Science.gov

Sample records for adiponectin protein degradation

  1. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    PubMed

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-01-01

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts. PMID:27428951

  2. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin

    PubMed Central

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-01-01

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts. PMID:27428951

  3. The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

    PubMed Central

    Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

  4. EFFECTS OF SOY PROTEIN AND ISOFLAVONES ON INSULIN RESISTANCE AND ADIPONECTIN IN MALE MONKEYS

    PubMed Central

    Wagner, Janice D.; Zhang, Li; Shadoan, Melanie K.; Kavanagh, Kylie; Chen, Haiying; Trenasari, Kristitianti; Kaplan, Jay R.; Adams, Michael R.

    2008-01-01

    Isoflavones may influence insulin action by means of their well-known receptor-mediated estrogenic activity. However, isoflavones also bind to PPAR’s which are strongly associated with insulin action. Soy protein with its isoflavones has previously been shown to improve glycemic control in diabetic postmenopausal women and to improve insulin sensitivity in ovariectomized monkeys. The purpose of the current report was to extend our studies of dietary soy protein to male monkeys and determine effects of the soy isoflavones on insulin resistance. Two studies are reported here. Study one involved 91 male monkeys consuming three diets differing only by the source of protein (casein-lactalbumin, soy protein with a low isoflavone concentration or soy protein with a high isoflavone concentration). Intravenous glucose tolerance tests (IVGTTs) were done and plasma adiponectin and lipoprotein concentrations were determined after 25 months of study. Samples of visceral fat were obtained at 31 months for assessment adiponectin and PPARγ expression. The second study involved 8 monkeys in a Latin square design that compared the effects of diets with either casein/lactalbumin, soy protein with a high isoflavone concentration, or soy protein that was alcohol-washed to deplete the isoflavones. After eight weeks of treatment, insulin sensitivity and plasma lipoproteins were assessed. At ten weeks, skeletal muscle was biopsied for determination of insulin receptor, PPARα and PPARγ content. The major findings were that consumption of isoflavone-containing soy protein dose-dependently increased insulin responses to the glucose challenge and decreased plasma adiponectin while isoflavone-depleted soy protein decreased body weight and had no effect on plasma adiponectin concentrations. Muscle PPARα and γ expression was also increased with the isoflavone-depleted soy relative to either casein or soy protein containing the isoflavones. Further studies are needed to determine the

  5. Adipocyte Spliced Form of X-Box–Binding Protein 1 Promotes Adiponectin Multimerization and Systemic Glucose Homeostasis

    PubMed Central

    Sha, Haibo; Yang, Liu; Liu, Meilian; Xia, Sheng; Liu, Yong; Liu, Feng; Kersten, Sander; Qi, Ling

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A–like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia. PMID:24241534

  6. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis.

    PubMed

    Sha, Haibo; Yang, Liu; Liu, Meilian; Xia, Sheng; Liu, Yong; Liu, Feng; Kersten, Sander; Qi, Ling

    2014-03-01

    The physiological role of the spliced form of X-box-binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A-like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia. PMID:24241534

  7. Retinol Binding Protein-4 and Adiponectin Levels in Thyroid Overt and Subclinical Dysfunction.

    PubMed

    Kokkinos, S; Papazoglou, D; Zisimopoulos, A; Papanas, N; Tiaka, E; Antonoglou, C; Maltezos, E

    2016-02-01

    Thyroid dysfunction is accompanied by numerous changes in intermediary metabolism. Retinol binding protein-4 (RBP-4) and adiponectin are 2 adipocytokines that have multiple metabolic functions. The aim of our study was to examine serum RBP4 and adiponectin levels in clinical (before and after therapy) and subclinical hyperthyroid and hypothyroid subjects as compared to controls.150 patients with thyroid dysfunction were recruited (65 hyperthyroid and 85 hypothyroid) while 28 euthyroid subjects served as a control group. We measured anthropometric, biochemical and hormonal (free T4, free T3, TSH, insulin) parameters in all participants. RBP-4 and adiponectin were measured using commercial ELISA kits.Mean baseline levels of RBP-4 were higher in patients with clinical hypothyroidism (29.0±10.2 ng/ml, 25.1±12.6 ng/ml, 38.8±16.5 ng/ml, 31.9±13.2 ng/ml, 20.4±8.2 ng/ml in patients with hyperthyroidism, subclinical hyperthryrodism, hypothyroidism, subclinical hypothyroidism and controls respectively, F=4.86, P<0.001) and decreased significantly in patients with clinical hyperthyroidism and hypothyroidism after normalization of thyroid hormones' levels (from 29.0±10.2 to 24.9±8.4 ng/ml, p=0.003 and from 38.8±16.5 to 29.0±10.8 ng/ml, p=0.001 respectively). We did not observe analogous changes in adiponectin levels in any of the studied groups.RBP-4 levels are higher in patients with clinical hypothyroidism and exhibit a marked decrease after normalization of thyroid function in both hyper and hypothyroid patients. We suggest that RBP-4 may play a role in the metabolic disturbances which accompany thyroid dysfunction. PMID:26575118

  8. Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling

    PubMed Central

    Zhao, Lin; Fan, Chongxi; Zhang, Yu; Yang, Yang; Wang, Dongjin; Deng, Chao; Hu, Wei; Ma, Zhiqiang; Jiang, Shuai; Di, Shouyi; Qin, Zhigang; Lv, Jianjun; Sun, Yang; Yi, Wei

    2016-01-01

    Adiponectin has been demonstrated to protect the cardiovascular system and bone marrow mesenchymal stem cells (BMSCs). However, it is unclear whether adiponectin can protect BMSCs against flow shear stress (FSS). In this study, our aim was to explore the effects of adiponectin on BMSCs and to explore the role of AMP-activated protein kinase (AMPK) signaling in this process. Shear stress significantly inhibits the survival and increases the apoptosis of BMSCs in an intensity-dependent manner. The expression levels of TGF-β, bFGF, VEGF, PDGF, and Bcl2 are simultaneously reduced, and the phosphorylation levels of AMPK and ACC, as well as the expression level of Bax, are increased. Supplementation with adiponectin promotes the survival of BMSCs; reverses the changes in the expression levels of TGF-β, bFGF, VEGF, PDGF, Bcl2, and Bax; and further amplifies the phosphorylation of AMPK and ACC. Furthermore, the protective effects of adiponectin can be partially neutralized by AMPK siRNA. In summary, we have demonstrated for the first time that adiponectin can effectively protect BMSCs from FSS and that this effect depends, at least in part, on the activation of AMPK signaling. PMID:27418435

  9. Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling.

    PubMed

    Zhao, Lin; Fan, Chongxi; Zhang, Yu; Yang, Yang; Wang, Dongjin; Deng, Chao; Hu, Wei; Ma, Zhiqiang; Jiang, Shuai; Di, Shouyi; Qin, Zhigang; Lv, Jianjun; Sun, Yang; Yi, Wei

    2016-01-01

    Adiponectin has been demonstrated to protect the cardiovascular system and bone marrow mesenchymal stem cells (BMSCs). However, it is unclear whether adiponectin can protect BMSCs against flow shear stress (FSS). In this study, our aim was to explore the effects of adiponectin on BMSCs and to explore the role of AMP-activated protein kinase (AMPK) signaling in this process. Shear stress significantly inhibits the survival and increases the apoptosis of BMSCs in an intensity-dependent manner. The expression levels of TGF-β, bFGF, VEGF, PDGF, and Bcl2 are simultaneously reduced, and the phosphorylation levels of AMPK and ACC, as well as the expression level of Bax, are increased. Supplementation with adiponectin promotes the survival of BMSCs; reverses the changes in the expression levels of TGF-β, bFGF, VEGF, PDGF, Bcl2, and Bax; and further amplifies the phosphorylation of AMPK and ACC. Furthermore, the protective effects of adiponectin can be partially neutralized by AMPK siRNA. In summary, we have demonstrated for the first time that adiponectin can effectively protect BMSCs from FSS and that this effect depends, at least in part, on the activation of AMPK signaling. PMID:27418435

  10. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  11. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  12. Tunable protein degradation in bacteria.

    PubMed

    Cameron, D Ewen; Collins, James J

    2014-12-01

    Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification. PMID:25402616

  13. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMP-activated protein kinase.

    PubMed

    Zakikhani, Mahvash; Dowling, Ryan J O; Sonenberg, Nahum; Pollak, Michael N

    2008-10-01

    Population studies provide evidence that obesity and insulin resistance are associated not only with elevated serum insulin levels and reduced serum adiponectin levels but also with increased risk of aggressive prostate and colon cancer. We show here that adiponectin activates AMP-activated protein kinase (AMPK) in colon (HT-29) and prostate (PC-3) cancer cells. These results are consistent with prior observations in myocytes, but we show that in epithelial cancer cells AMPK activation is associated with reduction in mammalian target of rapamycin activation as estimated by Ser(2448) phosphorylation, with reduction in p70S6 kinase activation as estimated by Thr(389) phosphorylation, with ribosomal protein S6 activation as estimated by Ser(235/236) phosphorylation, with reduction in protein translation as estimated by [(35)S]methionine incorporation, and with growth inhibition. Adiponectin-induced growth inhibition is significantly attenuated when AMPK level is reduced using small interfering RNA, indicating that AMPK is involved in mediating the antiproliferative action of this adipokine. Thus, adiponectin has the characteristics of a AMPK-dependent growth inhibitor that is deficient in obesity, and this may contribute to the adverse effects of obesity on neoplastic disease. Furthermore, metformin was observed to activate AMPK and to have growth inhibitory actions on prostate and colon cancer cells, suggesting that this compound may be of particular value in attenuating the adverse effects of obesity on neoplasia. PMID:19138981

  14. Protein Degradation and Iron Homeostasis

    PubMed Central

    Thompson, Joel W.; Bruick, Richard K.

    2013-01-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron’s privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. PMID:22349011

  15. Protein degradation and iron homeostasis.

    PubMed

    Thompson, Joel W; Bruick, Richard K

    2012-09-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals. PMID:22349011

  16. Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts

    SciTech Connect

    Kitahara, Kanako; Kusunoki, Natsuko; Kakiuchi, Terutaka; Suguro, Toru; Kawai, Shinichi

    2009-01-09

    The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.

  17. Effects of Adiponectin on Calcium Handling Proteins in Heart Failure with Preserved Ejection Fraction

    PubMed Central

    Tanaka, Komei; Wilson, Richard M.; Essick, Eric E.; Duffen, Jennifer L.; Scherer, Philipp E.; Ouchi, Noriyuki; Sam, Flora

    2014-01-01

    Background Despite the increasing prevalence of heart failure (HF) with preserved ejection fraction (HFpEF) in humans, there remains no therapeutic options for HFpEF. Adiponectin (APN), an adipocyte-derived cytokine exerts cardioprotective actions and its deficiency is implicated in the development of hypertension and HF with reduced ejection fraction. Similarly APN deficiency in HFpEF exacerbates left ventricular hypertrophy (LVH), diastolic dysfunction and HF. However, the therapeutic effects of APN in HFpEF remain unknown. We sought to test the hypothesis that chronic APN overexpression protects against the progression of HF in a murine model of HFpEF. Methods and Results APN transgenic (APNTG) and wild-type (WT) mice underwent uninephrectomy, a continuous saline or d-aldosterone infusion and given 1.0% sodium chloride drinking water for 4-weeks. Aldosterone-infused WT mice developed HFpEF with hypertension, LVH and diastolic dysfunction. Aldosterone infusion increased myocardial oxidative stress and decreased sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) protein expression in HFpEF. Although total phospholamban (PLN) protein expression was unchanged, there was decreased expression of PKA-dependent PLN phosphorylation at Ser16 and CaMKII-dependent PLN phosphorylation at Thr17. APN overexpression in aldosterone-infused mice ameliorated LVH, diastolic dysfunction, lung congestion and myocardial oxidative stress without affecting blood pressure (BP) and LVEF. This improvement in diastolic function parameters in aldosterone-infused APNTG mice was accompanied by preserved protein expression of PKA-dependent phosphorylation of PLN at Ser16. APN replacement prevented the progression of aldosterone-induced HFpEF, independent of BP, by improving diastolic dysfunction and modulating cardiac hypertrophy. Conclusions These findings suggest that APN may have therapeutic effects in patients with HFpEF. PMID:25149095

  18. The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy

    PubMed Central

    2012-01-01

    Introduction Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown. Methods Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR. Results Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ. Conclusions These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel

  19. Regulation of hepatic peroxisome proliferator-activated receptor-alpha (PPAR-a) expression but not adiponectin by dietary protein in finishing pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary soy protein reduction and supplemental leucine (Leu) have been found to decrease leanness and increase muscle lipid content of pig carcasses respectively. Soy protein regulates adiponectin and peroxisome proliferator activated receptor-alpha (PPAR-a) in some species, but the effect of dieta...

  20. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  1. Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization

    PubMed Central

    Decker, Caitlin G.; Maynard, Heather D.

    2015-01-01

    Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by 1H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable. PMID:25937643

  2. Adipocyte iron regulates adiponectin and insulin sensitivity

    PubMed Central

    Gabrielsen, J. Scott; Gao, Yan; Simcox, Judith A.; Huang, Jingyu; Thorup, David; Jones, Deborah; Cooksey, Robert C.; Gabrielsen, David; Adams, Ted D.; Hunt, Steven C.; Hopkins, Paul N.; Cefalu, William T.; McClain, Donald A.

