effect of water that exists in the rock pores. By comparison, the rock-mass heat capacity considers the heat capacity of both solids and pore

  17. Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence.

    Science.gov (United States)

    Cao, Huifang; Yang, Cuiting; Quan, Shu; Hu, Tianjian; Zhang, Lingzhi; Zhang, Yuanxing; Yang, Dahai; Liu, Qin

    2018-01-01

    Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host-pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N-terminal α-helix. We also found that EseK has an N-terminal chaperone-binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector-chaperone interaction and contributes to our understanding of bacterial pathogenesis. © 2017 John Wiley & Sons Ltd.

  18. An interaction network predicted from public data as a discovery tool: application to the Hsp90 molecular chaperone machine.

    Directory of Open Access Journals (Sweden)

    Pablo C Echeverría

    Full Text Available Understanding the functions of proteins requires information about their protein-protein interactions (PPI. The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 co-chaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community.

  19. Structure and Interactions of the TPR Domain of Sgt2 with Yeast Chaperones and Ybr137wp

    Directory of Open Access Journals (Sweden)

    Ewelina M. Krysztofinska

    2017-10-01

    Full Text Available Small glutamine-rich tetratricopeptide repeat-containing protein 2 (Sgt2 is a multi-module co-chaperone involved in several protein quality control pathways. The TPR domain of Sgt2 and several other proteins, including SGTA, Hop, and CHIP, is a highly conserved motif known to form transient complexes with molecular chaperones such as Hsp70 and Hsp90. In this work, we present the first high resolution crystal structures of Sgt2_TPR alone and in complex with a C-terminal peptide PTVEEVD from heat shock protein, Ssa1. Using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we demonstrate that Sgt2_TPR interacts with peptides corresponding to the C-termini of Ssa1, Hsc82, and Ybr137wp with similar binding modes and affinities.

  20. At the Start of the Sarcomere: A Previously Unrecognized Role for Myosin Chaperones and Associated Proteins during Early Myofibrillogenesis

    Directory of Open Access Journals (Sweden)

    J. Layne Myhre

    2012-01-01

    Full Text Available The development of striated muscle in vertebrates requires the assembly of contractile myofibrils, consisting of highly ordered bundles of protein filaments. Myofibril formation occurs by the stepwise addition of complex proteins, a process that is mediated by a variety of molecular chaperones and quality control factors. Most notably, myosin of the thick filament requires specialized chaperone activity during late myofibrillogenesis, including that of Hsp90 and its cofactor, Unc45b. Unc45b has been proposed to act exclusively as an adaptor molecule, stabilizing interactions between Hsp90 and myosin; however, recent discoveries in zebrafish and C. elegans suggest the possibility of an earlier role for Unc45b during myofibrillogenesis. This role may involve functional control of nonmuscle myosins during the earliest stages of myogenesis, when premyofibril scaffolds are first formed from dynamic cytoskeletal actin. This paper will outline several lines of evidence that converge to build a model for Unc45b activity during early myofibrillogenesis.

  1. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

    International Nuclear Information System (INIS)

    Umehara, Takashi; Otta, Yumi; Tsuganezawa, Keiko; Matsumoto, Takehisa; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2005-01-01

    The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1 + is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1 + -encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation

  2. Prospects for OPEC capacity

    International Nuclear Information System (INIS)

    Adelman, M.A.

    1995-01-01

    OPEC capacity is not exogenous, but responds to demand. Price increases have not been caused by capacity shortages. OPEC nations find it hard to set aside even very small portions of their revenues for oil investment, despite its extreme profitability. Foreign investors face high risks. Production sharing makes their after-tax return even more unstable. (author)

  3. Identification of VPA0451 as the specific chaperone for the Vibrio parahaemolyticus chromosome 1 type III-secreted effector VPA0450.

    Science.gov (United States)

    Waddell, Barbara; Southward, Carolyn M; McKenna, Neil; Devinney, Rebekah

    2014-04-01

    Vibrio parahaemolyticus is an important cause of gastroenteritis resulting from the consumption of raw or undercooked shellfish. The V. parahaemolyticus genome revealed the presence of two type III secretion systems (T3SS); one on each of the two chromosomes. To date, four effectors have been identified as secreted by the chromosome 1 T3SS (T3SS1). For some effectors, efficient secretion requires a cytosolic chaperone that is often encoded in close proximity to its cognate effector. In this study, we identified VPA0451 as the specific chaperone for the T3SS1 effector, VPA0450. VPA0451 is structurally similar to known T3SS chaperones. It is required for efficient VPA0450 secretion while not affecting the secretion of other T3SS1 effectors, suggesting it is a class 1A single cargo chaperone. VPA0450 translocation into the host cell membrane requires VPA0451. VPA0451 binds directly to VPA0450, and amino acids 25-100 contribute to this activity. Taken together, we conclude that VPA0451 is the cognate chaperone for the effector VPA0450 and is the second T3SS1 chaperone identified to date. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  4. Serum antibodies to Porphyromonas gingivalis chaperone HtpG predict health in periodontitis susceptible patients.

    Directory of Open Access Journals (Sweden)

    Charles E Shelburne

    2008-04-01

    Full Text Available Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG might be associated with protection/resistance against destructive periodontitis.ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043 but not anti-Fusobacterium nucleatum (an oral opportunistic commensal HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018. There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG.OUR RESULTS SUGGEST: 1 anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2 may augment the host defence to periodontitis and 3 a unique peptide of P. gingivalis HtpG offers significant potential as an

  5. SigE Is a Chaperone for the Salmonella enterica Serovar Typhimurium Invasion Protein SigD

    OpenAIRE

    Darwin, K. Heran; Robinson, Lloyd S.; Miller, Virginia L.

    2001-01-01

    SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His6 on a nickel column.

  6. Distinct Roles for the Hsp40 and Hsp90 Molecular Chaperones during Cystic Fibrosis Transmembrane Conductance Regulator Degradation in Yeast

    OpenAIRE

    Youker, Robert T.; Walsh, Peter; Beilharz, Traude; Lithgow, Trevor; Brodsky, Jeffrey L.

    2004-01-01

    Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydr...

  7. Using SANS to monitor the interaction of misfolding alcohol dehydrogenase with the molecular chaperone protein 14-3-3ζ

    International Nuclear Information System (INIS)

    Goodwin, Katy L.; Rekas, Agata; Wood, Katy; Knott, Robert B.; Carver, John A.

    2015-01-01

    14-3-3 is a family of acidic, dimeric proteins which are highly conserved across many species. Each monomer is approximately 30kDa in mass and contains 9 α-helices. Dimer formation is initiated at the N-terminal region of the protein as a result of the interaction between several buried polar and hydrophobic residues in this region. 14-3-3 proteins interact with a wide range of proteins to regulate many cellular processes, e.g. apoptosis and mitosis, as well as protein misfolding associated with conformational diseases such as Alzheimer’s and Parkinson’s Disease. A potential role of 14-3-3 in these diseases was discovered with the observation that 14-3-3ζ can act as a molecular chaperone, whereby it stabilises intermediately folded proteins to prevent their aggregation. The binding site and mechanism of the chaperone action of 14-3-3ζ are not known, despite being narrowed down in our NMR study. We produced deuterated 14-3-3ζ and used it in SANS experiments with a model misfolding protein, alcohol dehydrogenase (ADH). Contrast variation allowed us to monitor changes in each component separately after the initiation of ADH misfolding. The R g and D max values of ADH under stress show an increase in size with time, consistent with unfolding and aggregation. In the presence of 14-3-3ζ, the unfolding of ADH is reduced and the protein maintains a globular expanded conformation consistent with an adoption of an intermediately folded (molten globule) state. 14-3-3ζ whilst chaperoning showed a reduction in size, possibly due to dissociation. Ab initio models were also obtained. This is the first instance where conformational changes during chaperoning of either a partly folded target protein, or 14-3-3ζ, have been observed.

  8. Depletion of the C. elegans NAC engages the unfolded protein response, resulting in increased chaperone expression and apoptosis.

    Directory of Open Access Journals (Sweden)

    Paul T Arsenovic

    Full Text Available The nascent polypeptide-associated complex (NAC is a highly conserved heterodimer important for metazoan development, but its molecular function is not well understood. Recent evidence suggests the NAC is a component of the cytosolic chaperone network that interacts with ribosomal complexes and their emerging nascent peptides, such that the loss of the NAC in chaperone-depleted cells results in an increase in misfolded protein stress. We tested whether the NAC functions similarly in Caeonorhabditis (C. elegans and found that its homologous NAC subunits, i.e. ICD-1 and -2, have chaperone-like characteristics. Loss of the NAC appears to induce misfolded protein stress in the ER triggering the unfolded protein response (UPR. Depletion of the NAC altered the response to heat stress, and led to an up-regulation of hsp-4, a homologue of the human chaperone and ER stress sensor GRP78/BiP. Worms lacking both ICD-1 and the UPR transcription factor XBP-1 generated a higher proportion of defective embryos, showed increased embryonic apoptosis and had a diminished survival rate relative to ICD-1-depleted animals with an intact UPR. Up-regulation of hsp-4 in NAC-depleted animals was specific to certain regions of the embryo; in embryos lacking ICD-1, the posterior region of the embryo showed strong up-regulation of hsp-4, while the anterior region did not. Furthermore, loss of ICD-1 produced prominent lysosomes in the gut region of adults and embryos putatively containing lipofuscins, lipid/protein aggregates associated with cellular aging. These results are the first set of evidence consistent with a role for C. elegans NAC in protein folding and localization during translation. Further, these findings confirm C. elegans as a valuable model for studying organismal and cell-type specific responses to misfolded protein stress.

  9. Heat shock protein 90 (Hsp90) chaperone complex. A molecular target for enhancement of thermosensitivity and radiosensitivity

    International Nuclear Information System (INIS)

    Akimoto, Tetsuo; Nonaka, Tetsuo; Kitamoto, Yoshizumi; Sakurai, Hideyuki

    2002-01-01

    Heat shock protein 90 (Hsp90) is a highly conserved heat shock protein in animal and plants, and exists abundantly in the cytoplasm in unstressed condition, accounting for 1-2% in cytoplasmic proteins. Main difference of Hsp90 from other Hsps are its substrate that Hsp90 binds to. These substrates include various signal transduction proteins, kinase, steroid receptors and transcription factors, therefore, Hsp90 plays a key role in maintaining cellular signal transduction networks. Many chaperoned proteins (client proteins) of Hsp90 are associated with cellular proliferation or malignant transformation, thus Hsp90 chaperone complex has been focused as targets for cancer therapy. Among the client proteins, there are several molecules that have been defined as targets or factors for determination or enhancement of radiosensitivity or thermosensitivity. Thus, it is easily speculated that Hsp90 chaperone complex inhibitors that disrupt association of Hsp90 and client protein in combination with radiation or/and heat has potential effect on enhancement of radiosensitivity or thermosensitivity. In this paper, possible mechanisms in enhancing radiosensitivity or thermosensitivity according to the client proteins will be summarized. (author)

  10. PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum.

    Science.gov (United States)

    Florentin, Anat; Cobb, David W; Fishburn, Jillian D; Cipriano, Michael J; Kim, Paul S; Fierro, Manuel A; Striepen, Boris; Muralidharan, Vasant

    2017-11-14

    The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. The Hsp90 co-chaperones Sti1, Aha1, and P23 regulate adaptive responses to antifungal azoles

    Directory of Open Access Journals (Sweden)

    Xiaokui Gu

    2016-10-01

    Full Text Available Heat Shock Protein 90 (Hsp90 is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast, Aha1, and P23 (Sba1 in yeast were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

  12. HDAC6 inhibition enhances 17-AAG--mediated abrogation of hsp90 chaperone function in human leukemia cells.

    Science.gov (United States)

    Rao, Rekha; Fiskus, Warren; Yang, Yonghua; Lee, Pearl; Joshi, Rajeshree; Fernandez, Pravina; Mandawat, Aditya; Atadja, Peter; Bradner, James E; Bhalla, Kapil

    2008-09-01

    Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

  13. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks

    DEFF Research Database (Denmark)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia

    2015-01-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase......, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required...... for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling...

  14. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

    Science.gov (United States)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

  15. Cyanidin-3-O-glucoside functions like chemical chaperone and attenuates the glycation mediated amyloid formation in albumin.

    Science.gov (United States)

    Prasanna, Govindarajan; Jing, Pu

    2018-04-02

    In this study, chemical chaperone like function of cyanidin-3-O-glucoside (C3G) was investigated through fluorescence spectroscopy, UV-visible spectroscopy, circular dichroism spectroscopy, confocal microscopy, scanning electron microscopy and molecular docking studies. Early and advanced glycation inhibitory effect was evaluated by fluorescence spectroscopy and agarose gel electrophoresis. Amyloids were investigated based on their propensity to bind Congo Red (CR) and Thioflavin T (ThT) by multiple microscopic approaches. Circular dichroism studies were used to analyze the changes in the secondary structure due to glycation. C3G effectively inhibited early and advanced glycation by masking like function, carbonyl scavenging and chemical chaperone activity. C3G had molecular interaction with Glu186, Arg427, Ser428, Lys431, Arg435, and Arg458 of BSA. Based on the microscopic analysis, it is evident that C3G can inhibit protein aggregation and amyloid formation. Circular dichroism studies suggested that glycation had resulted in augmented β-sheet propensity, whereas C3G had a protective effect on the helical conformation of BSA. We conclude that C3G has a chemical chaperone like function on the event of glycation mediated amyloid formation in BSA. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation*

    Science.gov (United States)

    Monsellier, Elodie; Redeker, Virginie; Ruiz-Arlandis, Gemma; Bousset, Luc; Melki, Ronald

    2015-01-01

    The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease. PMID:25505179

  17. Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

    Directory of Open Access Journals (Sweden)

    Steven G. Griffiths

    2017-10-01

    Full Text Available The complexity of human tissue fluid precludes timely identification of cancer biomarkers by immunoassay or mass spectrometry. An increasingly attractive strategy is to primarily enrich extracellular vesicles (EVs released from cancer cells in an accelerated manner compared to normal cells. The Vn96 peptide was herein employed to recover a subset of EVs released into the media from cellular models of breast cancer. Vn96 has affinity for heat shock proteins (HSPs decorating the surface of EVs. Reflecting their cells of origin, cancer EVs displayed discrete differences from those of normal phenotype. GELFrEE LC/MS identified an extensive proteome from all three sources of EVs, the vast majority having been previously reported in the ExoCarta database. Pathway analysis of the Vn96-affinity proteome unequivocally distinguished EVs from tumorigenic cell lines (SKBR3 and MCF-7 relative to a non-tumorigenic source (MCF-10a, particularly with regard to altered metabolic enzymes, signaling, and chaperone proteins. The protein data sets provide valuable information from material shed by cultured cells. It is probable that a vast amount of biomarker identities may be collected from established and primary cell cultures using the approaches described here.

  18. Substrate-Activated Conformational Switch on Chaperones Encodes a Targeting Signal in Type III Secretion

    Directory of Open Access Journals (Sweden)

    Li Chen

    2013-03-01

    Full Text Available The targeting of type III secretion (TTS proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone, CesAB, to the EscN ATPase of the enteropathogenic E. coli (EPEC. We show that EscN selectively engages the EspA-loaded CesAB but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon the binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB-EspA complex and EscN resulted in severe secretion and infection defects. Additionally, we show that the targeting and secretion signals are distinct and that the two processes are likely regulated by different mechanisms.

  19. Possible involvement of the Sigma-1 receptor chaperone in chemotherapeutic-induced neuropathic pain.

    Science.gov (United States)

    Tomohisa, Mori; Junpei, Ohya; Aki, Masumoto; Masato, Harumiya; Mika, Fukase; Kazumi, Yoshizawa; Teruo, Hayashi; Tsutomu, Suzuki

    2015-11-01

    Previous studies have shown that ligands of the sigma-1 receptor chaperone (Sig-1R) regulate pain-related behaviors. Clinical use of chemotherapeutics is often compromised due to their adverse side effects, particularly those related to neuropathy. Previous studies have shown that repeated administration of oxaliplatin and paclitaxel produces neuropathy in rodents. Therefore, the aim of the present study was to clarify the involvement of the Sig-1R in chemotherapeutic-induced neuropathy by examining the effects of oxaliplatin and paclitaxel on the Sig-1R levels in the spinal cord, and by examining the effects of Sig-1R agonist and antagonist on oxaliplatin- and paclitaxel-induced neuropathy in rats. Chemotherapeutic-induced neuropathic pain was accompanied by a significant reduction of the Sig-1R level in the spinal cord. Furthermore, the administration of paclitaxel to CHO cells that stably overexpressed Sig-1Rs induced the clustering of Sig-1Rs. We also found that the Sig-1R agonist SA4503 potently inhibited the neuropathy induced by oxaliplatin- and paclitaxel, whereas this action was abolished by the Sig-1R antagonist NE-100. These results suggest that the reduction of Sig-1R activity is involved in chemotherapeutic-induced neuropathy, and the Sig-1R agonist SA4503 could serve as a potential candidate for the treatment of chemotherapeutic-induced neuropathy. © 2015 Wiley Periodicals, Inc.

  20. The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract

    DEFF Research Database (Denmark)

    Hansen, Lars; Comyn, Sophie; Mang, Yuan

    2014-01-01

    in this region, in ACACA and UNC45B. As alterations of the UNC45B protein have been shown to affect eye development in model organisms, effort was focused on the heterozygous UNC45B missense mutation. UNC45B encodes a myosin-specific chaperone that, together with the general heat shock protein HSP90, is involved...... in myosin assembly. The mutation changes p.Arg805 to Trp in the UCS domain, an amino acid that is highly conserved from yeast to human. UNC45B is strongly expressed in the heart and skeletal muscle tissue, but here we show expression in human embryo eye and zebrafish lens. The zebrafish mutant steif......, carrying an unc45b nonsense mutation, has smaller eyes than wild-type embryos and shows accumulation of nuclei in the lens. Injection of RNA encoding the human wild-type UNC45B protein into the steif homozygous embryo reduced the nuclei accumulation and injection of human mutant UNC45B cDNA in wild...