    2012-01-01

    Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores. PMID:22996660

  3. Association of Adiponectin Gene Polymorphism with Adiponectin Levels And Risk for Insulin Resistance Syndrome

    PubMed Central

    Prakash, Jai; Mittal, Balraj; Awasthi, Shally; Srivastava, Neena

    2015-01-01

    Background: Adiponectin is an abundant adipose tissue–derived protein with anti-atherogenic, anti-inflammatory and antidiabetic properties. Plasma adiponectin levels are decreased in obesity, type 2 diabetes, and coronary artery disease and low adiponectin levels also predict insulin resistance (IR). Methods: Case-control study in which 642 male and female subjects were participated from the North Indian population. Lipid, insulin, leptin and adiponectin level were estimated using standard protocols by commercially available test kits. Single nucleotide polymorphisms +45T>G and +276G>T of the AMP1 (adiponectin) gene was genotyped by polymerase chain reaction restriction fragment length polymorphism method. Results: Levels of adiponectin, insulin, homeostasis model assessment-IR index (HOMA-IR index), systolic blood pressure and fat mass showed significant differences between male and female subjects. Serum adiponectin level showed highly significant association with both the +45 and the +276 genotypes. The common haplotype triglyceride (TG) showed a significantly lower adiponectin value than other haplotypes (P = 0.0001). A clear trend of decreasing adiponectin levels per copy of the common haplotype was observed. Nonobese insulin sensitive subjects showed a higher adiponectin value (P = 0.0006) than nonobese insulin resistant subjects. The values of blood pressure, adiponectin, insulin, HOMA-IR, total-cholesterol, and low-density lipoprotein-cholesterol significantly associated with TG haplotype. Conclusions: We observed the very strong association of the adiponectin 45-276 genotypes and haplotypes with adiponectin levels in healthy north Indian population and TG haplotypes also associated with metabolic parameters of the IR syndrome. PMID:25949781

  4. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach

    PubMed Central

    Saleh, Md. Abu; Solayman, Md.; Paul, Sudip; Saha, Moumoni; Khalil, Md. Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  5. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach.

    PubMed

    Saleh, Md Abu; Solayman, Md; Paul, Sudip; Saha, Moumoni; Khalil, Md Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  6. Adiponectin Dysregulation and Insulin Resistance in Type 1 Diabetes

    PubMed Central

    Snell-Bergeon, Janet K.; Erickson, Christopher; Schauer, Irene E.; Bergman, Bryan C.; Rewers, Marian; Maahs, David M.

    2012-01-01

    Context: Type 1 diabetes (T1D) is associated with insulin resistance despite elevated levels of the insulin-sensitizing protein adiponectin. Whether the expected positive correlation between adiponectin and insulin sensitivity is preserved in a T1D population is unknown. Objective: We measured the correlation between total and high-molecular-weight (HMW) adiponectin and insulin sensitivity in T1D patients and nondiabetic controls and identified determinants of adiponectin levels in patients with T1D. Design and Participants: Fasting total and HMW adiponectin were measured in 86 subjects from the Coronary Artery Calcification in T1D (CACTI) cohort (39 T1D, 47 nondiabetic; age 45 ± 8 yr; 55% female). The association of adiponectin levels with insulin sensitivity was analyzed. Setting: The study was conducted at an academic research institute. Methods: Fasting total and HMW adiponectin were measured by RIA and ELISA, respectively. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp. Multivariate linear regression was used to identify determinants of adiponectin levels. Results: Adiponectin levels positively correlated with insulin sensitivity in both subject groups (total adiponectin, r = 0.33 P < 0.05 for T1D, r = 0.29 P < 0.05 controls), but insulin sensitivity was lower in T1D subjects at any given level of total or HMW adiponectin. Adiponectin levels were independently associated with age, gender, and trunk fat, but these variables did not account for increased adiponectin in patients with T1D. Conclusion: Adiponectin levels are positively correlated with insulin sensitivity in T1D patients. However, T1D patients have decreased insulin sensitivity compared with controls at every level of adiponectin, suggesting an important adaptive change of adiponectin set point. PMID:22278421

  7. Adiponectin in childhood.

    PubMed

    Jeffery, Alison N; Murphy, Michael J; Metcalf, Brad S; Hosking, Joanne; Voss, Linda D; English, Patrick; Sattar, Naveed; Wilkin, Terence J

    2008-01-01

    Adiponectin, a hormone produced and secreted by adipocytes, is present in circulation in high circulating concentrations, suggesting an important physiological role. An indirect regulator of glucose metabolism, adiponectin increases insulin sensitivity, improves glucose tolerance and inhibits inflammation. Plasma adiponectin relates inversely to adiposity and, importantly, reflects the sequelae of accumulation of excess adiposity. The role of adiponectin in adults has been explored in detail. Studies in children are now available and, given the increasing rates of childhood obesity, it is important to establish the role of adiponectin in mediating insulin resistance and cardiovascular disease in this age group. This paper reviews the regulation of adiponectin, its effect on body mass, glucose metabolism and cardiovascular risk in infants, children and adolescents. It demonstrates clear links between adiponectin and features of the metabolic syndrome in obese children and adolescents. However, adiponectin's role as a predictor of metabolic dysfunction in healthy, normal-weight youngsters is less clear. PMID:19086185

  8. Central adiponectin acutely improves glucose tolerance in male mice.

    PubMed

    Koch, Christiane E; Lowe, Chrishanthi; Legler, Karen; Benzler, Jonas; Boucsein, Alisa; Böttiger, Gregor; Grattan, David R; Williams, Lynda M; Tups, Alexander

    2014-05-01

    Adiponectin, an adipocyte-derived hormone, regulates glucose and lipid metabolism. It is also antiinflammatory. During obesity, adiponectin levels and sensitivity are reduced. Whereas the action of adiponectin in the periphery is well established the neuroendocrine role of adiponectin is largely unknown. To address this we analyzed the expression of adiponectin and the 2 adiponectin receptors (AdipoR1 and AdipoR2) in response to fasting and to diet-induced and genetic obesity. We also investigated the acute impact of adiponectin on central regulation of glucose homeostasis. Adiponectin (1 μg) was injected intracerebroventricularly (ICV), and glucose tolerance tests were performed in dietary and genetic obese mice. Finally, the influence of ICV adiponectin administration on central signaling cascades regulating glucose homeostasis and on markers of hypothalamic inflammation was assessed. Gene expression of adiponectin was down-regulated whereas AdipoR1 was up-regulated in the arcuate nucleus of fasted mice. High-fat (HF) feeding increased AdipoR1 and AdipoR2 gene expression in this region. In mice on a HF diet and in leptin-deficient mice acute ICV adiponectin improved glucose tolerance 60 minutes after injection, whereas normoglycemia in control mice was unaffected. ICV adiponectin increased pAKT, decreased phospho-AMP-activated protein kinase, and did not change phospho-signal transducer and activator of transcription 3 immunoreactivity. In HF-fed mice, ICV adiponectin reversed parameters of hypothalamic inflammation and insulin resistance as determined by the number of phospho-glycogen synthase kinase 3 β(Ser9) and phospho-c-Jun N-terminal kinase (Thr183/Tyr185) immunoreactive cells in the arcuate nucleus and ventromedial hypothalamus. This study demonstrates that the insulin-sensitizing properties of adiponectin are at least partially based on a neuroendocrine mechanism that involves centrally synthesized adiponectin. PMID:24564394

  9. Protein degradation and protection against misfolded or damaged proteins

    NASA Astrophysics Data System (ADS)

    Goldberg, Alfred L.

    2003-12-01

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

  10. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  11. Effect of adiponectin on the steroidogenic acute regulatory protein, P450 side chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase gene expression, progesterone and androstenedione production by the porcine uterus during early pregnancy.

    PubMed

    Smolinska, N; Dobrzyn, K; Kiezun, M; Szeszko, K; Maleszka, A; Kaminski, T

    2016-06-01

    Adiponectin and its receptors are expressed in the human and porcine uterus and this endocrine system has important role in the regulation of reproductive processes. The expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (HSD3B1) were observed in the human and porcine uterus during the oestrous cycle and pregnancy. The de novo synthesis of steroids in the uterus might be a crucial factor for effective implantation and maintenance of pregnancy. We hypothesized that adiponectin modulates the expression of key enzymes in the synthesis of the steroids: StAR, P450 side chain cleavage enzyme (CYP11A1) and HSD3B1, as well as progesterone (P4) and androstenedione (A4) secretion by the porcine uterus. Endometrial and myometrial explants harvested from gilts (n = 5) on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and on days 10 to 11 of the oestrous cycle were cultured in vitro in the presence of adiponectin (1, 10 μg/ml), adiponectin with insulin (10 ng/ml) and insulin alone (10 ng/ml). Gene expression was examined by real-time PCR, and the secretion of the steroids was determined by radioimmunoassay. The content of StAR, CYP11A1 and HSD3B1 mRNAs and the secretion of P4 and A4 was modulated by adiponectin in endometrial and myometrial tissue explants during early pregnancy and the oestrous cycle. In this action adiponectin interacted with insulin. Insulin itself also regulated the steroidogenic activity of the porcine uterus. ere we reported, for the first time, the expression of CYP11A1 genes in the porcine endometrium and myometrium. Our novel findings indicate that adiponectin affects basal and insulin-stimulated expression of key steroidogenic genes and production of steroid hormones by the porcine uterus during maternal recognition of pregnancy and implantation. PMID:27512005

  12. Degradation properties of protein and carbohydrate during sludge anaerobic digestion.

    PubMed

    Yang, Guang; Zhang, Panyue; Zhang, Guangming; Wang, Yuanyuan; Yang, Anqi

    2015-09-01

    Degradation of protein and carbohydrate is vital for sludge anaerobic digestion performance. However, few studies focused on degradation properties of protein and carbohydrate. This study investigated detailed degradation properties of sludge protein and carbohydrate in order to gain insight into organics removal during anaerobic digestion. Results showed that carbohydrate was more efficiently degraded than protein and was degraded prior to protein. The final removal efficiencies of carbohydrate and protein were 49.7% and 32.2%, respectively. The first 3 days were a lag phase for protein degradation since rapid carbohydrate degradation in this phase led to repression of protease formation. Kinetics results showed that, after initial lag phase, protein degradation followed the first-order kinetic with rate constants of 0.0197 and 0.0018 d(-1) during later rapid degradation phase and slow degradation phase, respectively. Carbohydrate degradation also followed the first-order kinetics with a rate constant of 0.007 d(-1) after initial quick degradation phase. PMID:26025350

  13. Tracing the movement of adiponectin in a parabiosis model of wild-type and adiponectin-knockout mice

    PubMed Central

    Nakatsuji, Hideaki; Kishida, Ken; Sekimoto, Ryohei; Funahashi, Tohru; Shimomura, Iichiro

    2014-01-01

    Adiponectin is exclusively synthesized by adipocytes and exhibits anti-diabetic, anti-atherosclerotic and anti-inflammatory properties. Hypoadiponectinemia is associated in obese individuals with insulin resistance and atherosclerosis. However, the mechanisms responsible for hypoadiponectinemia remain unclear. Here, we investigated adiponectin movement using hetero parabiosis model of wild type (WT) and adiponectin-deficient (KO) mice. WT mice were parabiosed with WT mice (WT–WT) or KO mice (WT–KO) and adiponectin levels were measured serially up to 63 days after surgery. In the WT–KO parabiosis model, circulating adiponectin levels of the WT partners decreased rapidly, on the other hand, those of KO partners increased, and then these reached comparable levels each other at day 7. Circulating adiponectin levels decreased further to the detection limit of assay, and remained low up to day 63. However, adiponectin protein was detected in the adipose tissues of not only the WT partner but also WT–KO mice. In the diet-induced obesity model, high adiponectin protein levels were detected in adipose stromal vascular fraction of diet-induced obese KO partner, without changes in its binding proteins. The use of parabiosis experiments shed light on movement of native adiponectin among different tissues such as the state of hypoadiponectinemia in obesity. PMID:24918039

  14. Urinary Adiponectin Excretion

    PubMed Central

    von Eynatten, Maximilian; Liu, Dan; Hock, Cornelia; Oikonomou, Dimitrios; Baumann, Marcus; Allolio, Bruno; Korosoglou, Grigorios; Morcos, Michael; Campean, Valentina; Amann, Kerstin; Lutz, Jens; Heemann, Uwe; Nawroth, Peter P.; Bierhaus, Angelika; Humpert, Per M.