  1. A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

    Directory of Open Access Journals (Sweden)

    Cécile M. Doyen

    2015-11-01

    Full Text Available During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG box, which we termed male-specific transcript (MST-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin.

  2. SIEVE ELEMENT-LINING CHAPERONE1 Restricts Aphid Feeding on Arabidopsis during Heat Stress.

    Science.gov (United States)

    Kloth, Karen J; Busscher-Lange, Jacqueline; Wiegers, Gerrie L; Kruijer, Willem; Buijs, Gonda; Meyer, Rhonda C; Albrectsen, Benedicte R; Bouwmeester, Harro J; Dicke, Marcel; Jongsma, Maarten A

    2017-10-01

    The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural Arabidopsis thaliana accessions, we identified the small heat shock-like SIEVE ELEMENT-LINING CHAPERONE1 ( SLI1 ). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On sli1 mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when SLI1 expression is upregulated. At this moderately high temperature, sli1 mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress. © 2017 American Society of Plant Biologists. All rights reserved.

  3. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

    Science.gov (United States)

    Niemiec, Moritz S; Weise, Christoph F; Wittung-Stafshede, Pernilla

    2012-01-01

    Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

  4. PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1.

    Directory of Open Access Journals (Sweden)

    Julia W Pridgeon

    2007-07-01

    Full Text Available Mutations in the PTEN induced putative kinase 1 (PINK1 gene cause an autosomal recessive form of Parkinson disease (PD. So far, no substrates of PINK1 have been reported, and the mechanism by which PINK1 mutations lead to neurodegeneration is unknown. Here we report the identification of TNF receptor-associated protein 1 (TRAP1, a mitochondrial molecular chaperone also known as heat shock protein 75 (Hsp75, as a cellular substrate for PINK1 kinase. PINK1 binds and colocalizes with TRAP1 in the mitochondria and phosphorylates TRAP1 both in vitro and in vivo. We show that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Moreover, we find that the ability of PINK1 to promote TRAP1 phosphorylation and cell survival is impaired by PD-linked PINK1 G309D, L347P, and W437X mutations. Our findings suggest a novel pathway by which PINK1 phosphorylates downstream effector TRAP1 to prevent oxidative-stress-induced apoptosis and implicate the dysregulation of this mitochondrial pathway in PD pathogenesis.

  5. Corticosteroid Receptors, Their Chaperones and Cochaperones: How Do They Modulate Adipogenesis?

    Directory of Open Access Journals (Sweden)

    Judith Toneatto

    2014-11-01

    Full Text Available It is well known that glucocorticoids and mineralocorticoids are part of the list of hormones that control adipogenesis as well as different aspects of the physiology of the adipose tissue. Their actions are mediated through their binding to the glucocorticoid and the mineralocorticoid receptors (GR and MR, respectively, in complex with heat shock proteins (Hsps and high molecular weight immunophilins (IMMs. Albeit many aspects of the molecular mechanism of the corticosteroid receptors are not fully elucidated yet, it was not until recently that the first evidences of the functional importance of Hsps and IMMs in the process of adipocyte differentiation have been described. Hsp90 and the high molecular weight IMM FKBP51 modulate GR and MR activity at multiple levels, that is, hormone binding affinity, their subcellular distribution, and the transcriptional status, among other aspects of the NR function. Interestingly, it has recently been described that Hsp90 and FKBP51 also participate in the control of PPARγ, a key transcription factor in the control of adipogenesis and the maintenance of the adipocyte phenotype. In addition, novel roles have been uncovered for FKBP51 in the organization of the nuclear architecture through its participation in the reorganization of the nuclear lamina and the control of the subnuclear distribution of GR. Thus, the aim of this review is to integrate and discuss the actual understanding of the role of corticosteroid receptors, their chaperones and cochaperones, in the process of adipocyte differentiation.

  6. Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone.

    Science.gov (United States)

    Ito, Shinya; Nagata, Kazuhiro

    2017-02-01

    Hsp47, a collagen-specific molecular chaperone that localizes in the endoplasmic reticulum (ER), is indispensable for molecular maturation of collagen. Hsp47, which is encoded by the SERPINH1 gene, belongs to the serpin family and has the serpin fold; however, it has no serine protease inhibitory activity. Hsp47 transiently binds to procollagen in the ER, dissociates in the cis-Golgi or ER-Golgi intermediate compartment (ERGIC) in a pH-dependent manner, and is then transported back to the ER via its RDEL retention sequence. Hsp47 recognizes collagenous (Gly-Xaa-Arg) repeats on triple-helical procollagen and can prevent local unfolding and/or aggregate formation of procollagen. Gene disruption of Hsp47 in mice causes embryonic lethality due to impairments in basement membrane and collagen fibril formation. In Hsp47-knockout cells, the type I collagen triple helix forms abnormally, resulting in thin and frequently branched fibrils. Secretion of type I collagens is slow and plausible in making aggregates of procollagens in the ER of hsp47-knocked out fibroblasts, which are ultimately degraded by autophagy. Mutations in Hsp47 are causally associated with osteogenesis imperfecta. Expression of Hsp47 is strongly correlated with expression of collagens in multiple types of cells and tissues. Therefore, Hsp47 represents a promising target for treatment of collagen-related disorders, including fibrosis of the liver, lung, and other organs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus.

    Science.gov (United States)

    Frees, Dorte; Gerth, Ulf; Ingmer, Hanne

    2014-03-01

    Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved biological processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cell of damaged proteins and by regulating abundance of functional proteins such as regulatory proteins. The ATP-dependent ClpP protease is highly conserved among eubacteria and in the chloroplasts and mitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation of clpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence. The contribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPases and the Clp protease are central players in stress responses required to cope with the adverse conditions met in the host. The ClpP protease has a dual role herein, as it both eliminates stress-damaged proteins as well as ensures the timely degradation of major stress regulators such as Spx, LexA and CtsR. Additionally, as we will summarize in this review, Clp proteases and Clp chaperones impact on such central processes as virulence gene expression, cell wall metabolism, survival in stationary phase, and cell division. These observations together with recent findings that Clp proteins contribute to adaptation to antibiotics highlights the importance of this interesting proteolytic machinery both for understanding pathogenicity of the organism and for treating staphylococcal infections. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

    Science.gov (United States)

    Tryggvesson, Anders; Ståhlberg, Frida M; Mogk, Axel; Zeth, Kornelius; Clarke, Adrian K

    2012-09-01

    The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

  9. Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.

    Directory of Open Access Journals (Sweden)

    Hermann Hämmerle

    Full Text Available In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A(15 and ADP were shown to bind to tripartite binding motifs (ARE circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65 in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.

  10. The RNA Chaperone Hfq Regulates Antibiotic Biosynthesis in the Rhizobacterium Pseudomonas aeruginosa M18

    Science.gov (United States)

    Wang, Guohao; Li, Sainan; Huang, Jiaofang; Wei, Xue; Li, Yaqian

    2012-01-01

    The rhizosphere microbe Pseudomonas aeruginosa M18 shows strong antifungal activities, mainly due to the biosynthesis of antibiotics like pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The ubiquitous RNA chaperone Hfq regulates bacterial virulence and stress tolerance through global posttranscriptional regulation. Here, we explored the molecular mechanism by which Hfq controls antibiotic biosynthesis in P. aeruginosa M18. The robust downregulation of Plt biosynthesis by Hfq was mediated exclusively by the posttranscriptional downregulation of the plt transcriptional activator PltR. Hfq posttranscriptionally repressed phzM expression and consequently reduced the conversion of PCA to pyocyanin. However, Hfq positively controlled the phz2 operon and PCA biosynthesis through both QscR-mediated transcriptional regulation at the promoter and an unknown regulation at the operator. Also, Hfq was shown to directly bind at the mRNA 5′ untranslated leaders of pltR, qscR, and phzM. These three negatively regulated target genes of Hfq shared a similar secondary structure with a short single-stranded AU-rich spacer (a potential Hfq-binding motif) linking two stem-loops. Taken together, these results indicate that Hfq, potentially in collaboration with unknown small noncoding RNAs (sRNAs), tightly controls antibiotic biosynthesis through both direct posttranscriptional inhibition and indirect transcriptional regulation. PMID:22427627

  11. Activation of sigma-1 receptor chaperone in the treatment of neuropsychiatric diseases and its clinical implication

    Directory of Open Access Journals (Sweden)

    Kenji Hashimoto

    2015-01-01

    Full Text Available Endoplasmic reticulum (ER protein sigma-1 receptor represents unique chaperone activity in the central nervous system, and it exerts a potent influence on a number of neurotransmitter systems. Several lines of evidence suggest that activation of sigma-1 receptor plays a role in the pathophysiology of neuropsychiatric diseases, as well as in the mechanisms of some therapeutic drugs and neurosteroids. Preclinical studies showed that some selective serotonin reuptake inhibitors (SSRIs; fluvoxamine, fluoxetine, excitalopram, donepezil, and ifenprodil act as sigma-1 receptor agonists. Furthermore, sigma-1 receptor agonists could improve the N-methyl-D-aspartate (NMDA antagonist phencyclidine (PCP-induced cognitive deficits in mice. A study using positron emission tomography have demonstrated that an oral administration of fluvoxamine or donepezil could bind to sigma-1 receptor in the healthy human brain, suggesting that sigma-1 receptor might be involved in the therapeutic mechanisms of these drugs. Moreover, case reports suggest that sigma-1 receptor agonists, including fluvoxamine, and ifenprodil, may be effective in the treatment of cognitive impairment in schizophrenia, delirium in elderly people, and flashbacks in post-traumatic stress disorder. In this review article, the author would like to discuss the clinical implication of sigma-1 receptor agonists, including endogenous neurosteroids, in the neuropsychiatric diseases.

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

    Directory of Open Access Journals (Sweden)

    Seung-Wook Shin

    2012-11-01

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

  13. Direct observation of the uptake of outer membrane proteins by the periplasmic chaperone Skp.

    Directory of Open Access Journals (Sweden)

    Zhi-Xin Lyu

    Full Text Available The transportation of membrane proteins through the aqueous subcellular space is an important and challenging process. Its molecular mechanism and the associated structural change are poorly understood. Periplasmic chaperones, such as Skp in Escherichia coli, play key roles in the transportation and protection of outer membrane proteins (OMPs in Gram-negative bacteria. The molecular mechanism through which Skp interacts with and protects OMPs remains mysterious. Here, a combined experimental and molecular dynamics simulation study was performed to gain the structural and dynamical information in the process of OMPs and Skp binding. Stopped-flow experiments on site specific mutated and labeled Skp and several OMPs, namely OmpC, the transmembrane domain of OmpA, and OmpF, allowed us to obtain the mechanism of OMP entering the Skp cavity, and molecular dynamics simulations yielded detailed molecular interactions responsible for this process. Both experiment and simulation show that the entrance of OMP into Skp is a highly directional process, which is initiated by the interaction between the N-terminus of OMP and the bottom "tentacle" domain of Skp. The opening of the more flexible tentacle of Skp, the non-specific electrostatic interactions between OMP and Skp, and the constant formation and breaking of salt bridges between Skp and its substrate together allow OMP to enter Skp and gradually "climb" into the Skp cavity in the absence of an external energy supply.

  14. Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis

    Science.gov (United States)

    Park, Sei-Kyoung; Hong, Joo Y.; Arslan, Fatih; Tietsort, Alex; Tank, Elizabeth M. H.; Li, Xingli

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS. PMID:28531192

  15. Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

    Science.gov (United States)

    Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

    2017-07-28

    Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

  16. Macroautophagy and Chaperone-Mediated Autophagy in Heart Failure: The Known and the Unknown

    Directory of Open Access Journals (Sweden)

    Rajeshwary Ghosh

    2018-01-01

    Full Text Available Cardiac diseases including hypertrophic and ischemic cardiomyopathies are increasingly being reported to accumulate misfolded proteins and damaged organelles. These findings have led to an increasing interest in protein degradation pathways, like autophagy, which are essential not only for normal protein turnover but also in the removal of misfolded and damaged proteins. Emerging evidence suggests a previously unprecedented role for autophagic processes in cardiac physiology and pathology. This review focuses on the major types of autophagic processes, the genes and protein complexes involved, and their regulation. It discusses the key similarities and differences between macroautophagy, chaperone-mediated autophagy, and selective mitophagy structures and functions. The genetic models available to study loss and gain of macroautophagy, mitophagy, and CMA are discussed. It defines the markers of autophagic processes, methods for measuring autophagic activities, and their interpretations. This review then summarizes the major studies of autophagy in the heart and their contribution to cardiac pathology. Some reports suggest macroautophagy imparts cardioprotection from heart failure pathology. Meanwhile, other studies find macroautophagy activation may be detrimental in cardiac pathology. An improved understanding of autophagic processes and their regulation may lead to a new genre of treatments for cardiac diseases.

  17. Expression of one important chaperone protein, heat shock protein 27, in neurodegenerative diseases.

    Science.gov (United States)

    Zhang, Xuekai; Shi, Jing; Tian, Jinzhou; Robinson, Andrew C; Davidson, Yvonne S; Mann, David M

    2014-01-01

    Many neurodegenerative diseases are characterised by accumulations of misfolded proteins that can colocalise with chaperone proteins (for example, heat shock protein 27 (HSP27)), which might act as modulators of protein aggregation. The role of HSP27 in the pathogenesis of neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD) and motor neuron disease (MND) was investigated. We used immunohistochemical and Western blot analysis to determine the distribution and amount of this protein in the frontal and temporal cortices of diseased and control subjects. HSP27 immunostaining presented as accumulations of granules within neuronal and glial cell perikarya. Patients with AD and FTLD were affected more often, and showed greater immunostaining for HSP27, than patients with MND and controls. In FTLD, there was no association between HSP27 and histological type. The neuropathological changes of FTLD, AD and MND were not immunoreactive to HSP27. Western blot analysis revealed higher HSP27 expression in FTLD than in controls, but without qualitative differences in banding patterns. The pattern of HSP27 immunostaining observed may reflect the extent of ongoing neurodegeneration in affected brain areas and is not specific to FTLD, AD or MND. It may represent an accumulation of misfolded, damaged or unwanted proteins, awaiting or undergoing degradation.

  18. Vedr.: Military capacity building

    DEFF Research Database (Denmark)

    Larsen, Josefine Kühnel; Struwe, Lars Bangert

    2013-01-01

    Military capacity building has increasingly become an integral part of Danish defence. Military capacity is a new way of thinking Danish defence and poses a new set of challenges and opportunities for the Danish military and the Political leadership. On the 12th of december, PhD. Candidate Josefine...... Kühnel Larsen and researcher Lars Bangert Struwe of CMS had organized a seminar in collaboration with Royal Danish Defense Colleg and the East African Security Governance Network. The seminar focused on some of the risks involved in Military capacity building and how these risks are dealt with from...

  19. Quality of Life and Functional Status Across the Life Course. Project 2: Investigating Mechanisms to Explain Age Associated Differences in Quality of Life Among Breast Cancer Patients

    National Research Council Canada - National Science Library

    Naughton, Michelle

    2002-01-01

    .... The study will examine psychosocial factors such as social support, coping strategies, resiliency, and the impact of cancer on life responsibilities as explanations of age-associated factors affecting HRQL...

  20. Capacity at Railway Stations

    DEFF Research Database (Denmark)

    Landex, Alex

    2011-01-01

    zone(s) the possible conflicts with other trains (also in the opposite direction) are taken into account leading to more trustworthy results. Although the UIC 406 methodology proposes that the railway network should be divided into line sections when trains turn around and when the train order...... is changed, this paper recommends that the railway lines are not always be divided. In case trains turn around on open (single track) line, the capacity consumption may be too low if a railway line is divided. The same can be the case if only few trains are overtaken at an overtaking station. For dead end......Stations do have other challenges regarding capacity than open lines as it is here the traffic is dispatched. The UIC 406 capacity method that can be used to analyse the capacity consumption can be exposed in different ways at stations which may lead to different results. Therefore, stations need...

  1. Visual attention capacity

    DEFF Research Database (Denmark)

    Habekost, Thomas; Starrfelt, Randi

    2009-01-01

    Psychophysical studies have identified two distinct limitations of visual attention capacity: processing speed and apprehension span. Using a simple test, these cognitive factors can be analyzed by Bundesen's Theory of Visual Attention (TVA). The method has strong specificity and sensitivity...

  2. Revisiting Absorptive Capacity

    DEFF Research Database (Denmark)

    de Araújo, Ana Luiza Lara; Ulhøi, John Parm; Lettl, Christopher

    learning processes of absorptive capacity, which comprise combinative and adaptive capabilities. Drawing on survey data (n=169), the study concludes that combinative capabilities primarily enhance transformative and exploratory learning processes, while adaptive capabilities strengthen all three learning......Absorptive capacity has mostly been perceived as a 'passive' outcome of R&D investments. Recently, however, a growing interest into its 'proactive' potentials has emerged. This paper taps into this development and proposes a dynamic model for conceptualizing the determinants of the complementary...

  3. Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B.

    Science.gov (United States)

    Materia, Stephanie; Cater, Michael A; Klomp, Leo W J; Mercer, Julian F B; La Fontaine, Sharon

    2011-03-25

    The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.

  4. Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato.

    Science.gov (United States)

    Li, Q B; Haskell, D W; Guy, C L

    1999-01-01

    Stress 70 molecular chaperones are found in all the major subcellular compartments of plant cells, and they are encoded by a multigene family. Twelve members of this family have been identified in spinach. The expression of the stress 70 molecular chaperones in response to heat shock is well-known and it appears that low temperature exposure can also stimulate their expression. However, it has been difficult to determine which member(s) of the family are specifically responsive to low temperature. This study was initiated to determine the levels of expression of the stress 70 family members and other selected chaperones in response to high and low temperature exposure. During heat shock of spinach, of the 10 stress 70 family members that were examined, all 10 showed increased RNA levels after one hour, and all showed down-regulation at longer durations of high temperature exposure. However, the response to low temperature was quite variable and complex. Some members were induced, some were transiently up-regulated, while others showed sustained up-regulation at a low non-freezing temperature. In comparison, the entirety of the molecular chaperone expression response of cold-sensitive tomato at the same low non-freezing temperature was even more dramatic with 11 of 15 molecular chaperones tested exhibiting elevated expression. The increased chaperone expression is consistent with the hypothesis that the biogenesis or stability of some proteins is compromised at low non-freezing temperatures. In contrast, mild freezing sufficient to cause injury of spinach did not materially activate chaperone expression.

  5. The impact of oxidative stress on chaperone-mediated human sperm-egg interaction.

    Science.gov (United States)

    Bromfield, Elizabeth G; Aitken, R John; Anderson, Amanda L; McLaughlin, Eileen A; Nixon, Brett

    2015-11-01

    How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability. An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients; however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation. Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h. Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine

  6. The Role of Sigma-1 Receptor, an Intracellular Chaperone in Neurodegenerative Diseases.

    Science.gov (United States)

    Penke, Botond; Fulop, Livia; Szucs, Maria; Frecska, Ede

    2018-01-01

    Widespread protein aggregation occurs in the living system under stress or during aging, owing to disturbance of endoplasmic reticulum (ER) proteostasis. Many neurodegenerative diseases may have a common mechanism: the failure of protein homeostasis. Perturbation of ER results in unfolded protein response (UPR). Prolonged chronical UPR may activate apoptotic pathways and cause cell death. Research articles on Sigma-1 receptor were reviewed. ER is associated to mitochondria by the mitochondria-associated ER-membrane, MAM. The sigma-1 receptor (Sig-1R), a well-known ER-chaperone localizes in the MAM. It serves for Ca2+-signaling between the ER and mitochondria, involved in ion channel activities and especially important during neuronal differentiation. Sig-1R acts as central modulator in inter-organelle signaling. Sig-1R helps cell survival by attenuating ER-stress. According to sequence based predictions Sig-1R is a 223 amino acid protein with two transmembrane (2TM) domains. The X-ray structure of the Sig-1R [1] showed a membrane-bound trimeric assembly with one transmembrane (1TM) region. Despite the in vitro determined assembly, the results of in vivo studies are rather consistent with the 2TM structure. The receptor has unique and versatile pharmacological profile. Dimethyl tryptamine (DMT) and neuroactive steroids are endogenous ligands that activate Sig-1R. The receptor has a plethora of interacting client proteins. Sig-1R exists in oligomeric structures (dimer-trimer-octamer-multimer) and this fact may explain interaction with diverse proteins. Sig-1R agonists have been used in the treatment of different neurodegenerative diseases, e.g. Alzheimer's and Parkinson's diseases (AD and PD) and amyotrophic lateral sclerosis. Utilization of Sig-1R agents early in AD and similar other diseases has remained an overlooked therapeutic opportunity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

    Science.gov (United States)

    Whitney, Spencer M; Birch, Rosemary; Kelso, Celine; Beck, Jennifer L; Kapralov, Maxim V

    2015-03-17

    Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.

  8. NAP-1, Nucleosome assembly protein 1, a histone chaperone involved in Drosophila telomeres.

    Science.gov (United States)

    López-Panadès, Elisenda; Casacuberta, Elena

    2016-03-01

    Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis.

    Science.gov (United States)

    Lo, Hui-Fen; Chen, Bo-En; Lin, Min-Guan; Chi, Meng-Chun; Wang, Tzu-Fan; Lin, Long-Liu

    2016-04-01

    Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg(2+) ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones

    Science.gov (United States)

    Burns, J. Nicole; Orwig, Susan D.; Harris, Julia L.; Watkins, J. Derrick; Vollrath, Douglas; Lieberman, Raquel L.

    2010-01-01

    Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228–504) fused to maltose binding protein (MBP) from E. coli. Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP, and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and ultimately delay the onset of myocilin glaucoma. PMID:20334347

  11. Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

    Science.gov (United States)

    Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J; Rogala, Kacper B; Deane, Charlotte M; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G

    2015-06-29

    Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh

    2007-06-01

    Full Text Available Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113FISREFHR(120 exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131LTITSSLSSDGV(142 and (156ERTIPITRE(164, corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113FISREFHR(120 and (156ERTIPITRE(164, inhibited microtubule disassembly by approximately 26-36%, and the peptides (113FISREFHR(120 and (131LTITSSLSSDGV(142 decreased the thermal aggregation of tubulin by approximately 42-44%. The (131LTITSSLSSDGV(142 and (156ERTIPITRE(164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first

  13. 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

  14. The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline ofCaenorhabditis elegans.

    Science.gov (United States)

    Lissemore, James L; Connors, Elyse; Liu, Ying; Qiao, Li; Yang, Bing; Edgley, Mark L; Flibotte, Stephane; Taylor, Jon; Au, Vinci; Moerman, Donald G; Maine, Eleanor M

    2018-03-05

    In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40 , defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90 , previously known as daf-21 , which encodes the C. elegans ortholog of the cytosolic form of HSP90 . This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90. RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40) , confirming the loss-of-function nature of hsp-90(om40) Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development. Copyright © 2018, G3: Genes, Genomes, Genetics.

  15. The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

    Directory of Open Access Journals (Sweden)

    Tamara Kakoschke

    Full Text Available To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin.

  16. The identification of age-associated cancer markers by an integrative analysis of dynamic DNA methylation changes.

    Science.gov (United States)

    Wang, Yihan; Zhang, Jingyu; Xiao, Xingjun; Liu, Hongbo; Wang, Fang; Li, Song; Wen, Yanhua; Wei, Yanjun; Su, Jianzhong; Zhang, Yunming; Zhang, Yan

    2016-03-07

    As one of the most widely studied epigenetic modifications, DNA methylation has an important influence on human traits and cancers. Dynamic variations in DNA methylation have been reported in malignant neoplasm and aging; however, the mechanisms remain poorly understood. By constructing an age-associated and cancer-related weighted network (ACWN) based on the correlation of the methylation level and the protein-protein interaction, we found that DNA methylation changes associated with age were closely related to the occurrence of cancer. Additional analysis of 102 module genes mined from the ACWN revealed discrimination based on two main patterns. One pattern involved methylation levels that increased with aging and were higher in cancer patients compared with normal controls (HH pattern). The other pattern involved methylation levels that decreased with aging and were lower in cancer compared with normal (LL pattern). Upon incorporation with gene expression levels, 25 genes were filtered based on negative regulation by DNA methylation. These genes were regarded as potential cancer risk markers that were influenced by age in the process of carcinogenesis. Our results will facilitate further studies regarding the impact of the epigenetic effects of aging on diseases and will aid in the development of tailored cancer preventive strategies.

  17. Pax6 interacts with Iba1 and shows age-associated alterations in brain of aging mice.

    Science.gov (United States)

    Maurya, Shashank Kumar; Mishra, Rajnikant

    2017-07-01

    The Pax6, a transcriptional regulator and multifunctional protein, has been found critical for neurogenesis, neuro-degeneration, mental retardation, neuroendocrine tumors, glioblastoma and astrocytomas. The age-associated alteration in the expression of Pax6 in neuron and glia has also been observed in the immunologically privileged brain. Therefore, it is presumed that Pax6 may modulate brain immunity by activation of microglia either directly interacting with genes or proteins of microglia or indirectly though inflammation associated with neurodegeneration. This report describes evaluation of expression, co-localization and interactions of Pax6 with Ionized binding protein1 (Iba1) in brain of aging mice by Immunohistochemistry, Chromatin Immuno-precipitation (ChIP) and Co-immunoprecipitation (Co-IP), respectively. The co-localization of Pax6 with Iba1 was observed in the cerebellum, cerebral cortex, hippocampus, midbrain and olfactory lobe. The Pax6 and Iba1 also interact physically. The age-dependent alteration in their expression and co-localization were also observed in mice. Results indicate Pax6-dependent activities of Iba1 in the remodelling of microglia during immunological surveillance of the brain. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Riluzole partially rescues age-associated, but not LPS-induced, loss of glutamate transporters and spatial memory.

    Science.gov (United States)

    Brothers, Holly M; Bardou, Isabelle; Hopp, Sarah C; Kaercher, Roxanne M; Corona, Angela W; Fenn, Ashley M; Godbout, Jonathan P; Wenk, Gary L

    2013-12-01

    Impaired memory may result from synaptic glutamatergic dysregulation related to chronic neuroinflammation. GLT1 is the primary excitatory amino acid transporter responsible for regulating extracellular glutamate levels in the hippocampus. We tested the hypothesis that if impaired spatial memory results from increased extracellular glutamate due to age or experimentally induced chronic neuroinflammation in the hippocampus, then pharmacological augmentation of the glutamate transporter GLT1 will attenuate deficits in a hippocampal-dependent spatial memory task. The profile of inflammation-related genes and proteins associated with normal aging, or chronic neuroinflammation experimentally-induced via a four-week LPS infusion into the IV(th) ventricle, were correlated with performance in the Morris water maze following treatment with Riluzole, a drug that can enhance glutamate clearance by increasing GLT1 expression. Age-associated inflammation was qualitatively different from LPS-induced neuro-inflammation in young rats. LPS produced a pro-inflammatory phenotype characterized by increased IL-1ß expression in the hippocampus, whereas aging was not associated with a strong central pro-inflammatory response but with a mixed peripheral immune phenotype. Riluzole attenuated the spatial memory impairment, the elevation of serum cytokines and the decrease in GLT1 gene expression in Aged rats, but had no effect on young rats infused with LPS. Our findings highlight the therapeutic potential of reducing glutamatergic function upon memory impairment in neurodegenerative diseases associated with aging.

  19. Weather and road capacity

    DEFF Research Database (Denmark)

    Jensen, Thomas Christian

    2014-01-01

    The paper presents estimations of the effect of bad weather on the observed speed on a Danish highway section; Køge Bugt Motorvejen. The paper concludes that weather, primarily precipitation and snow, has a clear negative effect on speed when the road is not in hypercongestion mode. Furthermore......, the capacity of the highway seems to be reduced in bad weather and there are indications that travel time variability is also increased, at least in free-flow conditions. Heavy precipitation reduces speed and capacity by around 5-8%, whereas snow primarily reduces capacity. Other weather variables......-parametrically against traffic density and in step 2 the residuals from step 1 are regressed linearly against the weather variables. The choice of a non-parametric method is made to avoid constricting ties from a parametric specification and because the focus here is not on the relationship between traffic flow...

  20. Dual capacity reciprocating compressor

    Science.gov (United States)

    Wolfe, Robert W.

    1984-01-01

    A multi-cylinder compressor 10 particularly useful in connection with northern climate heat pumps and in which different capacities are available in accordance with reversing motor 16 rotation is provided with an eccentric cam 38 on a crank pin 34 under a fraction of the connecting rods, and arranged for rotation upon the crank pin between opposite positions 180.degree. apart so that with cam rotation on the crank pin such that the crank throw is at its normal maximum value all pistons pump at full capacity, and with rotation of the crank shaft in the opposite direction the cam moves to a circumferential position on the crank pin such that the overall crank throw is zero. Pistons 24 whose connecting rods 30 ride on a crank pin 36 without a cam pump their normal rate with either crank rotational direction. Thus a small clearance volume is provided for any piston that moves when in either capacity mode of operation.

  1. Geothermal Plant Capacity Factors

    Energy Technology Data Exchange (ETDEWEB)

    Greg Mines; Jay Nathwani; Christopher Richard; Hillary Hanson; Rachel Wood

    2015-01-01

    The capacity factors recently provided by the Energy Information Administration (EIA) indicated this plant performance metric had declined for geothermal power plants since 2008. Though capacity factor is a term commonly used by geothermal stakeholders to express the ability of a plant to produce power, it is a term frequently misunderstood and in some instances incorrectly used. In this paper we discuss how this capacity factor is defined and utilized by the EIA, including discussion on the information that the EIA requests from operations in their 923 and 860 forms that are submitted both monthly and annually by geothermal operators. A discussion is also provided regarding the entities utilizing the information in the EIA reports, and how those entities can misinterpret the data being supplied by the operators. The intent of the paper is to inform the facility operators as the importance of the accuracy of the data that they provide, and the implications of not providing the correct information.

  2. Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

    Directory of Open Access Journals (Sweden)

    Colin A Cooper

    2010-02-01

    Full Text Available Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2 is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2 and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  3. Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

    Science.gov (United States)

    Kelley, Patrick B; Abraham, Edathara C

    2003-10-01

    alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

  4. Structural and Biochemical Characterization of SrcA, a Multi-cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, C.; Zhang, K; Andres, S; Fnag, Y; Kaniuk, N; Hannemann, M; Brumell, J; Foster, L; Junop, M; Coombes, B

    2010-01-01

    Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 {angstrom} revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  5. Value for railway capacity

    DEFF Research Database (Denmark)

    Sameni, Melody Khadem; Preston, John M.

    2012-01-01

    to analyze the efficiency of operators in transforming inputs of allocated capacity of infrastructure and franchise payments into valuable passenger service outputs while avoiding delays. By addressing operational and economic aspects of capacity utilization simultaneously, the paper deviates from existing...... DEA work on the economic efficiency of railways by considering a new combination of input-output that also incorporates quality of service. The constant and variable returns to scale models are applied to the case study of franchised passenger operators in Great Britain. The follow-up Tobit regression...

  6. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    Science.gov (United States)

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

  7. Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions during Translation.

    Science.gov (United States)

    Koubek, Jiří; Chang, Yi-Che; Yang, Sunny Yao-Chen; Huang, Joseph Jen-Tse

    2017-06-02

    Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner-Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

    Directory of Open Access Journals (Sweden)

    Jyoti K. Jha

    2017-04-01

    Full Text Available Replication of Vibrio cholerae chromosome 2 (Chr2 depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.

  9. The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

    Science.gov (United States)

    Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

    2014-02-01

    The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. The determinants and engagement patterns of chaperones and chauffeurs by Australian doctors in after-hours house-call services

    Directory of Open Access Journals (Sweden)

    Chris Onyebuchi Ifediora

    2017-04-01

    Full Text Available Objectives The use of escorts (chauffeurs and chaperones while on duty in after-hours-house-call (AHHC is one key protective option available to doctors in the service, and has been linked to low burnout and increased satisfaction in AHHC. This study aims to explore the patterns of engagement of escorts in Australian AHHC. Method This is a questionnaire-based, electronic survey of all 300 doctors involved in AHHC through the National Home Doctor Service (NHDS, Australia’s largest providers of the service. The survey explored the doctor’s experiences over the 12-month period from October 2013 to September 2014. Results This survey received a total of 168 valid responses, giving a response rate of 56%. Nearly 61% of the doctors involved in AHHC engaged escorts (chauffeurs and chaperones. Of those doctors that engage chauffeurs, three-quarters do so “all or most times”, while only one-quarter engaged chaperones to the same degree of frequency. Hiring escorts is very popular among Brisbane (91.7% and Sydney-based (88.2% practitioners, but is unpopular in the City of Gold Coast (26.1%. There were moderate patronages in Adelaide (52.9% and Melbourne Area (46.4%. Compared to males, females were less likely to drive themselves without escorts (OR 0.20; P < 0.01; CI [0.07–0.57], but more likely to engage chauffeurs (OR 5.87; P = 0.03; CI [1.16–29.77]. Practitioners who were apprehensive were three times more likely to either engage escorts as chauffeurs (OR 3.10; P = 0.04; CI [1.05–9.15] or as an accompanying chaperone if they self-drive (OR 3.03; P = 0.02; CI [1.16–7.89]. Conclusion More needs to be done to increase the engagement of escorts by doctors involved in the Australian AHHC, particularly given their proven benefits in the service. Future studies may be needed to fully explore the real reasons behind the significant associations identified in this study.

  11. The Intrinsically Disordered Domain of the Antitoxin Phd Chaperones the Toxin Doc against Irreversible Inactivation and Misfolding*

    Science.gov (United States)

    De Gieter, Steven; Konijnenberg, Albert; Talavera, Ariel; Butterer, Annika; Haesaerts, Sarah; De Greve, Henri; Sobott, Frank; Loris, Remy; Garcia-Pino, Abel

    2014-01-01

    The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site. PMID:25326388

  12. The Hydrophobic Region of the DmsA Twin-Arginine Leader Peptide Determines Specificity with Chaperone DmsD

    OpenAIRE

    Winstone, Tara M. L.; Tran, Vy A.; Turner, Raymond J.

    2013-01-01

    The system specific chaperone DmsD plays a role in the maturation of the catalytic subunit of dimethyl sulfoxide (DMSO) reductase, DmsA. Pre-DmsA contains a 45-amino acid twin-arginine leader peptide that is important for targeting and translocation of folded and cofactor-loaded DmsA by the twin-arginine translocase. DmsD has previously been shown to interact with the complete twin-arginine leader peptide of DmsA. In this study, isothermal titration calorimetry was used to investigate the the...

  13. The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

    DEFF Research Database (Denmark)

    Baek, Kristoffer T.; Bowman, Lisa; Millership, Charlotte

    2016-01-01

    Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise...... spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did...

  14. Evaluation of railway capacity

    DEFF Research Database (Denmark)

    Landex, Alex; Kaas, Anders H.; Schittenhelm, Bernd

    2006-01-01

    capacity consumptions. This paper describes the UIC 406 method and how it is expounded in Denmark. The paper describes the importance of choosing the right length of the line sections examined and how line sections with multiple track sections are examined. Furthermore, the possibility of using idle...