    2009-01-01

    OBJECTIVE Markers reliably identifying vascular damage and risk in diabetic patients are rare, and reports on associations of serum adiponectin with macrovascular disease have been inconsistent. In contrast to existing data on serum adiponectin, this study assesses whether urinary adiponectin excretion might represent a more consistent vascular damage marker in type 2 diabetes. RESEARCH DESIGN AND METHODS Adiponectin distribution in human kidney biopsies was assessed by immunohistochemistry, and urinary adiponectin isoforms were characterized by Western blot analysis. Total urinary adiponectin excretion rate was measured in 156 patients with type 2 diabetes who had a history of diabetic nephropathy and 40 healthy control subjects using enzyme-linked immunosorbent assay. Atherosclerotic burden was assessed by common carotid artery intima-media-thickness (IMT). RESULTS A homogenous staining of adiponectin was found on the endothelial surface of glomerular capillaries and intrarenal arterioles in nondiabetic kidneys, whereas staining was decreased in diabetic nephropathy. Low-molecular adiponectin isoforms (∼30–70 kDa) were detected in urine by Western blot analysis. Urinary adiponectin was significantly increased in type 2 diabetes (7.68 ± 14.26 vs. control subjects: 2.91 ± 3.85 μg/g creatinine, P = 0.008). Among type 2 diabetic patients, adiponectinuria was associated with IMT (r = 0.479, P < 0.001) and proved to be a powerful independent predictor of IMT (β = 0.360, P < 0.001) in multivariable regression analyses. In a risk prediction model including variables of the UK Prospective Diabetes Study coronary heart disease risk engine urinary adiponectin, but not the albumin excretion rate, added significant value for the prediction of increased IMT (P = 0.007). CONCLUSIONS Quantification of urinary adiponectin excretion appears to be an independent indicator of vascular damage potentially identifying an increased risk for vascular events. PMID:19509019

  15. Protein Degradation and the Stress Response

    PubMed Central

    Flick, Karin; Kaiser, Peter

    2012-01-01

    Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the Ubiquitin Proteasome System (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations. PMID:22414377

  16. Adiponectin--its role in metabolism and beyond.

    PubMed

    Stefan, N; Stumvoll, M

    2002-09-01

    Adiponectin is a recently identified adipose tissue-derived protein (adipocytokine) with important metabolic effects. It is exclusively expressed in adipose tissue and released into the circulation. Adiponectin expression and/or secretion is increased by insulin like growth factor-1 and ionomycin, and decreased by tumor necrosis factor-alpha, glucocorticoids, beta-adrenergic agonists and cAMP. Data for insulin are somewhat inconclusive. Moreover, adiponectin expression and secretion are increased by activators of peroxisome proliferator-activated receptor (PPAR)-gamma. Besides inhibiting inflammatory pathways, recombinant adiponectin increases insulin sensitivity and improves glucose tolerance in various animal models. This insulin-sensitizing effect appears to be mostly attributable to enhanced suppression of glucose production, but beneficial effects on muscle cannot be excluded. In humans, plasma adiponectin concentrations exceed those of any other hormone by a thousand times; they decrease with obesity and are positively associated with whole-body insulin sensitivity. Therefore, low adiponectin may contribute to the decrease in whole-body insulin sensitivity that accompanies obesity. Furthermore, there is increasing evidence that genetic variants in the adiponectin gene itself and/or in genes encoding adiponectin-regulatory proteins--such as PPAR-gamma--may be associated with hypoadiponectinemia, insulin resistance and type 2 diabetes. This suggests that adiponectin may reflect PPAR-gamma activity in vivo. Finally, reversal or alleviation of hypoadiponectinemia may represent a target for development of drugs improving insulin sensitivity and glucose tolerance. PMID:12384822

  17. Vascular smooth muscle cell-derived adiponectin: a paracrine regulator of contractile phenotype.

    PubMed

    Ding, Min; Carrão, Ana Catarina; Wagner, Robert J; Xie, Yi; Jin, Yu; Rzucidlo, Eva M; Yu, Jun; Li, Wei; Tellides, George; Hwa, John; Aprahamian, Tamar R; Martin, Kathleen A

    2012-02-01

    Adiponectin is a cardioprotective adipokine derived predominantly from visceral fat. We recently demonstrated that exogenous adiponectin induces vascular smooth muscle cell (VSMC) differentiation via repression of mTORC1 and FoxO4. Here we report for the first time that VSMC express and secrete adiponectin, which acts in an autocrine and paracrine manner to regulate VSMC contractile phenotype. Adiponectin was found to be expressed in human coronary artery and mouse aortic VSMC. Importantly, siRNA knock-down of endogenous adiponectin in VSMC significantly reduced the expression of VSMC contractile proteins. Contractile protein deficiency was also observed in primary VSMC isolated from Adiponectin(-/-) mice. This deficiency could be rescued by culturing Adiponectin(-/-) VSMC in conditioned media from wild type (WT) VSMC. Moreover, the paracrine effect of VSMC-derived adiponectin was confirmed as adiponectin neutralizing antibody blocked the rescue. Overexpressed adiponectin also exerted paracrine effects on neighboring untransfected VSMC, which was also blocked by adiponectin neutralizing antibody. Interestingly, adiponectin expression was inducible by the PPARγ agonist rosiglitazone. Our data support an important role for VSMC-derived adiponectin in maintaining VSMC contractile phenotype, contributing to critical cardioprotective functions in the vascular wall. This article is part of a Special Issue entitled "Local Signaling in Myocytes". PMID:21952104

  18. Postoperative Adiponectin Levels in Pediatric Patients Undergoing Open Heart Surgery

    PubMed Central

    Thaler, A.; Kanety, H.; Avni, T.; Mishali, D.; Hemi, R.; Yissaschar, E.; Pariente, C.; Paret, G.; Modan-Moses, D.

    2013-01-01

    Background. Adipose tissue is an important endocrine organ that secretes cytokines, including adiponectin, levels of which are negatively correlated with the severity of the inflammatory process. Aim. To assess the time course of adiponectin levels following open heart surgery with cardiopulmonary bypass and its correlation with early postoperative outcomes. Materials and Methods. Blood samples were obtained from 24 children undergoing cardiac surgery and analyzed for adiponectin, C-reactive protein, and other inflammatory markers. Results. Baseline adiponectin levels were negatively correlated with patients' preoperative weight and age. Postoperative adiponectin levels decreased compared to baseline (P = 0.01) and correlated negatively with duration of cardiopulmonary bypass (r = −0.438, P = 0.037), length of stay in the pediatric intensive care unit (r = −0.457, P = 0.025), and the inotropic score (r = −0.471, P = 0.02). Adiponectin levels were positively correlated with sVCAM 1 levels; however, there was no correlation between adiponectin levels and sP selectin, tPA, MCP1, and sCD40. Conclusions. The inflammatory response after open heart surgery with cardiopulmonary bypass is associated with a reduction in adiponectin levels. Prolonged or more complicated surgery induced a more substantial inflammatory process characterized by a significant reduction in adiponectin levels over time and a delayed return to baseline levels. PMID:24224162

  19. Degradation of SsrA-tagged proteins in streptococci.

    PubMed

    Tao, Liang; Biswas, Indranil

    2015-04-01

    In prokaryotes, a conserved small RNA molecule, called tmRNA, rescues ribosomes from proteins that are abnormally truncated due to the presence of rare codons or degraded mRNA. During the rescue process, a peptide tag (SsrA) encoded by tmRNA is cotranslationally added to the truncated polypeptides, thereby targeting these proteins for proteolytic degradation. In Escherichia coli, ClpXP and ClpAP proteases primarily degrade SsrA-tagged proteins. Other proteases such as Lon and FtsH also participate in the degradation in E. coli. However, in Bacillus subtilis, ClpXP is the major protease that degrades the SsrA-tagged proteins. Degradation of SsrA-tagged protein in streptococci is not well understood except that ClpXP is responsible for the majority of the degradation. Here we show that in Streptococcus mutans, in addition to ClpXP, two other Clp complexes, ClpCP and ClpEP, are also involved in the degradation. We also found that ClpCP- and ClpEP-mediated proteolysis of SsrA-tagged substrates is induced by heat stress. As ClpCP and ClpEP proteins are highly conserved in streptococci, we predicted that ClpEP- and ClpCP-mediated degradation of SsrA-tagged proteins might be operational in other streptococci. PMID:25645948

  20. Degradation of SsrA-tagged proteins in streptococci

    PubMed Central

    Tao, Liang

    2015-01-01

    In prokaryotes, a conserved small RNA molecule, called tmRNA, rescues ribosomes from proteins that are abnormally truncated due to the presence of rare codons or degraded mRNA. During the rescue process, a peptide tag (SsrA) encoded by tmRNA is cotranslationally added to the truncated polypeptides, thereby targeting these proteins for proteolytic degradation. In Escherichia coli, ClpXP and ClpAP proteases primarily degrade SsrA-tagged proteins. Other proteases such as Lon and FtsH also participate in the degradation in E. coli. However, in Bacillus subtilis, ClpXP is the major protease that degrades the SsrA-tagged proteins. Degradation of SsrA-tagged protein in streptococci is not well understood except that ClpXP is responsible for the majority of the degradation. Here we show that in Streptococcus mutans, in addition to ClpXP, two other Clp complexes, ClpCP and ClpEP, are also involved in the degradation. We also found that ClpCP- and ClpEP-mediated proteolysis of SsrA-tagged substrates is induced by heat stress. As ClpCP and ClpEP proteins are highly conserved in streptococci, we predicted that ClpEP- and ClpCP-mediated degradation of SsrA-tagged proteins might be operational in other streptococci. PMID:25645948

  1. Delivery Mode, Duration of Labor, and Cord Blood Adiponectin, Leptin, and C-Reactive Protein: Results of the Population-Based Ulm Birth Cohort Studies

    PubMed Central

    Logan, Chad A.; Thiel, Larissa; Bornemann, Rebecca; Koenig, Wolfgang; Reister, Frank; Brenner, Hermann; Rothenbacher, Dietrich; Genuneit, Jon

    2016-01-01

    Background Numerous studies have reported associations between delivery mode and health outcomes in infancy and later life. Previous smaller studies indicated a relationship between delivery mode and newborn inflammation potentially constituting a mediating factor. We aimed to determine the influence of delivery mode and duration of labor on cord blood concentrations of adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP). Methods In the Ulm SPATZ Health Study, 934 singleton newborns and their mothers were recruited during their hospital stay in the University Medical Center Ulm, Southern Germany, from 04/2012-05/2013. Inflammatory biomarkers were measured by ELISAs (n = 836). Delivery mode was analyzed categorically (elective cesarean (reference), active labor delivery: emergency cesarean, assisted vaginal, and spontaneous vaginal); duration of labor continuously. Following log-transformation, linear regression was used to estimate geometric means ratios (GMR) adjusted for potential confounders for the effects of delivery mode and duration of labor on each biomarker separately. Independent replication was sought in the similarly conducted Ulm Birth Cohort Study recruited from 11/2000-11/2001. Results Individually, active labor delivery modes as well as increasing duration of labor were associated with higher leptin and hs-CRP concentrations. After mutual adjustment, the associations with delivery modes were attenuated but those for duration of labor remained statistically significant (GMR (95%CI) 1.10 (1.00; 1.21) and 1.15 (1.04; 1.27) for leptin and hs-CRP per hour of labor, respectively). No significant adjusted associations were observed between delivery modes and adiponectin concentrations. These findings were replicated in an independent birth cohort study. Conclusions Cord blood leptin and hs-CRP concentrations were associated with duration of labor rather than delivery mode. Further research is warranted to investigate these associations

  2. Curcumin inhibits HIV-1 by promoting Tat protein degradation.

    PubMed

    Ali, Amjad; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  3. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    PubMed Central

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  4. Review: adiponectin in retinopathy.

    PubMed

    Fu, Zhongjie; Gong, Yan; Löfqvist, Chatarina; Hellström, Ann; Smith, Lois E H

    2016-08-01

    Neovascular eye diseases are a major cause of blindness including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration in which new vessel formation is driven by hypoxia or metabolic abnormalities affecting the fuel supply. White-adipose-tissue derived adipokines such as adiponectin modulate metabolic responses. Increasing evidence shows that lack of adiponectin may result in retinal neovascularization. Activation of the adiponectin pathway may in turn restore energy metabolism, to suppress the drive for compensatory but ultimately pathological neovessels of retinopathy. In this review, we will summarize our current knowledge of the role of adiponectin in eye diseases of premature infants, diabetic patients as well as the elderly. Further investigations in this field are likely to lead to new preventative approaches for these diseases. PMID:27155572

  5. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  6. Clipping or Extracting: Two Ways to Membrane Protein Degradation.

    PubMed

    Avci, Dönem; Lemberg, Marius K

    2015-10-01

    Protein degradation is a fundamentally important process that allows cells to recognize and remove damaged protein species and to regulate protein abundance according to functional need. A fundamental challenge is to understand how membrane proteins are recognized and removed from cellular organelles. While most of our understanding of this mechanism comes from studies on p97/Cdc48-mediated protein dislocation along the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, recent studies have revealed intramembrane proteolysis to be an additional mechanism that can extract transmembrane segments. Here, we review these two principles in membrane protein degradation and discuss how intramembrane proteolysis, which introduces an irreversible step in protein dislocation, is used to drive regulated protein turnover. PMID:26410407

  7. Underactivation of the adiponectin-adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression.

    PubMed

    Kleinmann, Nir; Duivenvoorden, Wilhelmina C M; Hopmans, Sarah N; Beatty, Laura K; Qiao, Shengjun; Gallino, Daniel; Lhotak, Sarka; Daya, Dean; Paschos, Athanasios; Austin, Richard C; Pinthus, Jehonathan H

    2014-02-01

    Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC. PMID:24096711

  8. Protein degradation in bovine milk caused by Streptococcus agalactiae.

    PubMed

    Åkerstedt, Maria; Wredle, Ewa; Lam, Vo; Johansson, Monika

    2012-08-01

    Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and β-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk. PMID:22850579

  9. Adiponectin gene polymorphisms: Association with childhood obesity

    PubMed Central

    Fraga, Vanêssa Gomes; Gomes, Karina Braga

    2014-01-01

    The current childhood obesity epidemic represents a particular challenge for public health. Understanding of the etiological mechanisms of obesity remains integral in treating this complex disorder. In recent years, studies have elucidated the influence of hormones secreted by adipose tissue named adipokines. Adiponectin is a adipokine that exhibits important anti-inflammatory, insulin-sensitizing and anti-atherogenic properties and it is strongly associated to obesity development. It is well known that adiponectin levels decrease with obesity. Furthermore, studies show that some single nucleotide polymorphisms in the gene encoding adiponectin, ADIPOQ, may influence the expression of this protein. The objective of this paper is to provide an up-to-date review of ADIPOQ polymorphisms in the context of childhood obesity.

  10. POLYPHENOL AND CONDITIONING EFFECTS ON FORAGE PROTEIN SOLUBILITY AND DEGRADABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing the degree of tissue disruption during mechanical harvesting of forages may augment protein interactions with polyphenols and other cellular constituents, enhancing protein utilization by reducing protein solubility and shifting its degradation from the rumen to the intestine. In 2002 and...

  11. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.

    2013-01-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

  12. mTORC1 activity as a determinant of cancer risk--rationalizing the cancer-preventive effects of adiponectin, metformin, rapamycin, and low-protein vegan diets.

    PubMed

    McCarty, Mark F

    2011-10-01

    Increased plasma levels of adiponectin, metformin therapy of diabetes, rapamycin administration in transplant patients, and lifelong consumption of low-protein plant-based diets have all been linked to decreased risk for various cancers. These benefits may be mediated, at least in part, by down-regulated activity of the mTORC1 complex, a key regulator of protein translation. By boosting the effective availability of the translation initiator eIF4E, mTORC1 activity promotes the translation of a number of "weak" mRNAs that code for proteins, often up-regulated in cancer, that promote cellular proliferation, invasiveness, and angiogenesis, and that abet cancer promotion and chemoresistance by opposing apoptosis. Measures which inhibit eIF4E activity, either directly or indirectly, may have utility not only for cancer prevention, but also for the treatment of many cancers in which eIF4E drives malignancy. Since eIF4E is overexpressed in many cancers, strategies which target eIF4E directly--some of which are now being assessed clinically--may have the broadest efficacy in this regard. Many of the "weak" mRNAs coding for proteins that promote malignant behavior or chemoresistance are regulated transcriptionally by NF-kappaB and/or Stat3, which are active in a high proportion of cancers; thus, regimens concurrently targeting eIF4E, NF-kappaB, and Stat3 may suppress these proteins at both the transcriptional and translational levels, potentially achieving a very marked reduction in their expression. PMID:21862237

  13. Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

    PubMed

    Buchanan, Bryce W; Lloyd, Michael E; Engle, Sarah M; Rubenstein, Eric M

    2016-01-01

    Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation. PMID:27167179

  14. Small-molecule control of protein degradation using split adaptors.

    PubMed

    Davis, Joseph H; Baker, Tania A; Sauer, Robert T

    2011-11-18

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  15. Small-molecule control of protein degradation using split adaptors

    PubMed Central

    Davis, Joseph H.; Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly-defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  16. Modulation of ovarian steroidogenesis by adiponectin during delayed embryonic development of Cynopterus sphinx.

    PubMed

    Anuradha; Krishna, Amitabh

    2014-09-01

    The aim of present study was to evaluate role of adiponectin in ovarian steroidogenesis during delayed embryonic development of Cynopterus sphinx. This study showed significantly low circulating adiponectin level and a decline in expression of adiponectin receptor 1 (AdipoR1) in the ovary during the period of delayed embryonic development as compared with the normal development. The adiponectin treatment in vivo during the period of delayed development caused significantly increased in circulating progesterone and estradiol levels together with increased expression of AdipoR1 in the ovary. The in vitro study confirmed the stimulatory effect of adiponectin on progesterone synthesis. Both in vivo and in vitro studies showed that the effects of adiponectin on ovarian steroidogenesis were mediated through increased expression of luteinizing hormone-receptor, steroidogenic acute regulatory protein and 3β-hydroxyl steroid dehydrogenase enzyme. The adiponectin treatment may also promote progesterone synthesis by modulating ovarian angiogenesis, cell survival and rate of apoptosis. PMID:24787661

  17. Protein-Linked Glycan Degradation in Infants Fed Human Milk

    PubMed Central

    Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

    2014-01-01

    Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

  18. Hydrolytically degradable thiol–ene hydrogels for protein release

    PubMed Central

    Rehmann, Matthew S.; Garibian, Andrew C.; Kloxin, April M.

    2014-01-01

    Summary A new degradable PEG-diester-dinorbornene/PEG-triester-trithiol hydrogel was evaluated for protein release. The hydrogel polymerized rapidly with seconds of UV irradiation and subsequently hydrolytically degraded in aqueous buffer over the course of approximately 3 weeks. Further, the hydrogel enabled the encapsulation and release of a model protein, bovine serum albumin (BSA), over 7 days with ~ 90% released at 48 h. This study serves as a proof-of-concept for the creation of hydrolytically degradable, PEG-ester-thiol-based hydrogels by a photoinitiated step growth mechanism for protein release. With this approach, degradation and release rates could be tuned by varying the monomer molecular weight and functionality in future studies. PMID:25309103

  19. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  20. The Cdc48 machine in endoplasmic reticulum associated protein degradation.

    PubMed

    Wolf, Dieter H; Stolz, Alexandra

    2012-01-01

    The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered process are subject of this review article. PMID:21945179

  1. Influence of androgens on circulating adiponectin in male and female rodents.

    PubMed

    Yarrow, Joshua F; Beggs, Luke A; Conover, Christine F; McCoy, Sean C; Beck, Darren T; Borst, Stephen E

    2012-01-01

    Several endocrine factors, including sex-steroid hormones are known to influence adiponectin secretion. Our purpose was to evaluate the influence of testosterone and of the synthetic non-aromatizable/non-5α reducible androgen 17β-hydroxyestra-4,9,11-trien-3-one (trenbolone) on circulating adiponectin and adiponectin protein expression within visceral fat. Young male and female F344 rats underwent sham surgery (SHAM), gonadectomy (GX), or GX plus supraphysiologic testosterone-enanthate (TE) administration. Total circulating adiponectin was 39% higher in intact SHAM females than SHAM males (p<0.05). GX increased total adiponectin by 29-34% in both sexes (p<0.05), while TE reduced adiponectin to concentrations that were 46-53% below respective SHAMs (p≤0.001) and ablated the difference in adiponectin between sexes. No differences in high molecular weight (HMW) adiponectin were observed between sexes or treatments. Adiponectin concentrations were highly and negatively associated with serum testosterone (males: r = -0.746 and females: r = -0.742, p≤0.001); however, no association was present between adiponectin and estradiol. In separate experiments, trenbolone-enanthate (TREN) prevented the GX-induced increase in serum adiponectin (p≤0.001) in young animals, with Low-dose TREN restoring adiponectin to the level of SHAMs and higher doses of TREN reducing adiponectin to below SHAM concentrations (p≤0.001). Similarly, TREN reduced adiponectin protein expression within visceral fat (p<0.05). In adult GX males, Low-dose TREN also reduced total adiponectin and visceral fat mass to a similar magnitude as TE, while increasing serum HMW adiponectin above SHAM and GX animals (p<0.05). Serum adiponectin was positively associated with visceral fat mass in young (r = 0.596, p≤0.001) and adult animals (r = 0.657, p≤0.001). Our results indicate that androgens reduce circulating total adiponectin concentrations in a dose-dependent manner, while maintaining HMW

  2. Protein Quality Control and Degradation in Cardiomyocytes

    PubMed Central

    Wang, Xuejun; Su, Huabo; Ranek, Mark J.

    2008-01-01

    The heart is constantly under stress and cardiomyocytes face enormous challenges to correctly fold nascent polypeptides and keep mature proteins from denaturing. To meet the challenge, cardiomyocytes have developed multi-layered protein quality control (PQC) mechanisms which are carried out primarily by chaperones and ubiquitin-proteasome system mediated proteolysis. Autophagy may also participate in PQC in cardiomyocytes, especially under pathological conditions. Cardiac PQC often becomes inadequate in heart disease, which may play an important role in the development of congestive heart failure. PMID:18495153

  3. Adiponectin, driver or passenger on the road to insulin sensitivity?

    PubMed Central

    Ye, Risheng; Scherer, Philipp E.

    2013-01-01

    Almost 20 years have passed since the first laboratory evidence emerged that an abundant message encoding a protein with homology to the C1q superfamily is highly specifically expressed in adipocytes. At this stage, we refer to this protein as adiponectin. Despite more than 10,000 reports in the literature since its initial description, we seem to have written only the first chapter in the textbook on adiponectin physiology. With every new aspect we learn about adiponectin, a host of new questions arise with respect to the underlying molecular mechanisms. Here, we aim to summarize recent findings in the field and bring the rodent studies that suggest a causal relationship between adiponectin levels in plasma and systemic insulin sensitivity in perspective with the currently available data on the clinical side. PMID:24049728

  4. Adiponectin and the control of female reproductive functions.

    PubMed

    Palin, Marie-France; Bordignon, V Vilceu; Murphy, Bruce D

    2012-01-01

    Adiponectin is the most abundant protein secreted by the white adipose tissue. It circulates at high levels in the bloodstream and its serum concentration is inversely correlated with body fat mass. The wide distribution of adiponectin receptors (AdipoR1, AdipoR2, and T-cadherin) in peripheral tissues and organs allows adiponectin to exert pleiotropic effects on whole-body metabolism. Besides its well-known antidiabetic, antiatherogenic, and anti-inflammatory properties, accumulating evidence suggests a direct role for adiponectin in reproductive tissues. The mammalian ovary and the ovarian follicle express AdipoR1 and AdipoR2, and treating pig granulosa cells with adiponectin induces changes characteristic of the periovulatory period. Moreover, additive effects are observed between adiponectin and insulin in induction of granulosa cell gene expression, thus suggesting that adiponectin actions on the ovary may be mediated through its insulin-sensitizing effects. Adiponectin receptors are also detected in the uterus. In women, higher AdipoR1 and AdipoR2 gene expression was observed during the mid-secretory phase of the menstrual cycle, suggesting that adiponectin is implicated in the endometrial changes in preparation for embryo implantation. Adiponectin receptors are found in oocytes and early developing pig, rabbit, and mice embryos, and it has been demonstrated that adiponectin can increase the success of porcine embryo development to the blastocyst stage in vitro. Moreover, adiponectin concentration is two to three times greater in human fetal circulation and in umbilical cord blood, compared to adult plasma. This further indicates a role for adiponectin in fetal growth. It has been further suggested that adipose-derived and locally produced adiponectin may act as a key neuromodulator of reproductive functions. For example, the inhibition of LH and GnRH release from rat pituitary and hypothalamic cells following treatment with adiponectin provides evidence that

  5. Regulation of protein degradation in muscle by calcium

    NASA Technical Reports Server (NTRS)

    Zeman, Richard J.; Kameyama, Tsuneo; Matsumoto, Kazue; Bernstein, Paul; Etlinger, Joseph D.

    1985-01-01

    Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. The effect of different classes of protease inhibitors was tested to determine the responsible proteolytic systems involved in calcium-dependent degradation. The results suggest that nonlysosomal leupetin- and E-64-c-sensitive proteases are resposible for calcium-dependent proteolysis in muscle.

  6. Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion*

    PubMed Central

    Ramezani-Moghadam, Mehdi; Wang, Jianhua; Ho, Vikki; Iseli, Tristan J.; Alzahrani, Badr; Xu, Aimin; Van der Poorten, David; Qiao, Liang; George, Jacob; Hebbard, Lionel

    2015-01-01

    Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin. PMID:25575598

  7. Prion protein degradation by lichens of the genus Cladonia

    USGS Publications Warehouse

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

    2012-01-01

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

  8. Adiponectin downregulation is associated with volume overload-induced myocyte dysfunction in rats

    PubMed Central

    Wang, Li-li; Miller, Dori; Wanders, Desiree; Nanayakkara, Gayani; Amin, Rajesh; Judd, Robert; Morrison, Edward E; Zhong, Ju-ming

    2016-01-01

    Aim: Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats. Methods: Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca2+ transient. Results: A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca2+ transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 μg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats. Conclusion: These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure. PMID:26616727

  9. Adiponectin in mice with altered GH action: links to insulin sensitivity and longevity?

    PubMed

    Lubbers, Ellen R; List, Edward O; Jara, Adam; Sackman-Sala, Lucila; Cordoba-Chacon, Jose; Gahete, Manuel D; Kineman, Rhonda D; Boparai, Ravneet; Bartke, Andrzej; Kopchick, John J; Berryman, Darlene E

    2013-03-01

    Adiponectin is positively correlated with longevity and negatively correlated with many obesity-related diseases. While there are several circulating forms of adiponectin, the high-molecular-weight (HMW) version has been suggested to have the predominant bioactivity. Adiponectin gene expression and cognate serum protein levels are of particular interest in mice with altered GH signaling as these mice exhibit extremes in obesity that are positively associated with insulin sensitivity and lifespan as opposed to the typical negative association of these factors. While a few studies have reported total adiponectin levels in young adult mice with altered GH signaling, much remains unresolved, including changes in adiponectin levels with advancing age, proportion of total adiponectin in the HMW form, adipose depot of origin, and differential effects of GH vs IGF1. Therefore, the purpose of this study was to address these issues using assorted mouse lines with altered GH signaling. Our results show that adiponectin is generally negatively associated with GH activity, regardless of age. Further, the amount of HMW adiponectin is consistently linked with the level of total adiponectin and not necessarily with previously reported lifespan or insulin sensitivity of these mice. Interestingly, circulating adiponectin levels correlated strongly with inguinal fat mass, implying that the effects of GH on adiponectin are depot specific. Interestingly, rbGH, but not IGF1, decreased circulating total and HMW adiponectin levels. Taken together, these results fill important gaps in the literature related to GH and adiponectin and question the frequently reported associations of total and HMW adiponectin with insulin sensitivity and longevity. PMID:23261955

  10. C1q/Tumor Necrosis Factor-Related Protein 9 Protects against Acute Myocardial Injury through an Adiponectin Receptor I-AMPK-Dependent Mechanism.