  15. Building capacity in Benin

    International Development Research Centre (IDRC) Digital Library (Canada)

    sigp1. Building capacity in. Benin. Training of technical staff of municipalities in the Okpara Basin of Benin in the use of GIS tools for water management, hydrological .... Practical exercises allowed participants to define the catchment area of the ...

  16. Testosterone Treatment and Cognitive Function in Older Men With Low Testosterone and Age-Associated Memory Impairment.

    Science.gov (United States)

    Resnick, Susan M; Matsumoto, Alvin M; Stephens-Shields, Alisa J; Ellenberg, Susan S; Gill, Thomas M; Shumaker, Sally A; Pleasants, Debbie D; Barrett-Connor, Elizabeth; Bhasin, Shalender; Cauley, Jane A; Cella, David; Crandall, Jill P; Cunningham, Glenn R; Ensrud, Kristine E; Farrar, John T; Lewis, Cora E; Molitch, Mark E; Pahor, Marco; Swerdloff, Ronald S; Cifelli, Denise; Anton, Stephen; Basaria, Shehzad; Diem, Susan J; Wang, Christina; Hou, Xiaoling; Snyder, Peter J

    2017-02-21

    Most cognitive functions decline with age. Prior studies suggest that testosterone treatment may improve these functions. To determine if testosterone treatment compared with placebo is associated with improved verbal memory and other cognitive functions in older men with low testosterone and age-associated memory impairment (AAMI). The Testosterone Trials (TTrials) were 7 trials to assess the efficacy of testosterone treatment in older men with low testosterone levels. The Cognitive Function Trial evaluated cognitive function in all TTrials participants. In 12 US academic medical centers, 788 men who were 65 years or older with a serum testosterone level less than 275 ng/mL and impaired sexual function, physical function, or vitality were allocated to testosterone treatment (n = 394) or placebo (n = 394). A subgroup of 493 men met criteria for AAMI based on baseline subjective memory complaints and objective memory performance. Enrollment in the TTrials began June 24, 2010; the final participant completed treatment and assessment in June 2014. Testosterone gel (adjusted to maintain the testosterone level within the normal range for young men) or placebo gel for 1 year. The primary outcome was the mean change from baseline to 6 months and 12 months for delayed paragraph recall (score range, 0 to 50) among men with AAMI. Secondary outcomes were mean changes in visual memory (Benton Visual Retention Test; score range, 0 to -26), executive function (Trail-Making Test B minus A; range, -290 to 290), and spatial ability (Card Rotation Test; score range, -80 to 80) among men with AAMI. Tests were administered at baseline, 6 months, and 12 months. Among the 493 men with AAMI (mean age, 72.3 years [SD, 5.8]; mean baseline testosterone, 234 ng/dL [SD, 65.1]), 247 were assigned to receive testosterone and 246 to receive placebo. Of these groups, 247 men in the testosterone group and 245 men in the placebo completed the memory study. There was no significant mean

  17. Age-Associated Decline in Dendritic Cell Function and the Impact of Mediterranean Diet Intervention in Elderly Subjects

    Directory of Open Access Journals (Sweden)

    Sarah J. Clements

    2017-12-01

    Full Text Available IntroductionAging is accompanied by increased susceptibility to infection and age-associated chronic diseases. It is also associated with reduced vaccine responses, which is often attributed to immunosenescence and the functional decline of the immune system. Immunosenescence is characterized by a chronic, low-grade, inflammatory state termed inflammaging. Habitants of Mediterranean (MED regions maintain good health into old age; often attributed to MED diets.HypothesisAdoption of a MED-diet by elderly subjects, in Norfolk (UK, may improve immune responses of these individuals and in particular, dendritic cell (DC function.Experimental approachA total of 120 elderly subjects (65–79 years old recruited onto the Nu-AGE study, a multicenter European dietary study specifically addressing the needs of the elderly, across five countries, and were randomized to the control or MED-diet groups, for one year. Blood samples were taken pre- and post-intervention for DC analysis and were compared with each other, and to samples obtained from 45 young (18–40 years old subjects. MED-diet compliance was assessed using high performance liquid chromatography-with tandem mass spectrometry analysis of urine samples. Immune cell and DC subset numbers and concentrations of secreted proteins were determined by flow cytometric analysis.ResultsAs expected, reduced myeloid DC numbers were observed in blood samples from elderly subjects compared with young. The elevated secretion of the adipokine, resistin, after ex vivo stimulation of peripheral blood mononuclear cells from elderly subjects, was significantly reduced after MED-diet intervention.ConclusionThis study provides further evidence of numerical and functional effects of aging on DCs. The MED-diet showed potential to impact on the aging immune cells investigated and could provide an economical approach to address problems associated with our aging population.

  18. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease.

    Directory of Open Access Journals (Sweden)

    Valentina Citro

    Full Text Available Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay.

  19. Chaperone-client interactions between Hsp21 and client proteins monitored in solution by small angle X-ray scattering and captured by crosslinking mass spectrometry

    DEFF Research Database (Denmark)

    Rutsdottir, Gudrun; I Rasmussen, Morten; Hojrup, Peter

    2018-01-01

    and six inwardly-facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone-client complexes were monitored and the Rg-values were found to increase......The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N- and C-terminal regions varying in length and sequence...... continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N-terminal arms of the Hsp21 dodecamer. No such increase in Rg-values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data...

  20. PGC-1alpha is required for training-induced prevention of age-associated decline in mitochondrial enzymes in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Leick, Lotte; Lyngby, Stine Secher; Wojtaszewski, Jørgen

    2010-01-01

    The aim of the present study was to test the hypothesis that exercise training prevents an age-associated decline in skeletal muscle mitochondrial enzymes through a PGC-1alpha dependent mechanism. Whole body PGC-1alpha knock-out (KO) and littermate wildtype (WT) mice were submitted to long term...... running wheel exercise training or a sedentary lifestyle from 2 to 13 month of age. Furthermore, a group of approximately 4-month-old mice was used as young untrained controls. There was in both genotypes an age-associated approximately 30% decrease in citrate synthase (CS) activity and superoxide...... dismutase (SOD)2 protein content in 13-month-old untrained mice compared with young untrained mice. However, training prevented the age-associated decrease in CS activity and SOD2 protein content only in WT mice, but long term exercise training did increase HKII protein content in both genotypes...

  1. Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Bao, Rui; Esser, Lothar; Poole, Steven; McVeigh, Annette; Chen, Yu Xing; Savarino, Stephen J; Xia, Di

    2014-02-01

    Understanding of pilus bioassembly in Gram-negative bacteria stems mainly from studies of P pili and type 1 fimbriae of uropathogenic Escherichia coli, which are mediated by the classic chaperone-usher pathway (CUP). However, CFA/I fimbriae, a class 5 fimbria and intestinal colonization factor for enterotoxigenic E. coli (ETEC), are proposed to assemble via the alternate chaperone pathway (ACP). Both CUP and ACP fimbrial bioassembly pathways require the function of a periplasmic chaperone, but their corresponding proteins share very low similarity in primary sequence. Here, the crystallization of the CFA/I periplasmic chaperone CfaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected from a native CfaA crystal to 2 Å resolution and to 1.8 and 2.8 Å resolution, respectively, from a lead and a platinum derivative. These crystals displayed the symmetry of space group C2, with unit-cell parameters a = 103.6, b = 28.68, c = 90.60 Å, β = 119.7°. Initial phases were derived from multiple isomorphous replacement with anomalous scattering experiments using the data from the platinum and lead derivatives. This resulted in an interpretable electron-density map showing one CfaA molecule in an asymmetric unit. Sequence assignments were aided by anomalous signals from the heavy-atom derivatives. Refinement of the atomic model of CfaA is ongoing, which is expected to further understanding of the essential aspects and allowable variations in tertiary structure of the greater family of chaperones involved in chaperone-usher mediated bioassembly.

  2. C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

    Science.gov (United States)

    Qualley, Dominic F; Stewart-Maynard, Kristen M; Wang, Fei; Mitra, Mithun; Gorelick, Robert J; Rouzina, Ioulia; Williams, Mark C; Musier-Forsyth, Karin

    2010-01-01

    Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability.

  3. Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking.

    Directory of Open Access Journals (Sweden)

    Maria E Palm-Espling

    Full Text Available Cisplatin (CisPt is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15 are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4, indicative of a possible drug detoxification mechanism.

  4. Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Čiplys, Evaldas; Aučynaitė, Agota; Slibinskas, Rimantas

    2014-02-11

    Human BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein. Expression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold. S. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast

  5. Regional deficiencies in chaperone-mediated autophagy underlie α-synuclein aggregation and neurodegeneration

    Science.gov (United States)

    Malkus, Kristen A.; Ischiropoulos, Harry

    2012-01-01

    In neurodegenerative diseases, it remains unclear why certain brain regions are selectively vulnerable to protein aggregation. In transgenic mice expressing human A53T α-synuclein, the brainstem and spinal cord develop the most prominent α-synuclein inclusions which correlate with age-dependent motor dysfunction. Herein we present the novel finding that this selective aggregation is in part dependent on the inability of chaperone-mediated autophagy (CMA) to effectively degrade α-synuclein in these brain regions. Lysosomal assays revealed that CMA activity was significantly decreased in aggregation-prone regions compared to the remainder of the brain. Previously, CMA activity has been shown to be proportional to levels of the CMA receptor Lamp-2a. Using antibodies, brain tissue from Lamp-2a null mice, enzymatic deglycosylation, and mass spectrometry, we identified Lamp2a as a novel 72 kDa glycoprotein in the mouse brain. Examination of Lamp-2a levels revealed differences in expression across brain regions. The brainstem and the spinal cord had a more than three-fold greater levels of Lamp-2a as compared to regions less vulnerable to aggregation and exhibited a selective upregulation of Lamp-2a during development of α-synuclein inclusions. Despite this dynamic response of Lamp-2a, the levels of substrates bound to the brain lysosomes as well as the rates of substrate uptake and degradation were not proportional to the levels of Lamp-2a. These regional differences in CMA activity and Lamp-2a expression were found in both non-transgenic mice as well as A53T α-syn mice. Therefore, these are inherent variations and not a transgene-specific effect. However, differences in CMA activity may render select brain regions vulnerable to homeostatic dysfunction in the presence of stressors such as overexpression of human A53T α-syn. Collectively, the data provide a potential mechanism to explain the dichotomy of vulnerability or resistance that underlies brain regions

  6. The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

    Directory of Open Access Journals (Sweden)

    Jiménez-Zurdo José I

    2010-03-01

    identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein. Conclusions Our results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

  7. Histone Chaperone Jun Dimerization Protein 2 (JDP2: Role in Cellular Senescence and Aging

    Directory of Open Access Journals (Sweden)

    Yu-Chang Huang

    2010-10-01

    Full Text Available Transcription factor Jun dimerization protein 2 (JDP2 binds directly to histones and DNA, and inhibits p300-mediated acetylation of core histones and reconstituted nucleosomes that contain JDP2-recognition DNA sequences. The region of JDP2 that encompasses its histone-binding domain and DNA-binding region is essential to inhibit histone acetylation by histone acetyltransferases. Moreover, assays of nucleosome assembly in vitro demonstrate that JDP2 also has histone-chaperone activity. The mutation of the region responsible for inhibition of histone acetyltransferase activity within JDP2 eliminates repression of transcription from the c-jun promoter by JDP2, as well as JDP2-mediated inhibition of retinoic-acid-induced differentiation. Thus JDP2 plays a key role as a repressor of cell differentiation by regulating the expression of genes with an activator protein 1 (AP-1 site via inhibition of histone acetylation and/or assembly and disassembly of nucleosomes. Senescent cells show a series of alterations, including flatten and enlarged morphology, increase in nonspecific acidic β-galactosidase activity, chromatin condensation, and changes in gene expression patterns. The onset and maintenance of senescence are regulated by two tumor suppressors, p53 and retinoblastoma proteins. The expression of p53 and retinoblastoma proteins is regulated by two distinct proteins, p16Ink4a and Arf, respectively, which are encoded by cdkn2a. JDP2 inhibits recruitment of the polycomb repressive complexes 1 and 2 (PRC-1 and PRC-2 to the promoter of the gene that encodes p16Ink4a and inhibits the methylation of lysine 27 of histone H3 (H3K27. The PRCs associate with the p16Ink4a/Arf locus in young proliferating cells and dissociate from it in senescent cells. Therefore, it seems that chromatin-remodeling factors that regulate association and dissociation of PRCs, and are controlled by JDP2, might play an important role in the senescence program. The molecular

  8. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    Science.gov (United States)

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  9. Co-expression with the Type 3 Secretion Chaperone CesT from EnterohemorrhagicE. coliIncreases Accumulation of Recombinant Tir in Plant Chloroplasts.

    Science.gov (United States)

    MacDonald, Jacqueline; Miletic, Sean; Gaildry, Typhanie; Chin-Fatt, Adam; Menassa, Rima

    2017-01-01

    Type 3 secretion systems (T3SSs) are utilized by pathogenic Escherichia coli to infect their hosts and many proteins from these systems are affected by chaperones specific to T3SS-containing bacteria. Toward developing a recombinant vaccine against enterohaemorrhagic E. coli (EHEC), we expressed recombinant T3SS and related proteins from predominant EHEC serotypes in Nicotiana chloroplasts. Nicotiana benthamiana were transiently transformed to express chloroplast-targeted Tir, NleA, and EspD from the EHEC serotype O157:H7; a fusion of EspA proteins from serotypes O157:H7 and O26:H11; and a fusion of epitopes of Tir (Tir-ep) from serotypes O157:H7, O26:H11, O45:H2, and O111:H8. C-terminal GFP reporter fusion constructs were also developed and transiently expressed to confirm subcellular localization and quantify relative expression levels in situ . Recombinant proteins were co-expressed with chaperones specific to each T3SS protein with the goal of increasing their accumulation in the chloroplast. We found that co-expression with the chloroplast-targeted chaperone CesT significantly increases accumulation of recombinant Tir when the latter is either transiently expressed in the nucleus and targeted to the chloroplast of N. benthamiana or stably expressed in transplastomic Nicotiana tabacum . CesT also helped maintain higher levels of Tir:GFP fusion protein over time both in vivo and ex vivo , indicating that the favorable effect of CesT on accumulation of Tir is not specific to a single time point or to fresh material. By contrast, T3SS chaperones CesT, CesAB, CesD, and CesD2 did not increase accumulation of NleA:GFP, EspA:GFP, or EspD:GFP, which suggests dissimilar functioning of these chaperone-substrate combinations. CesT did not increase accumulation of Tir-ep:GFP, which may be due to the absence of the CesT binding domain from this fusion protein. The fusion to GFP improved accumulation of Tir-ep relative to the unfused protein, but not for the other

  10. Seismic capacity of switchgear

    International Nuclear Information System (INIS)

    Bandyopadhyay, K.; Hofmayer, C.; Kassir, M.; Pepper, S.

    1989-01-01

    As part of a component fragility program sponsored by the USNRC, BNL has collected existing information on the seismic capacity of switchgear assemblies from major manufacturers. Existing seismic test data for both low and medium voltage switchgear assemblies have been evaluated and the generic results are presented in this paper. The failure modes are identified and the corresponding generic lower bound capacity levels are established. The test response spectra have been used as a measure of the test vibration input. The results indicate that relays chatter at a very low input level at the base of the switchgear cabinet. This change of state of devices including relays have been observed. Breaker tripping occurs at a higher vibration level. Although the structural failure of internal elements have been noticed, the overall switchgear cabinet structure withstands a high vibration level. 5 refs., 2 figs., 2 tabs

  11. Absorptive Capacity and Diversity

    DEFF Research Database (Denmark)

    Kristinsson, Kári

    international business, organizational economics, strategic management, technology management and last but not least neo-Schumpeterian economics. The goal of this dissertation is to examine what many consider as neglected arguments from the work by Cohen and Levinthal and thereby illuminate an otherwise......One of the most influential contributions to neo-Schumpeterian economics is Cohen and Levinthal‘s papers on absorptive capacity. Since their publication in the late 1980s and early 1990s the concept absorptive capacity has had substantial impact on research in economics and management, including...... overlooked area of research. Although research based on Cohen and Levinthal‘s work has made considerable impact, there is scarcity of research on certain fundamental points argued by Cohen and Levinthal. Among these is the importance of employee diversity as well as the type and nature of interaction between...

  12. Heat Capacity Mapping Mission

    Science.gov (United States)

    Nilsson, C. S.; Andrews, J. C.; Scully-Power, P.; Ball, S.; Speechley, G.; Latham, A. R. (Principal Investigator)

    1980-01-01

    The Tasman Front was delineated by airborne expendable bathythermograph survey; and an Heat Capacity Mapping Mission (HCMM) IR image on the same day shows the same principal features as determined from ground-truth. It is clear that digital enhancement of HCMM images is necessary to map ocean surface temperatures and when done, the Tasman Front and other oceanographic features can be mapped by this method, even through considerable scattered cloud cover.

  13. Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

    Directory of Open Access Journals (Sweden)

    Tomohisa Mori

    Full Text Available The membrane of the endoplasmic reticulum (ER of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

  14. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

    Science.gov (United States)

    Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

    2017-06-07

    Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

  15. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation.

    Science.gov (United States)

    Chai, Ye Jin; Sierecki, Emma; Tomatis, Vanesa M; Gormal, Rachel S; Giles, Nichole; Morrow, Isabel C; Xia, Di; Götz, Jürgen; Parton, Robert G; Collins, Brett M; Gambin, Yann; Meunier, Frédéric A

    2016-09-12

    Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. © 2016 Chai et al.