    PubMed

    Kambara, Takahiro; Shibata, Rei; Ohashi, Koji; Matsuo, Kazuhiro; Hiramatsu-Ito, Mizuho; Enomoto, Takashi; Yuasa, Daisuke; Ito, Masanori; Hayakawa, Satoko; Ogawa, Hayato; Aprahamian, Tamar; Walsh, Kenneth; Murohara, Toyoaki; Ouchi, Noriyuki

    2015-06-01

    Obesity is a risk factor for cardiovascular disease. C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine that is downregulated by obesity. We investigated the role of CTRP9 in cardiac injury with loss-of-function genetic manipulations and defined the receptor-mediated signaling pathway downstream of this adipokine. CTRP9-knockout (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from wild-type (WT) mice under basal conditions. CTRP9-KO mice had exacerbated contractile left ventricle dysfunction following intraperitoneal injection of lipopolysaccharide (LPS) compared to WT mice. Administration of LPS to CTRP9-KO mice also resulted in increased expression of proinflammatory cytokines and oxidative stress markers in the heart compared to WT mice. Likewise, CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory mediators in ischemic heart following ischemia and reperfusion compared to WT mice. Treatment of cardiac myocytes with CTRP9 protein led to suppression of LPS-induced expression of proinflammatory genes, which was reversed by blockade of AMPK or ablation of adiponectin receptor I (AdipoR1). Systemic delivery of CTRP9 attenuated LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK or in AdipoR1-knockout mice. CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AdipoR1/AMPK-dependent mechanisms. PMID:25870106

  11. Degradation of Akt Using Protein Catalyzed Capture Agents

    PubMed Central

    Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M.; Heath, James R.

    2016-01-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. PMID:26880702

  12. Adiponectin: an adipokine with protective features against metabolic syndrome

    PubMed Central

    Esfahani, Maryam; Movahedian, Ahmad; Baranchi, Mostafa; Goodarzi, Mohammad Taghi

    2015-01-01

    Metabolic syndrome (MetS) as a collection of obesity-associated disorders is associated with inflammation, oxidative stress, pro-thrombotic state, elevated risk of developing cardiovascular disease and type 2 diabetes. Adiponectin is one of the most abundant peptide hormones derived from adipose tissue. This protein plays a major role in glucose and lipid metabolism and prevents development of vascular changes. Anti-oxidative and anti-inflammatory effects are the other features of adiponectin. Hypoadiponectinemia is associated with hypertension and pro-thrombotic state. In this review, we discuss the crucial role of adiponectin in prevention of metabolic syndrome considering its effects on the components of this syndrome. Pharmacological interventions and lifestyle modification may increase plasma adiponectin level or tissue sensitivity which seems to be a promising target for prevention and therapeutic approaches of MetS and related diseases. PMID:26124928

  13. Protein unfolding and degradation by the AAA+ Lon protease.

    PubMed

    Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T

    2012-02-01

    AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases. PMID:22162032

  14. Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial

    PubMed Central

    Sriwijitkamol, A.; Wajcberg, E.; Tantiwong, P.; Li, M.; Prentki, M.; Madiraju, M.; Jenkinson, C. P.; Cersosimo, E.; Musi, N.; DeFronzo, R. A.

    2016-01-01

    Aims/hypothesis The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle. Methods A randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n=14) or (2) aggressive nutritional therapy (n=12) to reduce HbA1c to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic–hyperinsulinaemic (80 mU m−2 min−1) clamp. Results All patients in the pioglitazone (n=14) or nutritional therapy (n=12) group were included in the analysis. Pioglitazone significantly increased plasma adiponectin concentration by 79% and reduced fasting plasma NEFA by 35% (both p<0.01). Following pioglitazone, insulin-stimulated glucose disposal increased by 30% (p<0.01), and muscle AMPK and acetyl-CoA carboxylase (ACC) phosphorylation increased by 38% and 53%, respectively (p<0.05). Pioglitazone increased mRNA levels for adiponectin receptor 1 and 2 genes (ADIPOR1, ADIPOR2), peroxisome proliferator-activated receptor gamma, coactivator 1 gene (PPARGC1) and multiple genes involved in mitochondrial function and fat oxidation. Despite a similar reduction in HbA1c and similar improvement in insulin sensitivity with nutritional therapy, there were no significant changes in muscle AMPK and ACC phosphorylation, or the expression of ADIPOR1, ADIPOR2, PPARGC1 and genes involved in mitochondrial function and fat oxidation. No adverse (or unexpected) effects

  15. Role of adiponectin in delayed embryonic development of the short-nosed fruit bat, Cynopterus sphinx.

    PubMed

    Anuradha; Krishna, Amitabh

    2014-12-01

    The aim of this study was to evaluate the role of adiponectin in the delayed embryonic development of Cynopterus sphinx. Adiponectin receptor (ADIPOR1) abundance was first observed to be lower during the delayed versus non-delayed periods of utero-embryonic unit development. The effects of adiponectin treatment on embryonic development were then evaluated during the period of delayed development. Exogenous treatment increased the in vivo rate of embryonic development, as indicated by an increase in weight, ADIPOR1 levels in the utero-embryonic unit, and histological changes in embryonic development. Treatment with adiponectin during embryonic diapause showed a significant increase in circulating progesterone and estradiol concentrations, and in production of their receptors in the utero-embryonic unit. The adiponectin-induced increase in estradiol synthesis was correlated with increased cell survival (BCL2 protein levels) and cell proliferation (PCNA protein levels) in the utero-embryonic unit, suggesting an indirect effect of adiponectin via estradiol synthesis by the ovary. An in vitro study further confirmed the in vivo findings that adiponectin treatment increases PCNA levels together with increased uptake of glucose by increasing the abundance of glucose transporter 8 (GLUT8) in the utero-embryonic unit. The in vitro study also revealed that adiponectin, together with estradiol but not alone, significantly increased ADIPOR1 protein levels. Thus, adiponectin works in concert with estradiol to increase glucose transport to the utero-embryonic unit and promote cell proliferation, which together accelerate embryonic development. PMID:25295970

  16. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  17. Temperature compensation via cooperative stability in protein degradation

    NASA Astrophysics Data System (ADS)

    Peng, Yuanyuan; Hasegawa, Yoshihiko; Noman, Nasimul; Iba, Hitoshi

    2015-08-01

    Temperature compensation is a notable property of circadian oscillators that indicates the insensitivity of the oscillator system's period to temperature changes; the underlying mechanism, however, is still unclear. We investigated the influence of protein dimerization and cooperative stability in protein degradation on the temperature compensation ability of two oscillators. Here, cooperative stability means that high-order oligomers are more stable than their monomeric counterparts. The period of an oscillator is affected by the parameters of the dynamic system, which in turn are influenced by temperature. We adopted the Repressilator and the Atkinson oscillator to analyze the temperature sensitivity of their periods. Phase sensitivity analysis was employed to evaluate the period variations of different models induced by perturbations to the parameters. Furthermore, we used experimental data provided by other studies to determine the reasonable range of parameter temperature sensitivity. We then applied the linear programming method to the oscillatory systems to analyze the effects of protein dimerization and cooperative stability on the temperature sensitivity of their periods, which reflects the ability of temperature compensation in circadian rhythms. Our study explains the temperature compensation mechanism for circadian clocks. Compared with the no-dimer mathematical model and linear model for protein degradation, our theoretical results show that the nonlinear protein degradation caused by cooperative stability is more beneficial for realizing temperature compensation of the circadian clock.

  18. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  19. Crystal structures of the human adiponectin receptors.

    PubMed

    Tanabe, Hiroaki; Fujii, Yoshifumi; Okada-Iwabu, Miki; Iwabu, Masato; Nakamura, Yoshihiro; Hosaka, Toshiaki; Motoyama, Kanna; Ikeda, Mariko; Wakiyama, Motoaki; Terada, Takaho; Ohsawa, Noboru; Hato, Masakatsu; Ogasawara, Satoshi; Hino, Tomoya; Murata, Takeshi; Iwata, So; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yamauchi, Toshimasa; Kadowaki, Takashi; Yokoyama, Shigeyuki

    2015-04-16

    Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5' AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes. PMID:25855295

  20. Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice

    PubMed Central

    Correnti, Jason M.; Juskeviciute, Egle; Swarup, Aditi

    2014-01-01

    Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPAR-α) activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver and has been shown to reduce ceramide levels and activate AMPK and PPAR-α. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol-containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum alanine aminotransferase, and reduced liver TNF. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPAR-α DNA binding activity compared with control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting that alcohol-dependent deregulation of AMPK and PPAR-α in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis, and hepatomegaly. These data suggest that adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides, but its absence does not exacerbate alcohol-induced liver damage. PMID

  1. APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

    PubMed

    Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

    2016-09-15

    Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. PMID:27246173

  2. Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

    PubMed

    Du, Juan; Zhang, Junzheng; He, Tao; Li, Yajuan; Su, Ying; Tie, Feng; Liu, Min; Harte, Peter J; Zhu, Alan Jian

    2016-06-20

    Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity. PMID:27326929

  3. α-Synuclein and protein degradation systems: a reciprocal relationship.

    PubMed

    Xilouri, Maria; Brekk, Oystein Rod; Stefanis, Leonidas

    2013-04-01

    An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson's disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP)-mainly macroautophagy and chaperone-mediated autophagy-have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies. PMID:22941029

  4. IGFBP-3, hypoxia and TNF-{alpha} inhibit adiponectin transcription

    SciTech Connect

    Zappala, Giovanna; Rechler, Matthew M.

    2009-05-15

    The thiazolidinedione rosiglitazone, an agonist ligand for the nuclear receptor PPAR-{gamma}, improves insulin sensitivity in part by stimulating transcription of the insulin-sensitizing adipokine adiponectin. It activates PPAR-{gamma}-RXR-{alpha} heterodimers bound to PPAR-{gamma} response elements in the adiponectin promoter. Rosiglitazone-stimulated adiponectin protein synthesis in 3T3-L1 mouse adipocytes has been shown to be inhibited by IGFBP-3, which can be induced by hypoxia and the proinflammatory cytokine, TNF-{alpha}, two inhibitors of adiponectin transcription. The present study demonstrates that IGFBP-3, the hypoxia-mimetic agent cobalt chloride, and TNF-{alpha} inhibit rosiglitazone-induced adiponectin transcription in mouse embryo fibroblasts that stably express PPAR-{gamma}2. Native IGFBP-3 can bind RXR-{alpha} and inhibited rosiglitazone stimulated promoter activity, whereas an IGFBP-3 mutant that does not bind RXR-{alpha} did not. These results suggest that IGFBP-3 may mediate the inhibition of adiponectin transcription by hypoxia and TNF-{alpha}, and that IGFBP-3 binding to RXR-{alpha} may be required for the observed inhibition.

  5. Control of protein function through regulated protein degradation: biotechnological and biomedical applications.

    PubMed

    Nagpal, Jyotsna; Tan, Ju Lin; Truscott, Kaye N; Heras, Begoña; Dougan, David A

    2013-01-01

    Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest. PMID:23920496

  6. Endogenous and exogenous factors influencing the concentrations of adiponectin in body fluids and tissues in the bovine.

    PubMed

    Sauerwein, Helga; Häußler, Susanne

    2016-07-01

    Adiponectin, one of the messenger molecules secreted from adipose tissue that are collectively termed adipokines, has been demonstrated to play a central role in lipid and glucose metabolism in humans and laboratory rodents; it improves insulin sensitivity and exerts antidiabetic and antiinflammatory actions. Adiponectin is synthesized as a 28 kDa monomer but is not secreted as such; instead, it is glycosylated and undergoes multimerization to form different molecular weight multimers before secretion. Adiponectin is one of the most abundant adipokines (μg/mL range) in the circulation. The concentrations are negatively correlated with adipose depot size, in particular with visceral fat mass in humans. Adiponectin exerts its effects by activating a range of different signaling molecules via binding to 2 transmembrane receptors, adiponectin receptor 1 and adiponectin receptor 2. The adiponectin receptor 1 is expressed primarily in the skeletal muscle, whereas adiponectin receptor 2 is predominantly expressed in the liver. Many of the functions of adiponectin are relevant to growth, lactation, and health and are thus of interest in both beef and dairy production systems. Studies on the role of the adiponectin protein in cattle have been impeded by the lack of reliable assays for bovine adiponectin. Although there are species-specific bovine adiponectin assays commercially available, they suffer from a lack of scientific peer-review of validity. Quantitative data about the adiponectin protein in cattle available in the literature emerged only during the last 3 yr and were largely based on Western blotting using either antibodies against human adiponectin or partial peptides from the bovine sequence. Using native bovine high-molecular-weight adiponectin purified from serum, we were able to generate a polyclonal antiserum that can be used for Western blot but also in an ELISA system, which was recently validated. The objective of this review is to provide an overview

  7. Adiponectin-SOGA Dissociation in Type 1 Diabetes

    PubMed Central

    Snell-Bergeon, Janet K.; Maahs, David M.; Bergman, Bryan C.; Lamarche, Marie; Iberkleid, Laura; AbdelBaky, Omar; Tisch, Roland; Scherer, Philipp E.; Marliss, Errol B.

    2015-01-01

    Context: Circulating adiponectin is elevated in human type 1 diabetes (T1D) and nonobese diabetic (NOD) mice without the expected indications of adiponectin action, consistent with tissue resistance. Objective: Adiponectin stimulates hepatocyte production of the suppressor of glucose from autophagy (SOGA), a protein that inhibits glucose production. We postulated that due to tissue resistance, the elevation of adiponectin in T1D should fail to increase the levels of a surrogate marker for liver SOGA, the circulating C-terminal SOGA fragment. Main Outcome Measures: Liver and plasma SOGA were measured in NOD mice (n = 12) by Western blot. Serum adiponectin and SOGA were measured in T1D and control (Ctrl) participants undergoing a three-stage insulin clamp for the Coronary Artery Calcification in T1D study (n = 20). Glucose turnover was measured using 6,6[2H2]glucose (n = 12). Results: In diabetic NOD mice, the 13%–29% decrease of liver SOGA (P = .003) and the 30%–37% reduction of circulating SOGA (P < .001) were correlated (r = 0.826; P = .001). In T1D serum, adiponectin was 50%–60% higher than Ctrl, SOGA was 30%–50% lower and insulin was 3-fold higher (P < .05). At the low insulin infusion rate (4 mU/m2·min), the resulting glucose appearance correlated negatively with adiponectin in T1D (r = −0.985, P = .002) and SOGA in Ctrl and T1D (r = −0.837, P = .001). Glucose disappearance correlated with adiponectin in Ctrl (r = −0.757, P = .049) and SOGA in Ctrl and T1D (r = −0.709, P = .010). At 40 mU/m2·min, the lowered glucose appearance was similar in Ctrl and T1D. Glucose disappearance increased only in Ctrl (P = .005), requiring greater glucose infusion to maintain euglycemia (8.58 ± 1.29 vs 3.09 ± 0.87 mg/kg·min; P = .009). Conclusions: The correlation between liver and plasma SOGA in NOD mice supports the use of the latter as surrogate marker for liver concentration. Reduced SOGA in diabetic NOD mice suggests resistance to adiponectin. The

  8. Induction of human adiponectin gene transcription by telmisartan, angiotensin receptor blocker, independently on PPAR-{gamma} activation

    SciTech Connect

    Moriuchi, Akie ||. E-mail: f1195@cc.nagasaki-u-ac.jp; Shimamura, Mika; Kita, Atsushi; Kuwahara, Hironaga; Satoh, Tsuyoshi; Satoh, Tsuyoshi; Fujishima, Keiichiro; Fukushima, Keiko |; Hayakawa, Takao; Mizuguchi, Hiroyuki; Nagayama, Yuji; Kawasaki, Eiji

    2007-05-18

    Adiponectin, an adipose tissue-specific plasma protein, has been shown to ameliorate insulin resistance and inhibit the process of atherosclerosis. Recently, several reports have stated that angiotensin type 1 receptor blockers (ARBs), increase adiponectin plasma level, and ameliorate insulin resistance. Telmisartan, a subclass of ARBs, has been shown to be a partial agonist of the peroxisome proliferator-activated receptor (PPAR)-{gamma}, and to increase the plasma adiponectin level. However, the transcriptional regulation of the human adiponectin gene by telmisartan has not been determined yet. To elucidate the effect of telmisartan on adiponectin, the stimulatory regulation of human adiponectin gene by telmisartan was investigated in 3T3-L1 adipocytes, utilizing adenovirus-mediated luciferase reporter gene-transferring technique. This study indicates that telmisartan may stimulate adiponectin transcription independent of PPAR-{gamma}.

  9. Variants in the Adiponectin Gene and Serum Adiponectin: The Coronary Artery Development in Young Adults (CARDIA) Study

    PubMed Central

    Wassel, Christina L.; Pankow, James S.; Jacobs, David R.; Steffes, Michael W.; Li, Na; Schreiner, Pamela J.

    2016-01-01

    Circulating adiponectin is involved in the atherosclerotic process and has been associated with cardiovascular disease as well as obesity, insulin resistance, metabolic syndrome, and type 2 diabetes. The adiponectin gene (ADIPOQ) encodes the circulating protein adiponectin and affects its expression. Only a small proportion of all known ADIPOQ polymorphisms have been investigated in relation to circulating adiponectin concentrations. Using data from 3,355 African-American and white men and women aged 33–45 at the year 15 examination from the Coronary Artery Development in Young Adults (CARDIA) Study the association between 10 single-nucleotide polymorphisms (SNPs) within ADIPOQ and serum adiponectin was examined using linear regression. SNPs were chosen based on a tagSNP approach. Models were stratified by self-reported race to control for population stratification, and Bonferroni corrected for multiple comparisons. ADIPOQ SNPs rs17300539 (P < 0.0001), rs182052 (P = 0.0013), rs822393 (P = 0.0005), rs9882205 (P = 0.0001), and rs3774261 (P = 0.0001) were strongly associated with serum adiponectin concentrations in whites. In general, there was a dose–response relationship of adjusted mean adiponectin concentrations across genotypes. Only one SNP, rs17300539 was marginally associated with serum adiponectin concentrations (P = 0.0087) in African Americans. Significant interactions were found between waist and rs182052 (P = 0.0029) and between rs9882505 and smoking (P = 0.001) in whites. Many ADIPOQ SNPs have not yet been examined, and additional studies are needed to determine whether these may be functional variants. PMID:20395949

  10. Degradable thermoresponsive nanogels for protein encapsulation and controlled release.

    PubMed

    Bhuchar, Neha; Sunasee, Rajesh; Ishihara, Kazuhiko; Thundat, Thomas; Narain, Ravin

    2012-01-18

    Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h. PMID:22171688

  11. Association of Adiponectin with High-Sensitivity C-Reactive Protein and Clinical Outcomes in Peritoneal Dialysis Patients: A 3.5-Year Follow-Up Study

    PubMed Central

    Tung, Chun-Wu; Hsu, Yung-Chien; Shih, Ya-Hsueh; Lin, Chun-Liang

    2015-01-01

    Introduction Adiponectin (ADPN), one of most abundant fat-derived biologically active substances, plays an important role in anti-atherosclerotic process. There are conflicting results about the impact of ADPN on cardiovascular (CV) outcomes and mortality, particularly in patients undergoing peritoneal dialysis (PD). Moreover, the relationship between ADPN and inflammatory mediators has been seldom explored in this population. Therefore, we examined the relationship between ADPN and longitudinal high-sensitivity C-reactive protein (hs-CRP) changes and investigated whether ADPN or hs-CRP levels could predict CV outcomes and mortality in prevalent PD patients after comprehensive adjustment of possible confounders. Methods In this prospective cohort study, 78 PD patients were enrolled and followed from February 2009 to August 2012. During follow-up, CV events and all-cause mortality were recorded. Results The mean baseline ADPN value was 29.46±18.01 μg/ml and duration of PD treatment was 37.76±36.96 months. In multiple linear regression analysis, plasma ADPN levels positively correlated with high-density lipoprotein and negatively associated with hs-CRP, body mass index, D4/D0 glucose, triglyceride, and duration of PD treatment. After stratified by genders, the inverse association between baseline ADPN and hs-CRP was more significant in the female group. The hs-CRP levels were followed up annually and remained significantly lower in the high ADPN group in the first 2 years. Patients were then stratified into two groups according to the median ADPN value (23.8 μg/ml). The results of Kaplan-Meier survival analysis demonstrated less CV events and better survival in high ADPN group. On multivariate Cox regression analysis, only ADPN level (HR: 0.93, 95% CI: 0.88–0.98, p = 0.02), age and history of CV diseases were independent risk factors for future CV events. Furthermore, hs-CRP (HR: 1.11, 95% CI:1.001–1.22, p = 0.04) was identified as independent predictor of

  12. Unique profile of chicken adiponectin, a predominantly heavy molecular weight multimer, and relationship to visceral adiposity.

    PubMed

    Hendricks, Gilbert L; Hadley, Jill A; Krzysik-Walker, Susan M; Prabhu, K Sandeep; Vasilatos-Younken, Regina; Ramachandran, Ramesh

    2009-07-01

    Adiponectin, a 30-kDa adipokine hormone, circulates as heavy, medium, and light molecular weight isoforms in mammals. Plasma heavy molecular weight (HMW) adiponectin isoform levels are inversely correlated with the incidence of type 2 diabetes in humans. The objectives of the present study were to characterize adiponectin protein and quantify plasma adiponectin levels in chickens, which are naturally hyperglycemic relative to mammals. Using gel filtration column chromatography and Western blot analysis under nonreducing and non-heat-denaturing native conditions, adiponectin in chicken plasma, and adipose tissue is predominantly a multimeric HMW isoform that is larger than 669 kDa mass. Under reducing conditions and heating to 70-100 C, however, a majority of the multimeric adiponectin in chicken plasma and adipose tissue was reduced to oligomeric and/or monomeric forms. Immunoprecipitation and elution under neutral pH preserved the HMW adiponectin multimer, whereas brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the presence of hydroxyproline and differential glycosylation of hydroxylysine residues in the collagenous domain. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8- compared with 4-wk-old male chickens and inversely related to abdominal fat pad mass. Collectively, our results provide novel evidence that adiponectin in chicken plasma and tissues is predominantly a HMW multimer, suggesting the presence of unique multimerization and stabilization mechanisms in the chicken that favors preponderance of HMW adiponectin over other oligomers. PMID:19299452

  13. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  14. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    PubMed

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  15. Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

    PubMed Central

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  16. A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels

    PubMed Central

    Murthy, Niren; Xu, Mingcheng; Schuck, Stephany; Kunisawa, Jun; Shastri, Nilabh; Fréchet, Jean M. J.

    2003-01-01

    The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigen-presenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200–500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The acid-degradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. PMID:12704236

  17. Phytoferritin association induced by EGCG inhibits protein degradation by proteases.

    PubMed

    Wang, Aidong; Zhou, Kai; Qi, Xin; Zhao, Guanghua

    2014-12-01

    Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (-)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases. PMID:25384342

  18. Design Principles of a Universal Protein Degradation Machine

    PubMed Central

    Matyskiela, Mary E.; Martin, Andreas

    2012-01-01

    The 26S proteasome is a 2.5 MDa, 32-subunit ATP-dependent protease that is responsible for the degradation of ubiquitinated protein targets in all eukaryotic cells. This proteolytic machine consists of a barrel-shaped peptidase capped by a large regulatory particle, which contains a heterohexameric AAA+ unfoldase as well as several structural modules of previously unknown function. Recent electron microscopy studies have allowed major breakthroughs in understanding the architecture of the regulatory particle, revealing that the additional modules provide a structural framework to position critical, ubiquitin-interacting subunits and thus allow the 26S proteasome to function as a universal degradation machine for a wide variety of protein substrates. The electron microscopy studies have also uncovered surprising asymmetries in the spatial arrangement of proteasome subunits, yet the functional significance of these architectural features remains unclear. This review will summarize the recent findings on 26S proteasome structure and discuss the mechanistic implications for substrate binding, deubiquitination, unfolding, and degradation. PMID:23147216

  19. Intracellular protein degradation in mammalian cells: recent developments.

    PubMed

    Knecht, Erwin; Aguado, Carmen; Cárcel, Jaime; Esteban, Inmaculada; Esteve, Juan Miguel; Ghislat, Ghita; Moruno, José Félix; Vidal, José Manuel; Sáez, Rosana

    2009-08-01

    In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes. PMID:19399586

  20. Synthesis and protein degradation capacity of photoactivated enediynes.

    PubMed

    Fouad, Farid S; Wright, Justin M; Plourde, Gary; Purohit, Ajay D; Wyatt, Justin K; El-Shafey, Ahmed; Hynd, George; Crasto, Curtis F; Lin, Yiqing; Jones, Graham B

    2005-11-25

    [structure: see text] The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics. PMID:16292807

  1. Adiponectin-Mediated Analgesia and Anti-Inflammatory Effects in Rat

    PubMed Central

    Iannitti, Tommaso; Graham, Annette; Dolan, Sharron

    2015-01-01

    The adipose tissue-derived protein, adiponectin, has significant anti-inflammatory properties in a variety of disease conditions. Recent evidence that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in central nervous system, suggests that it may also have a central modulatory role in pain and inflammation. This study set out to investigate the effects of exogenously applied recombinant adiponectin (via intrathecal and intraplantar routes; 10–5000 ng) on the development of peripheral inflammation (paw oedema) and pain hypersensitivity in the rat carrageenan model of inflammation. Expression of adiponectin, AdipoR1 and AdipoR2 mRNA and protein was characterised in dorsal spinal cord using real-time polymerase chain reaction (PCR) and Western blotting. AdipoR1 and AdipoR2 mRNA and protein were found to be constitutively expressed in dorsal spinal cord, but no change in mRNA expression levels was detected in response to carrageenan-induced inflammation. Adiponectin mRNA, but not protein, was detected in dorsal spinal cord, although levels were very low. Intrathecal administration of adiponectin, both pre- and 3 hours post-carrageenan, significantly attenuated thermal hyperalgesia and mechanical hypersensitivity. Intrathecal administration of adiponectin post-carrageenan also reduced peripheral inflammation. Intraplantar administration of adiponectin pre-carrageenan dose-dependently reduced thermal hyperalgesia but had no effect on mechanical hypersensitivity and peripheral inflammation. These results show that adiponectin functions both peripherally and centrally at the spinal cord level, likely through activation of AdipoRs to modulate pain and peripheral inflammation. These data suggest that adiponectin receptors may be a novel therapeutic target for pain modulation. PMID:26352808

  2. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    PubMed

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  3. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

    PubMed Central

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K.; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-01

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  4. Evidence of oleuropein degradation by olive leaf protein extract.