  16. The Histone Chaperone NRP1 Interacts with WEREWOLF to ActivateGLABRA2in Arabidopsis Root Hair Development.

    Science.gov (United States)

    Zhu, Yan; Rong, Liang; Luo, Qiang; Wang, Baihui; Zhou, Nana; Yang, Yue; Zhang, Chi; Feng, Haiyang; Zheng, Lina; Shen, Wen-Hui; Ma, Jinbiao; Dong, Aiwu

    2017-02-01

    NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) defines an evolutionarily conserved family of histone chaperones and loss of function of the Arabidopsis thaliana NAP1 family genes NAP1-RELATED PROTEIN1 ( NRP1 ) and NRP2 causes abnormal root hair formation. Yet, the underlying molecular mechanisms remain unclear. Here, we show that NRP1 interacts with the transcription factor WEREWOLF (WER) in vitro and in vivo and enriches at the GLABRA2 ( GL2 ) promoter in a WER-dependent manner. Crystallographic analysis indicates that NRP1 forms a dimer via its N-terminal α-helix. Mutants of NRP1 that either disrupt the α-helix dimerization or remove the C-terminal acidic tail, impair its binding to histones and WER and concomitantly lead to failure to activate GL2 transcription and to rescue the nrp1-1 nrp2-1 mutant phenotype. Our results further demonstrate that WER-dependent enrichment of NRP1 at the GL2 promoter is involved in local histone eviction and nucleosome loss in vivo. Biochemical competition assays imply that the association between NRP1 and histones may counteract the inhibitory effect of histones on the WER-DNA interaction. Collectively, our study provides important insight into the molecular mechanisms by which histone chaperones are recruited to target chromatin via interaction with a gene-specific transcription factor to moderate chromatin structure for proper root hair development. © 2017 American Society of Plant Biologists. All rights reserved.

  17. Effect of leucine-to-methionine substitutions on the diffraction quality of histone chaperone SET/TAF-Iβ/INHAT crystals

    International Nuclear Information System (INIS)

    Senda, Miki; Muto, Shinsuke; Horikoshi, Masami; Senda, Toshiya

    2008-01-01

    The combination of leucine-to-methionine substitutions and optimization of cryoconditions improved the resolution of histone chaperone SET/TAF-Iβ/INHAT crystals from around 5.5 to 2.3 Å without changing the crystallization conditions, allowing successful structure determination of SET/TAF-Iβ/INHAT by the multiwavelength anomalous diffraction method. One of the most frequent problems in crystallization is poor quality of the crystals. In order to overcome this obstacle several methods have been utilized, including amino-acid substitutions of the target protein. Here, an example is presented of crystal-quality improvement by leucine-to-methionine substitutions. A variant protein with three amino-acid substitutions enabled improvement of the crystal quality of the histone chaperone SET/TAF-Iβ/INHAT when combined with optimization of the cryoconditions. This procedure improved the resolution of the SET/TAF-Iβ/INHAT crystals from around 5.5 to 2.3 Å without changing the crystallization conditions

  18. Enhanced soluble production of cholera toxin B subunit in Escherichia coli by co-expression of SKP chaperones.

    Science.gov (United States)

    Zhang, Yuanpeng; Qiao, Xuwen; Yu, Xiaoming; Chen, Jin; Hou, Liting; Bi, Zhixiang; Zheng, Qisheng; Hou, Jibo

    2017-10-01

    The cholera toxin B subunit (CTB) is a nontoxic portion of the cholera toxin that retains mucosal adjuvant properties. Expression of CTB in Escherichia coli is difficult as CTB aggregates and accumulates as insoluble inclusion bodies. To remedy this problem, the periplasmic chaperone, SKP, was investigated as possible co-expression partner to increase the solubility of recombinant CTB (rCTB) in E. coli. The result showed co-expression of SKP enhanced the soluble expression of rCTB in E. coli. Moreover, soluble rCTB was successfully expressed and secreted into the periplasmic space through the direction of the LTB leader signal. rCTB in periplasm was purified using an immobilized d-galactose resin; GM1-ELISA experiments showed that rCTB retains strong GM1 ganglioside-binding activity. Intranasal administration of ovalbumin (OVA) with rCTB significantly induced both mucosal and humoral immune responses specific to OVA. These data indicate that co-expression of the molecular chaperone SKP with CTB increased the solubility of rCTB while maintaining its function. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris.

    Science.gov (United States)

    Yang, Jun; Lu, Zhipeng; Chen, Jiawei; Chu, Pinpin; Cheng, Qingmei; Liu, Jie; Ming, Feiping; Huang, Chaoyuan; Xiao, Anji; Cai, Haiming; Zhang, Linghua

    2016-06-01

    Mammalian peptidoglycan recognition proteins (PGLYRPs) are highly conserved pattern-recognition molecules of the innate immune system with considerable bactericidal activity, which manifest their potential values for the application to food and pharmaceutical industry. However, the effective expression of porcine PGLYRP-1 in Pichia pastoris has not been reported so far. In this study, expression in P. pastoris was explored as an efficient way to produce functional porcine PGLYRP-1. Cooperation of chaperones co-expression and gene dosage (including protein disulfide isomerase (PDI)/binding protein (BiP) and pglyrp-1) were used to enhance functional expression of antimicrobial protein in P. pastoris. Overexpression of PDI was certainly able to increase secretion level of PGLYRP-1 protein because the increase in secreted PGLYRP-1 secretion was correlated with the copy numbers of PDI in high copy pglyrp-1 clones. However, co-expression of BiP was proved to be detrimental to PGLYRP-1 secretion. In addition, we also found that excessive expression of PDI and/or BiP could decrease the mRNA expression of pglyrp-1 gene. This showed that PDI and BiP as the target genes of unfolded protein response (UPR) might regulate the transcription of the target protein. These data demonstrated for the first time that the combination of chaperones and gene dosages could improve the yield of PGLYRP-1, which could facilitate the application to food and pharmaceutical industry.

  20. The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan.

    Science.gov (United States)

    Catalão, Maria João; Gil, Filipa; Moniz-Pereira, José; Pimentel, Madalena

    2010-08-01

    Like most double-stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin-endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone-like protein, in a holin-independent manner.

  1. Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

    International Nuclear Information System (INIS)

    Born, E J; Hartman, S V; Holstein, S A

    2013-01-01

    Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

  2. Hsp70 chaperones and type I PRMTs are sequestered at intranuclear inclusions caused by polyalanine expansions in PABPN1.

    Directory of Open Access Journals (Sweden)

    João Paulo Tavanez

    2009-07-01

    Full Text Available Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A-binding protein nuclear 1 (PABPN1 gene causes oculopharyngeal muscular dystrophy (OPMD. To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3 associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.

  3. Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

    Directory of Open Access Journals (Sweden)

    Helena Öhrvik

    2015-07-01

    Full Text Available The human copper (Cu chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

  4. CSTI High Capacity Power

    International Nuclear Information System (INIS)

    Winter, J.M.

    1989-01-01

    The SP-100 program was established in 1983 by DOD, DOE, and NASA as a joint program to develop the technology necessary for space nuclear power systems for military and civil application. During FY-86 and 87, the NASA SP-100 Advanced Technology Program was devised to maintain the momentum of promising technology advancement efforts started during Phase 1 of SP-100 and to strengthen, in key areas, the chances for successful development and growth capability of space nuclear reactor power systems for future space applications. In FY-88, the Advanced Technology Program was incorporated into NASA's new Civil Space Technology Initiative (CSTI). The CSTI Program was established to provide the foundation for technology development in automation and robotics, information, propulsion, and power. The CSTI High Capacity Power Program builds on the technology efforts of the SP-100 program, incorporates the previous NASA SP-100 Advanced Technology project, and provides a bridge to NASA Project Pathfinder. The elements of CSTI High Capacity Power development include Conversion Systems, Thermal Management, Power Management, System Diagnostics, and Environmental Interactions. Technology advancement in all areas, including materials, is required to assure the high reliability and 7 to 10 year lifetime demanded for future space nuclear power systems. The overall program will develop and demonstrate the technology base required to provide a wide range of modular power systems as well as allowing mission independence from solar and orbital attitude requirements. Several recent advancements in CSTI High Capacity power development will be discussed

  5. Pathological changes in hippocampal neuronal circuits underlie age-associated neurodegeneration and memory loss: positive clue toward SAD.

    Science.gov (United States)

    Moorthi, P; Premkumar, P; Priyanka, R; Jayachandran, K S; Anusuyadevi, M

    2015-08-20

    defect in neuronal-circuits of hippocampus (DG-CA4-CA1-Sub) that were significantly damaged leading to memory impairment. Interestingly, RSV was observed to culminate pathological events in the hippocampal neuronal circuit during aging, proving them as potent therapeutic drug against age-associated neurodegeneration and memory loss. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Competence building capacity shortage

    International Nuclear Information System (INIS)

    Doorman, Gerard; Wangensteen, Ivar; Bakken, Bjoern

    2005-02-01

    The objective of the project 'Competence Building Capacity Shortage' has been 'to increase knowledge about central approaches aimed at solving the peaking capacity problem in restructured power systems'. With respect to reserve markets, a model was developed in the project to analyze the relations between reserve requirements and prices in the spot and reserve markets respectively. A mathematical model was also developed and implemented, which also includes the balance market, and has a good ability to predict the relations between these markets under various assumptions. With some further development, this model can be used fore realistic analyses of these markets in a Nordic context. It was also concluded that certain system requirements with respect to frequency and time deviation can be relaxed without adverse effects. However, the requirements to system bias, Frequency Activated Operating Reserves and Frequency Activated Contingency Reserves cannot be relaxed, the latter because they must cover the dimensioning fault in the system. On the other hand, Fast Contingency Reserves can be reduced by removing requirements to national balances. Costs can furthermore be reduced by increasingly adapting a Nordic as opposed to national approach. A model for stepwise power flow was developed in the project, which is especially useful to analyze slow power system dynamics. This is relevant when analysing the effects of reserve requirements. A model for the analysis of the capacity balance in Norway and Sweden was also developed. This model is useful for looking at the future balance under various assumptions regarding e.g. weather conditions, demand growth and the development of the generation system. With respect to the present situation, if there is some price flexibility on the demand side and system operators are able to use reserves from the demand side, the probability for load shedding during the peak load hour is close to zero under the weather conditions after

  7. Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation

    Directory of Open Access Journals (Sweden)

    Dounia Djeghloul

    2016-06-01

    Full Text Available The capacity of hematopoietic stem cells (HSC to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

  8. Age-associated DNA methylation changes in naive CD4+T cells suggest an evolving autoimmune epigenotype in aging T cells.

    Science.gov (United States)

    Dozmorov, Mikhail G; Coit, Patrick; Maksimowicz-McKinnon, Kathleen; Sawalha, Amr H

    2017-04-01

    We sought to define age-associated DNA methylation changes in naive CD4 + T cells. Naive CD4 + T cells were collected from 74 healthy individuals (age 19-66 years), and age-related DNA methylation changes were characterized. We identified 11,431 age-associated CpG sites, 57% of which were hypermethylated with age. Hypermethylated sites were enriched in CpG islands and repressive transcription factor binding sites, while hypomethylated sites showed T cell specific enrichment in active enhancers marked by H3K27ac and H3K4me1. Our data emphasize cancer-related DNA methylation changes with age, and also reveal age-associated hypomethylation in immune-related pathways, such as T cell receptor signaling, FCγR-mediated phagocytosis, apoptosis and the mammalian target of rapamycin signaling pathway. The MAPK signaling pathway was hypermethylated with age, consistent with a defective MAPK signaling in aging T cells. Age-associated DNA methylation changes may alter regulatory mechanisms and signaling pathways that predispose to autoimmunity.

  9. 1992 Annual Capacity Report

    International Nuclear Information System (INIS)

    1993-05-01

    The Standard Contract for Disposal of Spent Nuclear Fuel and/or High-Level Radioactive Waste (10 CFR Part 961) requires the Department of Energy (DOE) to issue an Annual Capacity Report (ACR) for planning purposes. This report is the fifth in the series published by DOE. In May 1993, DOE published the 1992 Acceptance Priority Ranking (APR) that established the order in which DOE will allocate projected acceptance capacity. As required by the Standard Contract, the acceptance priority ranking is based on the date the spent nuclear fuel (SNF) was permanently discharged, with the owners of the oldest SNF, on an industry-wide basis, given the highest priority. The 1992 ACR applies the projected waste acceptance rates in Table 2.1 to the 1992 APR, resulting in individual allocations for the owners and generators of the SNF. These allocations are listed in detail in the Appendix, and summarized in Table 3.1. The projected waste acceptance rates for SNF presented in Table 2.1 are nominal and assume a site for a Monitored Retrievable Storage (MRS) facility will be obtained; the facility will initiate operations in 1998; and the statutory linkages between the MRS facility and the repository set forth in the Nuclear Waste Policy Act of 1982, as amended (NWPA), will be modified. During the first ten years following projected commencement of Civilian Radioactive Waste Management System (CRWMS) operation, the total quantity of SNF that could be accepted is projected to be 8,200 metric tons of uranium (MTU). This is consistent with the storage capacity licensing conditions imposed on an MRS facility by the NWPA. The annual acceptance rates provide an approximation of the system throughput and are subject to change as the program progresses

  10. Quantum reading capacity

    International Nuclear Information System (INIS)

    Pirandola, Stefano; Braunstein, Samuel L; Lupo, Cosmo; Mancini, Stefano; Giovannetti, Vittorio

    2011-01-01

    The readout of a classical memory can be modelled as a problem of quantum channel discrimination, where a decoder retrieves information by distinguishing the different quantum channels encoded in each cell of the memory (Pirandola 2011 Phys. Rev. Lett. 106 090504). In the case of optical memories, such as CDs and DVDs, this discrimination involves lossy bosonic channels and can be remarkably boosted by the use of nonclassical light (quantum reading). Here we generalize these concepts by extending the model of memory from single-cell to multi-cell encoding. In general, information is stored in a block of cells by using a channel-codeword, i.e. a sequence of channels chosen according to a classical code. Correspondingly, the readout of data is realized by a process of ‘parallel’ channel discrimination, where the entire block of cells is probed simultaneously and decoded via an optimal collective measurement. In the limit of a large block we define the quantum reading capacity of the memory, quantifying the maximum number of readable bits per cell. This notion of capacity is nontrivial when we suitably constrain the physical resources of the decoder. For optical memories (encoding bosonic channels), such a constraint is energetic and corresponds to fixing the mean total number of photons per cell. In this case, we are able to prove a separation between the quantum reading capacity and the maximum information rate achievable by classical transmitters, i.e. arbitrary classical mixtures of coherent states. In fact, we can easily construct nonclassical transmitters that are able to outperform any classical transmitter, thus showing that the advantages of quantum reading persist in the optimal multi-cell scenario. (paper)

  11. Geostationary orbit capacity study

    Science.gov (United States)

    Hansell, P. S.; Norris, P.; Walton, R.

    1982-04-01

    Factors influencing the communications satellite capacity of the geostationary orbit were analyzed to derive an interference model of the orbit environment. Comparison of the total orbit arc length required by each proposed planning method or by using different technology developments indicates that the orbit arc of most interest to Western Europe will not be saturated by the year 2000. The orbit arc occupied in the year 2000 by the satellites in the West European arc of interest can be approximately halved by using digital modulation techniques for TV program transfers which use FM at present, or by adopting an orbital planning method which assigns FM TV services to predefined orbit or spectrum segments.

  12. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of

  13. Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

    Science.gov (United States)

    Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

    2016-12-01

    The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

  14. Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

    Science.gov (United States)

    Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

    2017-07-01

    The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

  15. Importance of electrostatic interactions in the association of intrinsically disordered histone chaperone Chz1 and histone H2A.Z-H2B.

    Directory of Open Access Journals (Sweden)

    Xiakun Chu

    Full Text Available Histone chaperones facilitate assembly and disassembly of nucleosomes. Understanding the process of how histone chaperones associate and dissociate from the histones can help clarify their roles in chromosome metabolism. Some histone chaperones are intrinsically disordered proteins (IDPs. Recent studies of IDPs revealed that the recognition of the biomolecules is realized by the flexibility and dynamics, challenging the century-old structure-function paradigm. Here we investigate the binding between intrinsically disordered chaperone Chz1 and histone variant H2A.Z-H2B by developing a structure-based coarse-grained model, in which Debye-Hückel model is implemented for describing electrostatic interactions due to highly charged characteristic of Chz1 and H2A.Z-H2B. We find that major structural changes of Chz1 only occur after the rate-limiting electrostatic dominant transition state and Chz1 undergoes folding coupled binding through two parallel pathways. Interestingly, although the electrostatic interactions stabilize bound complex and facilitate the recognition at first stage, the rate for formation of the complex is not always accelerated due to slow escape of conformations with non-native electrostatic interactions at low salt concentrations. Our studies provide an ionic-strength-controlled binding/folding mechanism, leading to a cooperative mechanism of "local collapse or trapping" and "fly-casting" together and a new understanding of the roles of electrostatic interactions in IDPs' binding.

  16. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  17. Endoplasmic reticulum proteins SDF2 and SDF2L1 act as components of the BiP chaperone cycle to prevent protein aggregation.

    Science.gov (United States)

    Fujimori, Tsutomu; Suno, Ryoji; Iemura, Shun-Ichiro; Natsume, Tohru; Wada, Ikuo; Hosokawa, Nobuko

    2017-08-01

    The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  18. Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential

    NARCIS (Netherlands)

    Bross, Peter; Li, Zhijie; Hansen, Jakob; Hansen, Jens Jacob; Nielsen, Marit Nyholm; Corydon, Thomas Juhl; Georgopoulos, Costa; Ang, Debbie; Lundemose, Jytte Banner; Niezen-Koning, Klary; Eiberg, Hans; Yang, Huanming; Kolvraa, Steen; Bolund, Lars; Gregersen, Niels

    Molecular chaperones assist protein folding, and variations in their encoding genes may be disease-causing in themselves or influence the phenotypic expression of disease-associated or susceptibility-conferring variations in many different genes. We have screened three candidate patient groups for

  19. Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation

    NARCIS (Netherlands)

    Månsson, Cecilia; Arosio, Paolo; Hussein, Rasha; Kampinga, Harm H; Hashem, Reem M; Boelens, Wilbert C; Dobson, Christopher M; Knowles, Tuomas P J; Linse, Sara; Emanuelsson, Cecilia

    2014-01-01

    The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington's disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more

  20. HspB8 Participates in Protein Quality Control by a Non-chaperone-like Mechanism That Requires eIF2 alpha Phosphorylation

    NARCIS (Netherlands)

    Carra, Serena; Brunsting, Jeanette F.; Lambert, Herman; Landry, Jacques; Kampinga, Harm H.

    2009-01-01

    Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8 . Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as

  1. Deficiency of employability capacity

    Directory of Open Access Journals (Sweden)

    Pelse I.

    2012-10-01

    Full Text Available Young unemployed people have comprised one of the significantly largest groups of the unemployed people in Latvia in recent years. One of the reasons why young people have difficulty integrating into the labour market is the “expectation gap” that exists in the relations between employers and the new generation of workers. Employers focus on capacity-building for employability such individual factors as strength, patience, self-discipline, self-reliance, self-motivation, etc., which having a nature of habit and are developed in a long-term work socialization process, which begins even before the formal education and will continue throughout the life cycle. However, when the socialization is lost, these habits are depreciated faster than they can be restored. Currently a new generation is entering the labour market, which is missing the succession of work socialization. Factors, such as rising unemployment and poverty in the background over the past twenty years in Latvia have created a very unfavourable employability background of “personal circumstances” and “external factors”, which seriously have impaired formation of the skills and attitudes in a real work environment. The study reveals another paradox – the paradox of poverty. Common sense would want to argue that poverty can be overcome by the job. However, the real state of affairs shows that unfavourable coincidence of the individual, personal circumstances and external factors leads to deficit of employability capacity and possibility of marked social and employment deprivation.

  2. Thermal stress induced aggregation of aquaporin 0 (AQP0) and protection by α-crystallin via its chaperone function.

    Science.gov (United States)

    Swamy-Mruthinti, Satyanarayana; Srinivas, Volety; Hansen, John E; Rao, Ch Mohan

    2013-01-01

    Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.

  3. Thermal stress induced aggregation of aquaporin 0 (AQP0 and protection by α-crystallin via its chaperone function.

    Directory of Open Access Journals (Sweden)

    Satyanarayana Swamy-Mruthinti

    Full Text Available Aquaporin 0 (AQP0 formerly known as membrane intrinsic protein (MIP, is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin in close proximity to the lens membrane is physiologically relevant.

  4. Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts

    Directory of Open Access Journals (Sweden)

    Jacqueline MacDonald

    2017-03-01

    Full Text Available Type 3 secretion systems (T3SSs are utilized by pathogenic Escherichia coli to infect their hosts and many proteins from these systems are affected by chaperones specific to T3SS-containing bacteria. Toward developing a recombinant vaccine against enterohaemorrhagic E. coli (EHEC, we expressed recombinant T3SS and related proteins from predominant EHEC serotypes in Nicotiana chloroplasts. Nicotiana benthamiana were transiently transformed to express chloroplast-targeted Tir, NleA, and EspD from the EHEC serotype O157:H7; a fusion of EspA proteins from serotypes O157:H7 and O26:H11; and a fusion of epitopes of Tir (Tir-ep from serotypes O157:H7, O26:H11, O45:H2, and O111:H8. C-terminal GFP reporter fusion constructs were also developed and transiently expressed to confirm subcellular localization and quantify relative expression levels in situ. Recombinant proteins were co-expressed with chaperones specific to each T3SS protein with the goal of increasing their accumulation in the chloroplast. We found that co-expression with the chloroplast-targeted chaperone CesT significantly increases accumulation of recombinant Tir when the latter is either transiently expressed in the nucleus and targeted to the chloroplast of N. benthamiana or stably expressed in transplastomic Nicotiana tabacum. CesT also helped maintain higher levels of Tir:GFP fusion protein over time both in vivo and ex vivo, indicating that the favorable effect of CesT on accumulation of Tir is not specific to a single time point or to fresh material. By contrast, T3SS chaperones CesT, CesAB, CesD, and CesD2 did not increase accumulation of NleA:GFP, EspA:GFP, or EspD:GFP, which suggests dissimilar functioning of these chaperone–substrate combinations. CesT did not increase accumulation of Tir-ep:GFP, which may be due to the absence of the CesT binding domain from this fusion protein. The fusion to GFP improved accumulation of Tir-ep relative to the unfused protein, but not for the

  5. Workshop on moisture buffer capacity

    DEFF Research Database (Denmark)

    2003-01-01

    Summary report of a Nordtest workshop on moisture buffer capacity held at Copenhagen August 21-22 2003......Summary report of a Nordtest workshop on moisture buffer capacity held at Copenhagen August 21-22 2003...

  6. Building a capacity building manual

    CSIR Research Space (South Africa)

    Clinton, DD

    2010-02-01

    Full Text Available ? Capacity building in developed nations ? renewal stage HOW can capacity be developed and maintained? Through mentoring , training, education, physical projects, the infusion of financial and other resources and more importantly the motivation...

  7. Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

    Science.gov (United States)

    Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

    2016-05-01

    The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

  8. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

    Directory of Open Access Journals (Sweden)

    Ram H Nagaraj

    Full Text Available Methylglyoxal (MGO is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12 is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM, R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

  9. Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-Chaperone Sis1

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.

    2015-04-01

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at heir C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.

  10. 1990s: High Capacity Backbones

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. 1990s: High Capacity Backbones. Backbone capacities increased from 2.5 Gb/s to 100s of Gb/s during the 1990's. Wavelength division multiplexing with 160 waves of 10 Gb/s was commercially available. Several high-capacity backbones built in the US and Europe.

  11. Landfill Construction and Capacity Expansion

    NARCIS (Netherlands)

    Andre, F.J.; Cerda, E.

    2003-01-01

    We study the optimal capacity and lifetime of landfills taking into account their sequential nature.Such an optimal capacity is characterized by the so-called Optimal Capacity Condition.Particular versions of this condition are obtained for two alternative settings: first, if all the landfills are

  12. Capacity Ten-Seven

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-05-04

    The study was initiated to assemble UK solar experts who would define the parameters for the design of solar cell process plant with a capacity of about 10MWp per annum and establish the optimum substrate size, cell configuration and junction structure and show that these characteristics are suited to high volume low cost production. The study focused on proven materials and techniques rather than on innovative materials and processes. Peak production should deliver panels at a cost of less than $1 per peak watt. The study was reported under the headings of fundamental studies, front contact deposition, semiconductor deposition, rear contact deposition, cell isolation and initial design study. Since much of the work is commercially sensitive, not all findings are divulged in the report. The work, which achieved all its targets, was carried out by ICP Solar Technologies UK Ltd under contract to the DTI.

  13. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    . The negative effect of BSA on enzyme was not observed before. The residual activity of horseradish peroxidase was also improved by the reconstituted skim milk: addition of reconstituted skim milk prior to heat treatment resulted in higher residual activity of HRP compared to no addition (58±3% and 30......±1%, respectively) The findings of this study show that β-casein can influence the response of food enzymes to heat treatment. β-Casein is not a universal chaperone and its effect on different targets needs to be evaluated on a case-by-case basis. This study also shows that proteins as e.g. BSA may affect......ABSTRACT Activity of endogenous enzymes may cause browning of fruits and vegetables. These enzymes can be inactivated, for example by heat treatment, but the response of enzymes to heat treatment depends on many factors. Foods are very complex systems and the stability of enzymes...

  14. The intrinsically disordered domain of the antitoxin Phd chaperones the toxin Doc against irreversible inactivation and misfolding.

    Science.gov (United States)

    De Gieter, Steven; Konijnenberg, Albert; Talavera, Ariel; Butterer, Annika; Haesaerts, Sarah; De Greve, Henri; Sobott, Frank; Loris, Remy; Garcia-Pino, Abel

    2014-12-05

    The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. S-nitrosylation of the mitochondrial chaperone TRAP1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase

    DEFF Research Database (Denmark)

    Rizza, Salvatore; Montagna, Costanza; Cardaci, Simone

    2016-01-01

    degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S....... We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its......-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 1-13. ©2016 AACR....

  16. Sylvie Chaperon, La médecine du sexe et les femmes, Paris, La Musardine, 2008, 198 p.

    Directory of Open Access Journals (Sweden)

    Grégory Quin

    2010-07-01

    Full Text Available Après un travail fouillé sur les origines de la sexologie, Sylvie Chaperon, historienne spécialiste d’histoire des femmes et du genre, maîtresse de conférences à l’Université de Toulouse 2, propose dans son nouveau livre une anthologie de textes que des médecins du XIXe siècle ont produits sur la sexualité féminine et ses diverses pathologies. L’ouvrage est construit autour d’une cinquantaine de cas de quelques-unes de ces pathologies associées à la sexualité des femmes : l’onanisme, la nymph...

  17. Sylvie Chaperon, La médecine du sexe et les femmes, Paris, La Musardine, 2008, 198 p.

    OpenAIRE

    Quin, Grégory

    2010-01-01

    Après un travail fouillé sur les origines de la sexologie, Sylvie Chaperon, historienne spécialiste d’histoire des femmes et du genre, maîtresse de conférences à l’Université de Toulouse 2, propose dans son nouveau livre une anthologie de textes que des médecins du XIXe siècle ont produits sur la sexualité féminine et ses diverses pathologies. L’ouvrage est construit autour d’une cinquantaine de cas de quelques-unes de ces pathologies associées à la sexualité des femmes : l’onanisme, la nymph...

  18. Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA.

    Science.gov (United States)

    Adam, Salomé; Polo, Sophie E; Almouzni, Geneviève

    2013-09-26

    Understanding how to recover fully functional and transcriptionally active chromatin when its integrity has been challenged by genotoxic stress is a critical issue. Here, by investigating how chromatin dynamics regulate transcriptional activity in response to DNA damage in human cells, we identify a pathway involving the histone chaperone histone regulator A (HIRA) to promote transcription restart after UVC damage. Our mechanistic studies reveal that HIRA accumulates at sites of UVC irradiation upon detection of DNA damage prior to repair and deposits newly synthesized H3.3 histones. This local action of HIRA depends on ubiquitylation events associated with damage recognition. Furthermore, we demonstrate that the early and transient function of HIRA in response to DNA damage primes chromatin for later reactivation of transcription. We propose that HIRA-dependent histone deposition serves as a chromatin bookmarking system to facilitate transcription recovery after genotoxic stress. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. [Association of cytochrome P450 2B4 with molecular chaperones in heterological expression in E coli].

    Science.gov (United States)

    Prozorovskiĭ, T V; Schunck, W H; Archakov, A I

    2001-01-01

    To produce a water-soluble form of microsomal P450 2B4, fusion proteins with glutathione-S-transpherase were genetically engineered. Specific proteolitic sites recognized by Factor Xa and Thrombin have been introduced into N-terminus of P450 2B4 (46-49), lacking signal anchor sequence (2-27). It was supposed that proteolysis at this site could give the possibility to produce protein lacking hydrophobic N-terminus sequence (1-49). However, it was shown that given region in P450 2B4 his resistant against specific proteinase action. Positive result has been obtained at specific proteolysis with IgA endoproteinase, recognizing the native sequence PPGP (31-34) in P450 2B4. Thus, at first time truncated form of cytochrome 2B4, lacking its 33 N-terminal amino acid residues has been created. It was found that the expression of genetically engineered variants of GST-2B4 in Escherichia coli is accompanied by tight complex formation with molecular chaperones GroEL and DnaK. Dissociation of the complex occurred after proteolysis in: linker sequence (position 6-7) between C-terminal part of GST domain and N-terminal part of 2B4, and also before N-terminal methionine 2B4 and at position 33-34 (2B4). These results suggest the possibility that interaction with a GroEL/DnaK molecular chaperones may be requirement for correct folding of eukaryotic cytochrome 2B4 during its biosynthesis in E. coli.

  20. The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

    Science.gov (United States)

    Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria; Biggiogera, Marco; Leikin, Sergey; Fisher, Shannon; Tenedini, Elena; Yorgan, Timur A; Schinke, Thorsten; Tian, Kun; Schwartz, Jean-Marc; Forte, Fabiana; Wagener, Raimund; Villani, Simona; Rossi, Antonio; Forlino, Antonella

    2017-08-01

    Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment. © The Author 2017. Published by Oxford University Press.

  1. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  2. Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Jeong, Jae-Hoon [Division of Radiotherapy, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Kang, Seongman [Division of Life Sciences, Korea University, Seoul 136-701 (Korea, Republic of); Lim, Young-Bin, E-mail: yblim@kirams.re.kr [Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2014-07-25

    Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

  3. Interaction between SGT1 and Cytosolic/Nuclear HSC70 Chaperones Regulates Arabidopsis Immune Responses[W

    Science.gov (United States)

    Noël, Laurent D.; Cagna, Giuseppe; Stuttmann, Johannes; Wirthmüller, Lennart; Betsuyaku, Shigeyuki; Witte, Claus-Peter; Bhat, Riyaz; Pochon, Nathalie; Colby, Thomas; Parker, Jane E.

    2007-01-01

    The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene–triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70–SGT1 association. PMID:18065690

  4. North American fertilizer capacity data

    Energy Technology Data Exchange (ETDEWEB)

    1991-01-01

    This listing of producers and their fertilizer production capacities was compiled in January 1991 with the cooperation of the US and Canadian fertilizer industry. Capacity is only an indicator of supply. Nameplate capacity differs from planned production levels or actual production because plants often operate above or below design capacity. Unless reported otherwise, plant capacities are based on 340 days per year of operation. No adjustment is made for partial year operation. Numerical data for the production of ammonia, ammonium nitrate, nitrogen solutions, urea, phosphate rock, phosphoric acid and ammonium phosphates is included.

  5. Radio-capacity of ecosystems

    International Nuclear Information System (INIS)

    Kultakhmedov, Yu.; Kultakhmedova-Vyshnyakova, V.

    1997-01-01

    This paper consider a universal approach to ecosystems of different types, based on representation of their radio-capacity. The concept of ecosystem includes reproduction of components (bio-productivity) and conditions such as maintaining of environment quality. Radio-capacity in the case of radionuclide pollution appears in accumulation and redistribution of radionuclides in the ecosystem. As a result the radionuclides are redistributed and buried in soil or lake bottom sediments. Estimation models for the radio-capacity of water and terrestrial ecosystems are represented. The calculations of the radio-capacity factor of water ecosystems are performed, and the high radio-capacity of a freshwater reservoir (F=0.6-0.8) and extremely high radio-capacity of a reservoir cascade (F c =0.99) is shown material from the Dnieper's cascade reservoirs. The methods of radio-capacity estimation of agroecosystems, wood and marine ecosystems are developed. (authors)

  6. Quality of Life and Functional Status Across the Life Course. Project 2: Investigating Mechanisms to Explain Age Associated Differences in Quality of Life Among Breast Cancer Patients

    Science.gov (United States)

    2002-10-01

    4.3.7 Symptoms Questionnaire 4.3.8 Quality of Life Form....... 4.3.9 Spirituality ....................... 4.3.10 BriefCOPE Scale...AD Award Nuimber: DAMD17-01-1-0447 TITLE: Quality of Life and Functional Status Across the Life Course Project 2: Investigating Mechanisms to...Explain Age Associated Differences in Quality of Life Among Breast Cancer Patients PRINCIPAL INVESTIGATOR: Michelle J. Naughton, Ph.D. Nancy E

  7. Sex-specific age associations of ankle proprioception test performance in older adults: results from the Baltimore Longitudinal Study of Aging.

    Science.gov (United States)

    Ko, Seung-Uk; Simonsick, Eleanor; Deshpande, Nandini; Ferrucci, Luigi

    2015-05-01

    this study was aimed to test the hypothesis that ankle proprioception assessed by custom-designed proprioception testing equipment changes with ageing in men and women. ankle proprioception was assessed in 289 participants (131 women) of the Baltimore Longitudinal Study of Aging (BLSA); the participants aged 51-95 years and were blinded during testing. the average minimum perceived ankle rotation was 1.11° (SE = 0.07) in women and 1.00° (SE = 0.06) in men, and it increased with ageing in both sexes (P proprioception was introduced in this study using a customized test instrument, software and test protocol. Age-associated reduction in ankle proprioception was confirmed from two subtests of threshold and tracking separately for women and men. Findings in this study prompt future studies to determine whether these age-associated differences in the threshold for passive motion detection and movement tracking are evident in longitudinal study and how these specific deficits in ankle proprioception are related to age-associated chronic conditions such as knee or hip osteoarthritis and type II diabetes and affect daily activities such as gait. © The Author 2015. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

    Directory of Open Access Journals (Sweden)

    Yun-Long Tsai

    Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

  9. Available transmission capacity assessment

    Directory of Open Access Journals (Sweden)

    Škokljev Ivan

    2012-01-01

    Full Text Available Effective power system operation requires the analysis of vast amounts of information. Power market activities expose power transmission networks to high-level power transactions that threaten normal, secure operation of the power system. When there are service requests for a specific sink/source pair in a transmission system, the transmission system operator (TSO must allocate the available transfer capacity (ATC. It is common that ATC has a single numerical value. Additionally, the ATC must be calculated for the base case configuration of the system, while generation dispatch and topology remain unchanged during the calculation. Posting ATC on the internet should benefit prospective users by aiding them in formulating their requests. However, a single numerical value of ATC offers little for prospect for analysis, planning, what-if combinations, etc. A symbolic approach to the power flow problem (DC power flow and ATC offers a numerical computation at the very end, whilst the calculation beforehand is performed by using symbols for the general topology of the electrical network. Qualitative analysis of the ATC using only qualitative values, such as increase, decrease or no change, offers some new insights into ATC evaluation, multiple transactions evaluation, value of counter-flows and their impact etc. Symbolic analysis in this paper is performed after the execution of the linear, symbolic DC power flow. As control variables, the mathematical model comprises linear security constraints, ATC, PTDFs and transactions. The aim is to perform an ATC sensitivity study on a five nodes/seven lines transmission network, used for zonal market activities tests. A relatively complicated environment with twenty possible bilateral transactions is observed.