    PubMed

    De Leonardis, Antonella; Macciola, Vincenzo; Cuomo, Francesca; Lopez, Francesco

    2015-05-15

    The enzymatic activity of raw protein olive leaf extract has been investigated in vivo, on olive leaf homogenate and, in vitro with pure oleuropein and other phenolic substrates. At least two types of enzymes were found to be involved in the degradation of endogenous oleuropein in olive leaves. As for the in vitro experiments, the presence of active polyphenoloxidase and β-glucosidase was determined by HPLC and UV-Visible spectroscopy. Interestingly, both the enzymatic activities were found to change during the storage of olive leaves. Specifically, the protein extracts obtained from fresh leaves showed the presence of both the enzymatic activities, because oleuropein depletion occurred simultaneously with the formation of the oleuropein aglycon, 3,4-DHPEA-EA. In comparison leaves subjected to the drying process showed a polyphenoloxidase activity leading exclusively to the formation of oxidation products responsible for the typical brown coloration of the reaction solution. PMID:25577121

  5. Differential effects of leptin on adiponectin expression with weight gain versus obesity

    PubMed Central

    Singh, Prachi; Sharma, Pragya; Sahakyan, Karine R.; Davison, Diane E.; Sert-Kuniyoshi, Fatima H; Romero-Corral, Abel; Swain, James M.; Jensen, Michael D.; Lopez-Jimenez, Francisco; Kara, Tomas; Somers, Virend K.

    2015-01-01

    Background/Objective Adiponectin exerts beneficial effects by reducing inflammation, and improving lipid metabolism and insulin-sensitivity. Although adiponectin is lower in obese individuals, whether weight gain reduces adiponectin expression in humans is controversial. We sought to investigate the role of weight gain, and consequent changes in leptin, on altering adiponectin expression in humans. Methods/Results Forty four normal-weight healthy subjects were recruited (mean age 29 years; 14 women) and randomized to either gain 5% of body weight by 8-weeks of overfeeding (n=34) or maintain weight (n=10). Modest weight gain of 3.8 ± 1.2 kg resulted in increased adiponectin (p=0.03) while weight maintenance resulted in no changes in adiponectin. Further, changes in adiponectin correlated positively with changes in leptin (p=0.0085). In-vitro experiments using differentiated human white preadipocytes showed that leptin increased adiponectin mRNA and protein expression, while a leptin-antagonist had opposite effects. To understand the role of leptin in established obesity, we compared adipose tissue samples obtained from normal weight versus obese subjects. We noted, first, that leptin activated cellular signaling pathways and increased adiponectin mRNA in adipose tissue from normal-weight participants, but did not do so in adipose tissue from obese participants; and second, that obese subjects had increased caveolin-1 expression, which attenuates leptin-dependent increases in adiponectin. Conclusions Modest weight gain in healthy individuals is associated with increases in adiponectin, which correlate positively with changes in leptin. In-vitro, leptin induces adiponectin expression which is attenuated by increased caveolin-1 expression. Additionally, adipose tissue from obese subjects shows increased caveolin-1 expression, and impaired leptin signaling. This leptin signal impairment may prevent concordant increases in adiponectin in obese subjects despite their

  6. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2—dependent mechanisms

    PubMed Central

    Shibata, Rei; Sato, Kaori; Pimentel, David R; Takemura, Yukihiro; Kihara, Shinji; Ohashi, Koji; Funahashi, Tohru; Ouchi, Noriyuki; Walsh, Kenneth

    2010-01-01

    Obesity-related disorders are associated with the development of ischemic heart disease. Adiponectin is a circulating adipose-derived cytokine that is downregulated in obese individuals and after myocardial infarction. Here, we examine the role of adiponectin in myocardial remodeling in response to acute injury. Ischemia-reperfusion in adiponectin-deficient (APN-KO) mice resulted in increased myocardial infarct size, myocardial apoptosis and tumor necrosis factor (TNF)-α expression compared with wild-type mice. Administration of adiponectin diminished infarct size, apoptosis and TNF-α production in both APN-KO and wild-type mice. In cultured cardiac cells, adiponectin inhibited apoptosis and TNF-α production. Dominant negative AMP-activated protein kinase (AMPK) reversed the inhibitory effects of adiponectin on apoptosis but had no effect on the suppressive effect of adiponectin on TNF-α production. Adiponectin induced cyclooxygenase (COX)-2–dependent synthesis of prostaglandin E2 in cardiac cells, and COX-2 inhibition reversed the inhibitory effects of adiponectin on TNF-α production and infarct size. These data suggest that adiponectin protects the heart from ischemia-reperfusion injury through both AMPK- and COX-2–dependent mechanisms. PMID:16155579

  7. Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation.

    PubMed

    Liu, Xiaming; Han, Weiwei; Gulla, Sarah; Simon, Nicholas I; Gao, Yanfei; Cai, Changmeng; Yang, Hongmei; Zhang, Xiaoping; Liu, Jihong; Balk, Steven P; Chen, Shaoyong

    2016-01-12

    The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4-2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC). PMID:26636645

  8. Induced oligomerization targets Golgi proteins for degradation in lysosomes

    PubMed Central

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

    2015-01-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

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

    PubMed

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

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  10. Effect of repeated US stimulation on adiponectin secretion by adipocytes of obese human subjects

    NASA Astrophysics Data System (ADS)

    Fujii, Yasutomo; Taniguchi, Nobuyuki; Satoh, Masaaki; Irie, Takasuke; Itoh, Kouichi

    2006-05-01

    To clarify the effect of the repeated sonication on the adiponectin secretion by adipocytes obtained from obese subjects. Using 1-MHz continuous-wave ultrasound at an intensity of 0.50 or 2.1 W/cm2, we sonicated culture flasks of subcutaneous adipocytes obtained from obese human subjects, in a series of 3 sessions of US stimulation applied for a daily total of 15 min. For the measurement of adiponectin secretion, 50 μl of the culture medium was collected from each flask every 24 h after the 1st stimulation. Quantification of adiponectin protein levels in cell culture supernatants was performed with a commercially available ELISA kit recommended by the manufacturer. The adiponectin concentrations in the culture medium of the US stimulation groups rose significantly (p<0.05). Repeated US stimulation may accelerate adiponectin secretion in obese human adipocytes.

  11. Chemical methods for degradation of target proteins using designed light-activatable organic molecules.

    PubMed

    Tanimoto, Shuho; Takahashi, Daisuke; Toshima, Kazunobu

    2012-08-11

    Molecular design, chemical synthesis, and biological evaluation of several designed organic molecules, which target-selectively degrade proteins upon photo-irradiation, are introduced. The designed molecules for protein photo-degradation include 2-phenylquinoline-steroid hormone hybrids and porphyrin derivatives, both of which selectively photo-degrade estrogen receptor-α, and fullerene-sugar and -sulfonic acid hybrids, which selectively photo-degrade HIV-1 protease and amyloid β, respectively. The information will provide a novel and effective way to control specific functions of proteins, and contribute to the molecular design of novel protein photo-degrading agents, which should find wide application in chemistry, biology, and medicine. PMID:22739361

  12. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  13. Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However,...

  14. Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.

    PubMed

    Singh, S P; Häussler, S; Gross, J J; Schwarz, F J; Bruckmaier, R M; Sauerwein, H

    2014-03-01

    Adiponectin, one of the most abundant adipokines in circulation, is known for its role in regulation of body metabolism. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages of lactation (lactational NEB at the onset of lactation and an induced NEB by feed restriction near 100 d of lactation) on circulating adiponectin concentrations. We also investigated the effect of feed restriction on adiponectin concentrations in milk and the relationships of blood and milk adiponectin with selected plasma or milk variables and with measures of body condition. Plasma adiponectin was measured in 50 multiparous Holstein dairy cows throughout 3 experimental periods [i.e., period 1=3 wk antepartum up to 12 wk postpartum, period 2=3 wk of feed restriction starting at around 100 d in milk with a control (n=25) and feed-restricted group (50% of energy requirements; n=25), and period 3=subsequent realimentation period for 8 wk]. Milk adiponectin was investigated among 21 multiparous cows at wk 2 and wk 12 of period 1 and wk 2 of period 2. Adiponectin concentrations in plasma and skim milk were measured using an in-house ELISA specific for bovine adiponectin. Major changes in circulating adiponectin concentrations were observed during the periparturient period, whereas energy deficiency during established lactation at around 100 d in milk and subsequent refeeding did not affect plasma adiponectin. Together with lower adiponectin concentrations in milk (µg/mL), the reduction in milk yield led to decreased adiponectin secretion via milk (mg/d) at the second week of feed restriction. Irrespective of time and treatment, milk adiponectin represented about 0.002% of total milk protein. Mean adiponectin concentrations in milk (0.61 ± 0.03 µg/mL) were about 92% lower than the mean plasma adiponectin concentrations (32.1 ± 1.0 µg/mL). The proportion of the steady-state plasma adiponectin pool secreted daily via milk was 2.7%. In view of the

  15. ASSOCIATION BETWEEN ADIPONECTIN, INSULIN RESISTANCE, AND ENDOMETRIAL CANCER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Obesity is a well-known risk factor for the development of endometrial cancer; however, weight alone does not account for all cases. The authors hypothesized that insulin resistance also contributes to an increased risk for endometrial cancer. Adiponectin is a protein secreted by adipose...

  16. MicroRNA-378 Regulates Adiponectin Expression in Adipose Tissue: A New Plausible Mechanism

    PubMed Central

    Ishida, Masayoshi; Shimabukuro, Michio; Yagi, Shusuke; Nishimoto, Sachiko; Kozuka, Chisayo; Fukuda, Daiju; Soeki, Takeshi; Masuzaki, Hiroaki; Tsutsui, Masato; Sata, Masataka

    2014-01-01

    Aims Mechanisms regulating adiponectin expression have not been fully clarified. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, are involved in biological processes, including obesity and insulin resistance. We evaluated whether the miRNA-378 pathway is involved in regulating adiponectin expression. Methods and Results First, we determined a putative target site for miRNA-378 in the 3 prime untranslated region (3'UTR) of the adiponectin gene by in silico analysis. The levels of adiponectin mRNA and protein were decreased in 3T3-L1 cells overexpressing the mimic of miRNA-378. Luminescence activity in HEK293T cells expressing a renilla-luciferase-adiponectin-3'UTR sequence was inhibited by overexpressing the mimic of miRNA-378, and the decrease was reversed by adding the inhibitor of miRNA-378. Moreover, we confirmed the inhibitory effects of the mimic were cancelled in a deleted mutant of the miR-378 3′-UTR binding site. Addition of tumor necrosis factor-α (TNFα) led a upregulation of miR-378 and downregulation of adiponectin at mRNA and protein levels in 3T3-L1 cells. Level of miR-378 was higher and mRNA level of adiponectin was lower in diabetic ob/ob mice than those of normal C57BL/6 mice and levels of miR378 and adiponectin were negatively well correlated (r = −0.624, p = 0.004). Conclusions We found that levels of miRNA-378 could modulate adiponectin expression via the 3'UTR sequence-binding site. Our findings warrant further investigations into the role of miRNAs in regulating the adiponectin expression. PMID:25379946

  17. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  18. Lack of response to addition of degradable protein to a low protein diet fed to midlactation dairy cows.