  10. Perioperative functional residual capacity.

    Science.gov (United States)

    Wahba, R W

    1991-04-01

    The literature dealing with the magnitude, mechanism and effects of reduced FRC in the perioperative period is reviewed. During general anaesthesia FRC is reduced by approximately 20%. The reduction is greater in the obese and in patients with COPD. The most likely mechanism is the loss of inspiratory muscle tone of the muscles acting on the rib cage. Gas trapping is an additional mechanism. Lung compliance decreases and airways resistance increases, in large part, due to decreased FRC. The larynx is displaced anteriorly and elongated, making laryngoscopy and intubation more difficult. The change in FRC creates or increases intrapulmonary shunt and areas of low ventilation to perfusion. This is due to the occurrence of compression atelectasis, and to regional changes in mechanics and airway closure which tend to reduce ventilation to dependent lung zones which are still well perfused. Abdominal and thoracic operations tend to increase shunting further. Large tidal volume but not PEEP will improve oxygenation, although both increase FRC. Both FRC and vital capacity are reduced following abdominal and thoracic surgery in a predictable pattern. The mechanism is the combined effect of incisional pain and reflex dysfunction of the diaphragm. Additional effects of thoracic surgery include pleural effusion, cooling of the phrenic nerve and mediastinal widening. Postoperative hypoxaemia is a function of reduced FRC and airway closure. There is no real difference among the various methods of active lung expansion in terms of the speed of restoration of lung function, or in preventing postoperative atelectasis/pneumonia. Epidural analgesia does not influence the rate of recovery of lung function, nor does it prevent atelectasis/pneumonia.

  11. Algorithmic complexity of quantum capacity

    Science.gov (United States)

    Oskouei, Samad Khabbazi; Mancini, Stefano

    2018-04-01

    We analyze the notion of quantum capacity from the perspective of algorithmic (descriptive) complexity. To this end, we resort to the concept of semi-computability in order to describe quantum states and quantum channel maps. We introduce algorithmic entropies (like algorithmic quantum coherent information) and derive relevant properties for them. Then we show that quantum capacity based on semi-computable concept equals the entropy rate of algorithmic coherent information, which in turn equals the standard quantum capacity. Thanks to this, we finally prove that the quantum capacity, for a given semi-computable channel, is limit computable.

  12. Capacity Measurement with the UIC 406 Capacity Method

    DEFF Research Database (Denmark)

    Landex, Alex; Schittenhelm, Bernd; Kaas, Anders H.

    2008-01-01

    This article describes the fast and effective UIC 406 method for calculating capacity consumption on railway lines. It is possible to expound the UIC 406 method in different ways which can lead to different capacity consumptions. Therefore, this article describes how the methodology is expounded...

  13. High Aerobic Capacity Mitigates Changes in the Plasma Metabolomic Profile Associated with Aging.

    Science.gov (United States)

    Falegan, Oluyemi S; Vogel, Hans J; Hittel, Dustin S; Koch, Lauren G; Britton, Steven L; Hepple, Russ T; Shearer, Jane

    2017-02-03

    Advancing age is associated with declines in maximal oxygen consumption. Declines in aerobic capacity not only contribute to the aging process but also are an independent risk factor for morbidity, cardiovascular disease, and all-cause mortality. Although statistically convincing, the relationships between aerobic capacity, aging, and disease risk remain largely unresolved. To this end, we employed sensitive, system-based metabolomics approach to determine whether enhanced aerobic capacity could mitigate some of the changes seen in the plasma metabolomic profile associated with aging. Metabolomic profiles of plasma samples obtained from young (13 month) and old (26 month) rats bred for low (LCR) or high (HCR) running capacity using proton nuclear magnetic resonance spectroscopy ( 1 H NMR) were examined. Results demonstrated strong profile separation in old and low aerobic capacity rats, whereas young and high aerobic capacity rat models were less predictive. Significantly differential metabolites between the groups include taurine, acetone, valine, and trimethylamine-N-oxide among other metabolites, specifically citrate, succinate, isovalerate, and proline, were differentially increased in older HCR animals compared with their younger counterparts. When interactions between age and aerobic capacity were examined, results demonstrated that enhanced aerobic capacity could mitigate some but not all age-associated alterations in the metabolomic profile.

  14. Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

    Science.gov (United States)

    Carver, John A; Ecroyd, Heath; Truscott, Roger J W; Thorn, David C; Holt, Carl

    2018-03-20

    Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in

  15. Modulation of chaperone-like and membranolytic activities of major horse seminal plasma protein HSP-1/2 by L-carnitine.

    Science.gov (United States)

    Sudheer Kumar, C; Swamy, Musti J

    2017-09-01

    The major protein of horse seminal plasma, HSP-1/2, exhibits membranolytic and chaperone-like activities and plays a crucial role in regulating sperm capacitation. L-Carnitine is a small polar molecule present in high concentrations in mammalian seminal plasma. The present results demonstrate that L-carnitine binds to HSP-1/2 and increases its thermal stability, enhances cooperativity of its chemical unfolding and decreases both chaperone-like and membranolytic activities of this protein. The HSP-1/2-L-carnitine complex exhibits anti-oxidative behaviour by inhibiting the production of hydroxyl radicals, suggesting that it can protect other constituents of seminal plasma from damage by hydroxyl radicals. As HSP-1/2 and L-carnitine share the same spatiotemporal location in the horse reproductive tract, this interaction is physiologically significant and may prevent premature interaction of HSP-1/2 with sperm, which in turn regulates the sperm capacitation.

  16. The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

    DEFF Research Database (Denmark)

    Pagh, Rasmus; Duus, Karen; Laursen, Inga

    2008-01-01

    was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding...... with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion......The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL...

  17. Conformational Analysis of a High-Mannose-Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR-Validated Molecular Dynamics Simulation.

    Science.gov (United States)

    Suzuki, Tatsuya; Kajino, Megumi; Yanaka, Saeko; Zhu, Tong; Yagi, Hirokazu; Satoh, Tadashi; Yamaguchi, Takumi; Kato, Koichi

    2017-02-16

    Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses 13 C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.

    Directory of Open Access Journals (Sweden)

    Luiz Felipe M Prota

    Full Text Available Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR. Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression.Dose-response (1 nM to 10 µM and time course (3 to 48 h curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90 binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70 binding decreased 0.30 fold in an immunoprecipitation assay.Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

  19. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

    Directory of Open Access Journals (Sweden)

    Ana Paço

    Full Text Available The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials. The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants

  20. Homozygosity for a Missense Mutation in SERPINH1, which Encodes the Collagen Chaperone Protein HSP47, Results in Severe Recessive Osteogenesis Imperfecta

    OpenAIRE

    Christiansen, Helena E.; Schwarze, Ulrike; Pyott, Shawna M.; AlSwaid, Abdulrahman; Al Balwi, Mohammed; Alrasheed, Shatha; Pepin, Melanie G.; Weis, Mary Ann; Eyre, David R.; Byers, Peter H.

    2010-01-01

    Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missens...

  1. The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

    Science.gov (United States)

    Leandro, João; Saraste, Jaakko; Leandro, Paula; Flatmark, Torgeir

    2011-01-01

    Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

    OpenAIRE

    Taniguchi, Naohiro; Tokuda, Hajime

    2008-01-01

    An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

  3. Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa.

    Science.gov (United States)

    Banerjee, Anindyajit; Dey, Supratim; Chakraborty, Abhijit; Datta, Aohona; Basu, Abhishek; Chakrabarti, Saikat; Datta, Saumen

    2014-12-01

    Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. © 2014 Wiley Periodicals, Inc.

  4. LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease

    OpenAIRE

    Rothaug, Michelle; Stroobants, Stijn; Schweizer, Michaela; Peters, Judith; Zunke, Friederike; Allerding, Mirka; D?Hooge, Rudi; Saftig, Paul; Blanz, Judith

    2015-01-01

    The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with ...

  5. RPS3a over-expressed in HBV-associated hepatocellular carcinoma enhances the HBx-induced NF-κB signaling via its novel chaperoning function.

    Directory of Open Access Journals (Sweden)

    Keo-Heun Lim

    Full Text Available Hepatitis B virus (HBV infection is one of the major causes of hepatocellular carcinoma (HCC development. Hepatitis B virus X protein (HBx is known to play a key role in the development of hepatocellular carcinoma (HCC. Several cellular proteins have been reported to be over-expressed in HBV-associated HCC tissues, but their role in the HBV-mediated oncogenesis remains largely unknown. Here, we explored the effect of the over-expressed cellular protein, a ribosomal protein S3a (RPS3a, on the HBx-induced NF-κB signaling as a critical step for HCC development. The enhancement of HBx-induced NF-κB signaling by RPS3a was investigated by its ability to translocate NF-κB (p65 into the nucleus and the knock-down analysis of RPS3a. Notably, further study revealed that the enhancement of NF-κB by RPS3a is mediated by its novel chaperoning activity toward physiological HBx. The over-expression of RPS3a significantly increased the solubility of highly aggregation-prone HBx. This chaperoning function of RPS3a for HBx is closely correlated with the enhanced NF-κB activity by RPS3a. In addition, the mutational study of RPS3a showed that its N-terminal domain (1-50 amino acids is important for the chaperoning function and interaction with HBx. The results suggest that RPS3a, via extra-ribosomal chaperoning function for HBx, contributes to virally induced oncogenesis by enhancing HBx-induced NF-κB signaling pathway.

  6. Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone

    Directory of Open Access Journals (Sweden)

    Carmela Garcia-Doval

    2015-12-01

    Full Text Available Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala, in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396 consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.

  7. Installed capacity in New York

    International Nuclear Information System (INIS)

    Charlton, J.

    2006-01-01

    This presentation discussed capacity issues related to the New York Independent System Operator (NYISO). The NYISO's market volume was approximately $11 billion in 2005, and it was responsible for providing 32,075 MW of electricity at peak load to its users. Regulatory uncertainty is currently discouraging investment in new generating resources. All load serving entities are required to contract for sufficient capacity in order to meet their capacity obligations. Market participants currently determine capacity and energy revenues. The NYISO market allows suppliers to recover variable costs for providing ancillary services, and the economic value of the revenue source governs decisions made in the wholesale electricity market. The installed capacity market was designed as a spot auction deficiency auction. Phased-in demand curves are used to modify the installed capacity market's design. A sloped demand curve mechanism is used to value capacity above the minimum requirement for both reliability and competition. Participation in the day-ahead market enhances competition and exerts downward pressure on energy and ancillary service market prices. It was concluded that the market structures and design features of the installed capacity markets recognize the need for system reliability in addition to encouraging robust competition and recognizing energy price caps and regulatory oversights. tabs., figs

  8. Checking Capacity for MIMO Configurations

    DEFF Research Database (Denmark)

    Thaysen, Jesper; Jakobsen, Kaj Bjarne

    2007-01-01

    Wireless system capacity can be added by increasing the number of antennas in a MIMO setup or by carefully optimizing the performance of a smaller number of antennas.......Wireless system capacity can be added by increasing the number of antennas in a MIMO setup or by carefully optimizing the performance of a smaller number of antennas....

  9. Estimation of capacities on Florida freeways.

    Science.gov (United States)

    2014-09-01

    Current capacity estimates within Floridas travel time reliability tools rely on the Highway Capacity Manual (HCM 2010) to : estimate capacity under various conditions. Field measurements show that the capacities of Florida freeways are noticeably...

  10. Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a.

    Science.gov (United States)

    Singh, Jagmohan; Boopathi, Ettickan; Addya, Sankar; Phillips, Benjamin; Rigoutsos, Isidore; Penn, Raymond B; Rattan, Satish

    2016-11-01

    A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A 2 analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence. Copyright © 2016 the American Physiological Society.

  11. North American fertilizer capacity data

    Energy Technology Data Exchange (ETDEWEB)

    1993-02-01

    This listing of fertilizer producers and their production capacities was compiled in February 1993 with the cooperation of the US and Canadian fertilizer industry. TVA does not guarantee the completeness or accuracy of the information. Capacity is only an indicator of supply. Nameplate capacity differs from planned production levels or actual production because plants often operate above or below design capacity. Unless reported otherwise, plant capacities are based on 340 days per year of operation. No adjustment is made for partial year operation. Information is given on the following types of fertilizers: ammonia, ammonium nitrate, nitrogen solutions, urea, ammonium sulfate, phosphate rock, wet-process phosphoric acid, ammonium phosphates, concentrated superphosphates, potash, nitric acid, superphosphoric acid, upgraded phosphoric acids, normal superphosphate, elemental phosphorus, potassium sulfate, and sulfate of potash/magnesia.

  12. Capacity Building in Land Administration

    DEFF Research Database (Denmark)

    Enemark, Stig; Williamson, I

    2004-01-01

    Capacity building increasingly seen as a key component of land administration projects in developing and countries in transition undertaken by the international development banks and individual country development assistance agencies. However, the capacity building concept is often used within...... infrastructures for implementing land policies in a sustainable way. Where a project is established to create land administration infrastructures in developing or transition countries, it is critical that capacity building is a mainstream component, not as an add-on, which is often the case. In fact such projects...... should be dealt with as capacity building projects in themselves.    The article introduces a conceptual analytical framework that provides some guidance when dealing with capacity building for land administration in support of a broader land policy agenda....

  13. One gene, two proteins: coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli.

    Science.gov (United States)

    Drees, Steffen L; Klinkert, Birgit; Helling, Stefan; Beyer, Dominik F; Marcus, Katrin; Narberhaus, Franz; Lübben, Mathias

    2017-11-01

    Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P 1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P 1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock. © 2017 John Wiley & Sons Ltd.

  14. Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism.

    Science.gov (United States)

    Anglès, Fréderic; Castanié-Cornet, Marie-Pierre; Slama, Nawel; Dinclaux, Mickael; Cirinesi, Anne-Marie; Portais, Jean-Charles; Létisse, Fabien; Genevaux, Pierre

    2017-01-27

    Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo. Using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproducts production and flux rearrangements, we demonstrate that DnaK significantly impacts the hierarchical order of carbon sources utilization, the excretion of main coproducts and the distribution of metabolic fluxes, thus revealing a multilevel interaction of DnaK with the central metabolism.

  15. Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

    Directory of Open Access Journals (Sweden)

    Coral Y. Zhou

    2017-06-01

    Full Text Available The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs are essential for diverse processes ranging from metabolic signaling to chromatin remodeling, but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain in the Ino80 ATPase subunit of the INO80 chromatin-remodeling complex, termed Ino80INS. Ino80INS stimulates Rvbs’ ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients such as Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly.

  16. Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

    2015-05-01

    We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

  17. Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization.

    Science.gov (United States)

    Emelyanov, Alexander V; Rabbani, Joshua; Mehta, Monika; Vershilova, Elena; Keogh, Michael C; Fyodorov, Dmitry V

    2014-09-15

    Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells. © 2014 Emelyanov et al.; Published by Cold Spring Harbor Laboratory Press.

  18. The Influence of Chemical Chaperones on Enzymatic Activity under Thermal and Chemical Stresses: Common Features and Variation among Diverse Chemical Families

    Science.gov (United States)

    Feibish, Nir; Sharon, Noa; Schnaider, Lee; Shmul, Guy; Amir, Yaniv; Buzhansky, Ludmila; Gazit, Ehud

    2014-01-01

    Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups. PMID:24520396

  19. Inhibition of chaperone-mediated autophagy prevents glucotoxicity in the Caenorhabditis elegans mev-1 mutant by activation of the proteasome.

    Science.gov (United States)

    Eisermann, Dorothé Jenni; Wenzel, Uwe; Fitzenberger, Elena

    2017-02-26

    Chronic hyperglycemia is a hallmark of diabetes mellitus and the main cause of diabetes-associated complications. Increased intracellular glucose levels lead to damaged proteins and in consequence disturb cellular proteostasis. As an important contributor to the maintenance and restoration of proteostasis, autophagy mediates the lysosomal degradation of damaged proteins or entire cellular organelles. In the present study we used the stress-sensitive mev-1 mutant of the nematode Caenorhabditis elegans in order to assess the role of lmp-2, a homologue of the lysosome associated membrane protein type 2A, in the context of glucotoxicity, which was achieved by feeding glucose in a liquid medium. Knockdown of lmp-2 by RNA interference completely prevented the survival reduction caused by glucose under heat stress. Those effects were associated with the prevention of (1) increased lysosome formation and (2) reduction of proteasomal activity, which were observed under glucose feeding. Finally, the survival reduction due to knockdown of ubiquitin remained unaffected by the additional lmp-2 knockdown in the absence or presence of glucose. In conclusion, our study provides evidence that lmp-2, a key player in chaperone-mediated autophagy, is functional in C. elegans, too. Inhibition of lmp-2 prevents the reduction of proteasomal activity by glucose and thereby prevents also glucotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.

    Science.gov (United States)

    Ito, Shinya; Ogawa, Koji; Takeuchi, Koh; Takagi, Motoki; Yoshida, Masahito; Hirokawa, Takatsugu; Hirayama, Shoshiro; Shin-Ya, Kazuo; Shimada, Ichio; Doi, Takayuki; Goshima, Naoki; Natsume, Tohru; Nagata, Kazuhiro

    2017-12-08

    Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

    Directory of Open Access Journals (Sweden)

    Bogen Bjarne

    2010-02-01

    Full Text Available Abstract Background Efficient expression systems exist for antibody (Ab molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs. Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc TCRs. Results The effect of 1 over-expression of the periplasmic chaperon FkpA, 2 culture conditions and 3 molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1 high yield recovery sufficient for biophysical characterization and 2 high throughput screening of such molecules following molecular engineering.

  2. Restoration of mp53 to wtp53 by chemical chaperones restores p53-dependent apoptosis after radiotherapy

    International Nuclear Information System (INIS)

    Ohnishi, T.; Asakawa, I.; Tamamoto, T.; Takahashi, A.; Ohnishi, K.

    2003-01-01

    The mutations of many kinds of cancer related genes have been investigated for the predictive assay against cancer therapy by the application of molecular biology. A tumor suppressor gene product of wtp53 plays important roles in cancer suppression through the induction of cell growth arrest, DNA repair or apoptosis. The p53 exerts its function by induction of downstream genes and/or interaction to various proteins. Mutations in the p53 gene (mp53) cause conformational alterations in the p53 protein, the majority of which can no longer induce expression of the downstream genes. The genetic status of p53 gene has been focused as the most important candidate among them for cancer therapy. The gene therapy of p53 has been already applied. We reported that the transfection of mp53 gene increased the radio-, thermo- and chemo-resistance, and depressed apoptosis introduced with them through bax-induction and proteolysis of PARP and caspase-3. From these results, we propose that the gene therapy of wtp53 to p53-deleted cancer cells may be very useful for cancer therapy by the combination with radiotherapy. Even in the case of mp53 cancer cells, we succeeded the restoration of mp53 to wtp53 by glycerol or C-terminal peptide of p53 as chemical chaperones. These experimental progresses might support effective cancer therapy against individual patients bearing with different p53 gene status by the use of the most suitable treatment to them in the near future

  3. Binary Supramolecular Gel of Achiral Azobenzene with a Chaperone Gelator: Chirality Transfer, Tuned Morphology, and Chiroptical Property.

    Science.gov (United States)

    Ji, Lukang; Ouyang, Guanghui; Liu, Minghua

    2017-10-31

    Binary supramolecular gels based on achiral azobenzene derivatives and a chiral chaperone gelator, long-alkyl-chain-substituted L-Histidine (abbreviated as LHC18) that could assist many nongelling acids in forming gels, were investigated in order to fabricate the chiroptical gel materials in a simple way. It was found that although the carboxylic acid-terminated achiral azobenzene derivatives could not form gels in any solvents, when mixed with LHC18 they formed the co-gels and self-assembled into various morphologies ranging from nanotubes and loose nanotubes to nanosheets, depending on the substituent groups on the azobenzene moiety. The ether linkage and the number of carboxylic acid groups attached to the azobenzene moiety played important roles. Upon gel formation, the localized molecular chirality in LHC18 could be transferred to the azobenzene moiety. Combined with the trans-cis isomerization of the azobenzene, optically and chiroptically reversible gels were generated. It was found that the gel based on azobenzene with two carboxylic acid groups and ether linkages showed clear optical reversibility but less chiroptical reversibility, whereas the gel based on azobenzene with one carboxylic acid and an ether linkage showed both optical and chiroptical reversibility. Thus, new insights into the relationship among the molecular structures of the azobenzene, self-assembled nanostructures in the gel and the optical and chiroptical reversibility were disclosed.

  4. The therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins for treatment of preeclampsia

    Directory of Open Access Journals (Sweden)

    Tereza eCindrova-Davies

    2014-05-01

    Full Text Available Preeclampsia is a complex multifactorial disease. Placental oxidative stress, a result of deficient spiral artery remodeling, plays an important role in the pathophysiology of preeclampsia. Antiangiogenic factors secreted from malperfused placenta are instrumental in mediating maternal endothelial dysfunction and consequent symptoms of preeclampsia; the mechanism is likely to involve increased ET-1 secretion and reduced NO bioavailability. Therapeutic interventions so far remain only experimental and there is no established remedy for the treatment of preeclampsia. This review concentrates on the evidence for the therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins. These compounds display pleitropic antioxidant, anti-inflammatory, and pro-angiogenic effects in animal and in vitro studies. Although clinical trials on the use of antioxidant vitamins in pregnancy proved largely unsuccessful, the scope for their use still exists given the beneficial cardioprotective effects of antioxidant-rich Mediterranean diet, periconceptual vitamin use and the synergistic effect of vitamin C and l-arginine. Encouraging clinical evidence exists for the use of NO donors, and a clinical trial is underway testing the effect of statins in treatment of preeclampsia. H2S recently emerged as a novel therapeutic agent for cardiovascular disease, and its beneficial effects were also tested in animal models of preeclampsia. It is risky to prescribe any medication to pregnant women on a large scale, and any future therapeutic intervention has to be well tested and safe. Many of the compounds discussed could be potential candidates.

  5. Sulforaphane Potentiates the Efficacy of 17-Allylamino 17-Demethoxygeldanamycin Against Pancreatic Cancer Through Enhanced Abrogation of Hsp90 Chaperone Function

    Science.gov (United States)

    Li, Yanyan; Zhang, Tao; Schwartz, Steven J.; Sun, Duxin

    2013-01-01

    Heat shock protein 90 (Hsp90), an essential molecular chaperone that regulates the stability of a wide range of oncogenic proteins, is a promising target for cancer therapeutics. We investigated the combination efficacy and potential mechanisms of sulforaphane, a dietary component from broccoli and broccoli sprouts, and 17-allylamino 17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, in pancreatic cancer. MTS assay demonstrated that sulforaphane sensitized pancreatic cancer cells to 17-AAG in vitro. Caspase-3 was activated to 6.4-fold in response to simultaneous treatment with sulforaphane and 17-AAG, whereas 17-AAG alone induced caspase-3 activity to 2-fold compared to control. ATP binding assay and coimmunoprecipitation revealed that sulforaphane disrupted Hsp90-p50Cdc37 interaction, whereas 17-AAG inhibited ATP binding to Hsp90. Concomitant use of sulforaphane and 17-AAG synergistically downregulated Hsp90 client proteins in Mia Paca-2 cells. Co-administration of sulforaphane and 17-AAG in pancreatic cancer xenograft model led to more than 70% inhibition of the tumor growth, whereas 17-AAG alone only suppressed the tumor growth by 50%. Our data suggest that sulforaphane potentiates the efficacy of 17-AAG against pancreatic cancer through enhanced abrogation of Hsp90 function. These findings provide a rationale for further evaluation of broccoli/broccoli sprout preparations combined with 17-AAG for better efficacy and lower dose-limiting toxicity in pancreatic cancer. PMID:21875325

  6. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

    Science.gov (United States)

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-01-01

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies. PMID:27387855

  7. The Stress Protein/Chaperone Grp94 Counteracts Muscle Disuse Atrophy by Stabilizing Subsarcolemmal Neuronal Nitric Oxide Synthase

    Science.gov (United States)

    Vitadello, Maurizio; Gherardini, Jennifer

    2014-01-01

    Abstract Aims: Redox and growth-factor imbalance fosters muscle disuse atrophy. Since the endoplasmic-reticulum chaperone Grp94 is required for folding insulin-like growth factors (IGFs) and for antioxidant cytoprotection, we investigated its involvement in muscle mass loss due to inactivity. Results: Rat soleus muscles were transfected in vivo and analyzed after 7 days of hindlimb unloading, an experimental model of muscle disuse atrophy, or standard caging. Increased muscle protein carbonylation and decreased Grp94 protein levels (pmuscle atrophy regulators identified 160 kDa neuronal nitric oxide synthase (nNOS) as a new Grp94 partner. Unloading was demonstrated to untether nNOS from myofiber subsarcolemma; here, we show that such nNOS localization, revealed by means of NADPH-diaphorase histochemistry, appeared preserved in unloaded myofibers expressing recombinant Grp94, compared to those transfected with the empty vector or deleted Grp94 cDNA (p<0.02). Innovation: Grp94 interacts with nNOS and prevents its untethering from sarcolemma in unloaded myofibers. Conclusion: Maintenance of Grp94 expression is sufficient to counter unloading atrophy and oxidative stress by mechanistically stabilizing nNOS-multiprotein complex at the myofiber sarcolemma. Antioxid. Redox Signal. 20, 2479–2496. PMID:24093939

  8. Over-expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons.

    Science.gov (United States)

    Ness, Frederique; Cox, Brian S; Wongwigkarn, Jintana; Naeimi, Wesley R; Tuite, Mick F

    2017-04-01

    The ability of a yeast cell to propagate [PSI + ], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI + ] propagation in dividing cells and they get diluted out as cells multiply. Over-expression of Hsp104 also leads to [PSI + ] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over-expression in [PSI + ] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion-free [psi - ] cells present, no change in the molecular weight of Sup35-containing SDS-resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over-expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over-expression of Hsp104 causes malpartition of [PSI + ] propagons between mother and daughter cells in a sub-population of cells during cell division thereby generating prion-free [psi - ] cells. © 2017 John Wiley & Sons Ltd.

  9. Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

    Science.gov (United States)

    Wang, Jie; Pareja, Kristeen A; Kaiser, Chris A; Sevier, Carolyn S

    2014-01-01

    Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress. DOI: http://dx.doi.org/10.7554/eLife.03496.001 PMID:25053742

  10. Changes in macroautophagy, chaperone-mediated autophagy, and mitochondrial metabolism in murine skeletal and cardiac muscle during aging.

    Science.gov (United States)

    Zhou, Jin; Chong, Shu Yun; Lim, Andrea; Singh, Brijesh K; Sinha, Rohit A; Salmon, Adam B; Yen, Paul M

    2017-02-26

    Aging causes a general decline in cellular metabolic activity, and function in different tissues and whole body homeostasis. However, the understanding about the metabolomic and autophagy changes in skeletal muscle and heart during aging is still limited. We thus examined markers for macroautophagy, chaperone-mediated autophagy (CMA), mitochondrial quality control, as well as cellular metabolites in skeletal and cardiac muscle from young (5 months old) and aged (27 months old) mice. We found decreased autophagic degradation of p62 and increased ubiquitinated proteins in both tissues from aged mice, suggesting a decline in macroautophagy during aging. In skeletal muscle from aged mice, there also was a decline in LC3B-I conjugation to phosphatidylethanolamine (PE) possibly due to decreased protein levels of ATG3 and ATG12-ATG5. The CMA markers, LAMP-2A and Hsc70, and mitochondrial turnover markers, Drp1, PINK1 and PGC1α also were decreased. Metabolomics analysis showed impaired β-oxidation in heart of aged mice, whereas increased branched-chain amino acids (BCAAs) and ceramide levels were found in skeletal muscle of aged mice that in turn, may contribute to insulin resistance in muscle. Taken together, our studies showed similar declines in macroautophagy but distinct effects on CMA, mitochondrial turnover, and metabolic dysfunction in muscle vs. heart during aging.

  11. High-dimensional analysis of the aging immune system: verification of age-associated differences in immune signaling responses in healthy donors.

    Science.gov (United States)

    Longo, Diane M; Louie, Brent; Ptacek, Jason; Friedland, Greg; Evensen, Erik; Putta, Santosh; Atallah, Michelle; Spellmeyer, David; Wang, Ena; Pos, Zoltan; Marincola, Francesco M; Schaeffer, Andrea; Lukac, Suzanne; Railkar, Radha; Beals, Chan R; Cesano, Alessandra; Carayannopoulos, Leonidas N; Hawtin, Rachael E

    2014-06-21

    Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.

  12. The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney

    Science.gov (United States)

    O’Brown, Zach K.; Van Nostrand, Eric L.; Higgins, John P.; Kim, Stuart K.

    2015-01-01

    Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney. PMID:26678048

  13. Dietary (-)-Epigallocatechin-3-gallate Supplementation Counteracts Aging-Associated Skeletal Muscle Insulin Resistance and Fatty Liver in Senescence-Accelerated Mouse.

    Science.gov (United States)

    Liu, Hung-Wen; Chan, Yin-Ching; Wang, Ming-Fu; Wei, Chu-Chun; Chang, Sue-Joan

    2015-09-30

    Aging is accompanied by pathophysiological changes including insulin resistance and fatty liver. Dietary supplementation with (-)-epigallocatechin-3-gallate (EGCG) improves insulin sensitivity and attenuates fatty liver disease. We hypothesized that EGCG could effectively modulate aging-associated changes in glucose and lipid metabolism in senescence-accelerated mice (SAM) prone 8 (SAMP8). Higher levels of glucose, insulin, and free fatty acid, inhibited Akt activity, and decreased glucose transporter 4 (GLUT4) expression were observed in SAMP8 mice compared to the normal aging group, SAM resistant 1 mice. EGCG supplementation for 12 weeks successfully decreased blood glucose and insulin levels via restoring Akt activity and GLUT4 expression and stimulating AMPKα activation in skeletal muscle. EGCG up-regulated genes involved in mitochondrial biogenesis and subsequently restored mitochondrial DNA copy number in skeletal muscle of SAMP8 mice. Decreased adipose triglyceride lipase and increased sterol regulatory element binding proteins-1c (SREBP-1c) and carbohydrate responsive element binding protein at mRNA levels were observed in SAMP8 mice in accordance with hepatocellular ballooning and excess lipid accumulation. The pevention of hepatic lipid accumulation by EGCG was mainly attributed to down-regulation of mTOR and SREBP-1c-mediated lipid biosynthesis via suppression of the positive regulator, Akt, and activation of the negative regulator, AMPKα, in the liver. EGCG beneficially modulates glucose and lipid homeostasis in skeletal muscle and liver, leading to alleviation of aging-associated metabolic disorders.

  14. Capacity Measurement with the UIC 406 Capacity Method

    DEFF Research Database (Denmark)

    Landex, Alex; Schittenhelm, Bernd; Kaas, Anders H.

    2008-01-01

    This article describes the fast and effective UIC 406 method for calculating capacity consumption on railway lines. It is possible to expound the UIC 406 method in different ways which can lead to different capacity consumptions. Therefore, this article describes how the methodology is expounded...... in Denmark. This includes how and where to divide the railway lines into line sections, how to analyze stations and junctions, and how to examine line sections with different amounts of tracks....

  15. Regular capacities on metrizable spaces

    Directory of Open Access Journals (Sweden)

    T. M. Cherkovskyi

    2014-07-01

    Full Text Available It is proved that for a (not necessarily compact metric space: the metrics on the space of capacities in the sense of Zarichnyi and Prokhorov are equal; completeness of the space of capacities is equivalent to completeness of the original space. It is shown that for the capacities on metrizable spaces the properties of $\\omega$-smoothness and of $\\tau$-smoothness are equivalent precisely on the separable spaces, and the properties of $\\omega$-smoothness and of regularity w.r.t. some (then w.r.t. any admissible metric are equivalent precisely on the compact spaces.

  16. Capacity Building in Land Management

    DEFF Research Database (Denmark)

    Enemark, Stig; Ahene, Rexford

    2003-01-01

    There is a significant need for capacity building in the interdisciplinary area of land management especially in developing countries and countries in transition, to deal with the complex issues of building efficient land information systems and sustainable institutional infrastructures. Capacity...... development in this area. Furthermore, capacity building should ensure that the focus is on building sound institutions and governance rather than just high-level IT-infrastructures.    This overall approach to capacity building in land management is used for implementing a new land policy reform in Malawi...... building in land management is not only a question of establishing a sufficient technological level or sufficient economic resources. It is mainly a question of understanding the interdisciplinary and cross-sectoral nature of land administration systems, and understanding the need for human resource...

  17. Building Capacity in Ethical Review

    DEFF Research Database (Denmark)

    Douglas-Jones, Rachel

    2017-01-01

    Capacity building in biomedical research ethics review has been a European priority since the early 2000s. Prompted by the increase in data originating in internationally sponsored trials in emerging economies, a range of capacity building initiatives were put in place in the field of ethical...... review to ensure the protection of human subjects participating in research. Drawing on fieldwork with the Forum for Ethical Review Committees in the Asian and Western Pacific Region, I explore two distinct forms taken by capacity building within that organization to support and train members of ethics...... review committees. The first, with an emphasis on standards and measurability, takes as its priority international accountability for clinical trial research. e second explores how the organization goes about persuading trainees to see and do ‘ethics’ differently. is distinction between forms of capacity...

  18. Capacity Markets and Market Stability

    International Nuclear Information System (INIS)

    Stauffer, Hoff

    2006-01-01

    The good news is that market stability can be achieved through a combination of longer-term contracts, auctions for far enough in the future to permit new entry, a capacity management system, and a demand curve. The bad news is that if and when stable capacity markets are designed, the markets may seem to be relatively close to where we started - with integrated resource planning. Market ideologues will find this anathema. (author)

  19. Research projects and capacity building

    CSIR Research Space (South Africa)

    Breen, CM

    2004-10-01

    Full Text Available - knowledged as necessary and urgent, but is this sufficient? Are ?head counts? and/or ?degree counts? of individuals from marginalised sectors, including gender, participating in research projects appro- priate measures for capacity building? Much depends... numbers of people (including race and gender) gaining higher degrees and papers published in peer-reviewed journals may provide some measure of capacity building, they do not measure the extent to which fundamental enabling conditions have been fostered...

  20. Maximizing the optical network capacity.

    Science.gov (United States)

    Bayvel, Polina; Maher, Robert; Xu, Tianhua; Liga, Gabriele; Shevchenko, Nikita A; Lavery, Domaniç; Alvarado, Alex; Killey, Robert I

    2016-03-06

    Most of the digital data transmitted are carried by optical fibres, forming the great part of the national and international communication infrastructure. The information-carrying capacity of these networks has increased vastly over the past decades through the introduction of wavelength division multiplexing, advanced modulation formats, digital signal processing and improved optical fibre and amplifier technology. These developments sparked the communication revolution and the growth of the Internet, and have created an illusion of infinite capacity being available. But as the volume of data continues to increase, is there a limit to the capacity of an optical fibre communication channel? The optical fibre channel is nonlinear, and the intensity-dependent Kerr nonlinearity limit has been suggested as a fundamental limit to optical fibre capacity. Current research is focused on whether this is the case, and on linear and nonlinear techniques, both optical and electronic, to understand, unlock and maximize the capacity of optical communications in the nonlinear regime. This paper describes some of them and discusses future prospects for success in the quest for capacity. © 2016 The Authors.