    PubMed

    Armentano, L E; Bertics, S J; Riesterer, J

    1993-12-01

    Midlactation Holstein cows (n = 24, 12 primiparous) were subjected to four dietary treatments arranged in six Latin squares. Cows were assigned to squares according to parity and previous production within parity. Diets contained 53 to 55% DM from corn silage, and the remaining DM was from concentrates. The basal diet contained 13.9% CP with 9.5% rumen-degraded and 4.4% undegraded intake protein. Three other treatments were formulated to give one diet with more degradable true protein than the basal (11.9% degraded intake protein, 4.3% undegraded intake protein); another with urea added to the basal (12.2% degraded intake protein, 4.5% undegraded intake protein); and a third with additional undegraded protein added to the basal (8.3% degraded intake protein, 7.2% undegraded intake protein). Milk fat and protein concentration were unaffected by diet in all squares. Only the highest producing cows responded significantly to increased undegraded intake protein; milk production was 30.8, 30.9, 31.6, and 33.2 kg/d for basal, added degradable true protein, added urea, and added undegraded protein, respectively. Corresponding protein productions were 913, 929, 927, and 1004 g/d for these cows. Neither degradable true protein nor degradable N increased milk production in the highest producing cows, suggesting that microbial protein production was not limited by the amount of degradable protein in the basal diet. For midlactation, multiparous, and primiparous cows producing < 30 and 25 kg of milk/d, respectively, the protein content of the basal ration appeared to be adequate. PMID:8132882

  19. Nudix hydrolases degrade protein-conjugated ADP-ribose.

    PubMed

    Daniels, Casey M; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  20. Nudix hydrolases degrade protein-conjugated ADP-ribose

    PubMed Central

    Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  1. Amyloid precursor protein modulates β-catenin degradation

    PubMed Central

    Chen, Yuzhi; Bodles, Angela M

    2007-01-01

    Background The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. Results We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. Conclusion We have provided strong evidence that APP modulates β-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation. PMID:18070361

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

    SciTech Connect

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

    1986-06-01

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

  3. GnRH decreases adiponectin expression in pituitary gonadotropes via the calcium and PKA pathways.

    PubMed

    Kim, Jonathan; Zheng, Weiming; Grafer, Constance; Mann, Merry Lynn; Halvorson, Lisa M

    2013-08-01

    As endocrinologically active cells, adipocytes are capable of secreting various adipocytokines such as leptin, resistin, and adiponectin to impact metabolic function. Although adipocytes remain to be the primary site of synthesis and secretion, there is now growing evidence that supports the presence of adiponectin and its receptors within the hypothalamic-pituitary-gonadal axis, providing a possible link between obesity and abnormal reproductive physiology. It has been demonstrated that adiponectin may reduce gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus as well as modulate gonadal steroid hormone production. Furthermore, prior data indicate that adiponectin may play a role in decreasing luteinizing hormone secretion from pituitary gonadotropes. We aimed to identify the hormonal regulators of adiponectin and its receptors, AdipoR1 and AdipoR2, in pituitary gonadotropes using immortalized gonadotropic LβT2 cells and primary rat pituitary cells. Our study shows significant alterations in adiponectin expression across the estrous cycle. In addition, we present a novel finding that GnRH suppresses pituitary adiponectin expression via the calcium and protein kinase A intracellular pathways in both cultured rat primary pituitary cells and the LβT2 gonadotrope cell line. The GnRH did not alter expression of the adiponectin receptors, AdipoR1 and AdipoR2, in cultured gonadotropes. Expression of the adiponectin receptors, AdipoR1 and AdipoR2, was not altered by GnRH in cell culture but in vivo or in vitro. Our data suggest that gonadotrope function may be modulated by GnRH-mediated changes in adiponectin expression. PMID:23239819

  4. Serum Adiponectin and Cardiometabolic Risk in Patients with Acute Coronary Syndromes

    PubMed Central

    Oliveira, Gustavo Bernardes de Figueiredo; França, João Ítalo Dias; Piegas, Leopoldo Soares

    2013-01-01

    Background The adipose tissue is considered not only a storable energy source, but mainly an endocrine organ that secretes several cytokines. Adiponectin, a novel protein similar to collagen, has been found to be an adipocyte-specific cytokine and a promising cardiovascular risk marker. Objectives To evaluate the association between serum adiponectin levels and the risk for cardiovascular events in patients with acute coronary syndromes (ACS), as well as the correlations between adiponectin and metabolic, inflammatory, and myocardial biomarkers. Methods We recruited 114 patients with ACS and a mean 1.13-year follow-up to measure clinical outcomes. Clinical characteristics and biomarkers were compared according to adiponectin quartiles. Cox proportional hazard regression models with Firth's penalization were applied to assess the independent association between adiponectin and the subsequent risk for both primary (composite of cardiovascular death/non-fatal acute myocardial infarction (AMI)/non-fatal stroke) and co-primary outcomes (composite of cardiovascular death/non-fatal AMI/non-fatal stroke/ rehospitalization requiring revascularization). Results There were significant direct correlations between adiponectin and age, HDL-cholesterol, and B-type natriuretic peptide (BNP), and significant inverse correlations between adiponectin and waist circumference, body weight, body mass index, Homeostasis Model Assessment (HOMA) index, triglycerides, and insulin. Adiponectin was associated with higher risk for primary and co-primary outcomes (adjusted HR 1.08 and 1.07/increment of 1000; p = 0.01 and p = 0.02, respectively). Conclusion In ACS patients, serum adiponectin was an independent predictor of cardiovascular events. In addition to the anthropometric and metabolic correlations, there was a significant direct correlation between adiponectin and BNP. PMID:24029961

  5. Small Molecule-facilitated Degradation of ANO1 Protein

    PubMed Central

    Bill, Anke; Hall, Michelle Lynn; Borawski, Jason; Hodgson, Catherine; Jenkins, Jeremy; Piechon, Philippe; Popa, Oana; Rothwell, Christopher; Tranter, Pamela; Tria, Scott; Wagner, Trixie; Whitehead, Lewis; Gaither, L. Alex

    2014-01-01

    ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers. PMID:24599954

  6. Preliminary evidence of genetic determinants of adiponectin response to fenofibrate in the Genetics of Lipid Lowering Drugs and Diet Network

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adiponectin is an adipose-secreted protein that has been linked to changes in insulin sensitivity, high-density lipoprotein cholesterol levels, and inflammatory patterns. Although fenofibrate therapy can raise adiponectin levels, treatment response is heterogeneous and heritable, suggesting a role f...

  7. Adiponectin: a manifold therapeutic target for metabolic syndrome, diabetes, and coronary disease?

    PubMed Central

    2014-01-01

    Adiponectin is the most abundant peptide secreted by adipocytes, being a key component in the interrelationship between adiposity, insulin resistance and inflammation. Central obesity accompanied by insulin resistance is a key factor in the development of metabolic syndrome (MS) and future macrovascular complications. Moreover, the remarkable correlation between coronary artery disease (CAD) and alterations in glucose metabolism has raised the likelihood that atherosclerosis and type 2 diabetes mellitus (T2DM) may share a common biological background. We summarize here the current knowledge about the influence of adiponectin on insulin sensitivity and endothelial function, discussing its forthcoming prospects and potential role as a therapeutic target for MS, T2DM, and cardiovascular disease. Adiponectin is present in the circulation as a dimer, trimer or protein complex of high molecular weight hexamers, >400 kDa. AdipoR1 and AdipoR2 are its major receptors in vivo mediating the metabolic actions. Adiponectin stimulates phosphorylation and AMP (adenosin mono phosphate) kinase activation, exerting direct effects on vascular endothelium, diminishing the inflammatory response to mechanical injury and enhancing endothelium protection in cases of apolipoprotein E deficiency. Hypoadiponectinemia is consistently associated with obesity, MS, atherosclerosis, CAD, T2DM. Lifestyle correction helps to favorably modify plasma adiponectin levels. Low adiponectinemia in obese patients is raised via continued weight loss programs in both diabetic and nondiabetic individuals and is also accompanied by reductions in pro-inflammatory factors. Diet modifications, like intake of fish, omega-3 supplementation, adherence to a Mediterranean dietary pattern and coffee consumption also increase adiponectin levels. Antidiabetic and cardiovascular pharmacological agents, like glitazones, glimepiride, angiotensin converting enzyme inhibitors and angiotensin receptor blockers are also able to

  8. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

    PubMed Central

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

    2015-01-01

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. PMID:25995378

  9. Heat-induced Protein Structure and Subfractions in Relation to Protein Degradation Kinetics and Intestinal Availability in Dairy Cattle

    SciTech Connect

    Doiron, K.; Yu, P; McKinnon, J; Christensen, D

    2009-01-01

    The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included (1) protein structure a-helix to e-sheet ratio; (2) protein subfractions profiles; (3) protein degradation kinetics and effective degradability; (4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120C for 40 and 60 min increased the protein structure a-helix to e-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences

  10. Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase, TOPP4

    PubMed Central

    Qin, Qianqian; Wang, Wei; Guo, Xiaola; Yue, Jing; Huang, Yan; Xu, Xiufei; Li, Jia; Hou, Suiwen

    2014-01-01

    Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway. PMID:25010794

  11. An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

    PubMed Central

    Wilmington, Shameika R.; Matouschek, Andreas

    2016-01-01

    A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels. PMID:27043013

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

    PubMed Central

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

    2015-01-01

    Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases. PMID:26415220

  13. [The 2004 Nobel Prize in Chemistry for the discovery of ubiquitin-mediated protein degradation].

    PubMed

    Neefjes, J; Groothuis, T A M; Dantuma, N P

    2004-12-25

    This year's Nobel Prize in Chemistry has been awarded to Aaron Ciechanover, Avram Herskho and Irwin Rose for the discovery of ubiquitin-mediated protein degradation. In a series of groundbreaking experiments these scientists described the basic principles for a unique posttranslational modification based on the conjugation of the small protein ubiquitin to proteins deemed for degradation. Although ubiquitin started in 1980 as an unusual modification of certain proteins, it is now clear that it functions as a signal for degradation when it forms a polymer. Hundreds of proteins are involved in the controlled destruction of ubiquitin-labelled proteins in the cell. And hundreds of other proteins are involved in protein modification by mono-ubiquitin, so that other processes, such as the formation of another degradation compartment, the lysosome, can proceed normally. PMID:15646859

  14. Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

    PubMed

    Gorovits, Rena; Fridman, Lilia; Kolot, Mikhail; Rotem, Or; Ghanim, Murad; Shriki, Oz; Czosnek, Henryk

    2016-02-01

    Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins. PMID:26654789

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

    PubMed Central

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-01-01

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons. PMID:25766616

  16. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

    PubMed Central

    Sekar, Karthik; Gentile, Andrew M.; Bostick, John W.; Tyo, Keith E. J.

    2016-01-01

    Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits. PMID:26900850

  17. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  18. Genetic Architecture of Plasma Adiponectin Overlaps With the Genetics of Metabolic Syndrome–Related Traits

    PubMed Central

    Henneman, Peter; Aulchenko, Yurii S.; Frants, Rune R.; Zorkoltseva, Irina V.; Zillikens, M. Carola; Frolich, Marijke; Oostra, Ben A.; van Dijk, Ko Willems; van Duijn, Cornelia M.

    2010-01-01

    OBJECTIVE Adiponectin, a hormone secreted by adipose tissue, is of particular interest in metabolic syndrome, because it is inversely correlated with obesity and insulin sensitivity. However, it is not known to what extent the genetics of plasma adiponectin and the genetics of obesity and insulin sensitivity are interrelated. We aimed to evaluate the heritability of plasma adiponectin and its genetic correlation with the metabolic syndrome and metabolic syndrome–related traits and the association between these traits and 10 ADIPOQ single nucleotide polymorphisms (SNPs). RESEARCH DESIGN AND METHODS We made use of a family-based population, the Erasmus Rucphen Family study (1,258 women and 967 men). Heritability analysis was performed using a polygenic model. Genetic correlations were estimated using bivariate heritability analyses. Genetic association analysis was performed using a mixed model. RESULTS Plasma adiponectin showed a heritability of 55.1%. Genetic correlations between plasma adiponectin HDL cholesterol and plasma insulin ranged from 15 to 24% but were not significant for fasting glucose, triglycerides, blood pressure, homeostasis model assessment of insulin resistance (HOMA-IR), and C-reactive protein. A significant association with plasma adiponectin was found for ADIPOQ variants rs17300539 and rs182052. A nominally significant association was found with plasma insulin and HOMA-IR and ADIPOQ variant rs17300539 after adjustment for plasma adiponectin. CONCLUSIONS The significant genetic correlation between plasma adiponectin and HDL cholesterol and plasma insulin should be taken into account in the interpretation of genome-wide association studies. Association of ADIPOQ SNPs with plasma adiponectin was replicated, and we showed association between one ADIPOQ SNP and plasma insulin and HOMA-IR. PMID:20067957

  19. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  20. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H2O2 or TNFα was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  1. POLYPHENOLS AND MECHANICAL MACERATION SHIFT PROTEIN FRACTIONS IN LEGUME HAYS FROM RAPIDLY TO SLOWLY DEGRADED FORMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid proteolysis of forage protein during rumen fermentation can impair protein use by dairy cattle. The severity of conditioning at harvest may influence protein degradability in forages, particularly if protein-binding polyphenols are present. In 2002 and 2003, first and second cuttings of alfalf...

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

    PubMed

    Mozziconacci, Olivier; Schöneich, Christian

    2015-10-01

    Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light. PMID:25481682

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

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

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

    PubMed

    Comyn, Sophie A; Young, Barry P; Loewen, Christopher J; Mayor, Thibault

    2016-07-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  5. The prevention and treatment of hypoadiponectinemia-associated human diseases by up-regulation of plasma adiponectin.

    PubMed

    Hossain, Md Murad; Mukheem, Abdul; Kamarul, Tunku

    2015-08-15

    Hypoadiponectinemia is characterized by low plasma adiponectin levels that can be caused by genetic factors, such as single nucleotide polymorphisms (SNPs) and mutations in the adiponectin gene or by visceral fat deposition/obesity. Reports have suggested that hypoadiponectinemia is associated with dyslipidemia, hypertension, hyperuricemia, metabolic syndrome, atherosclerosis, type 2 diabetes mellitus and various cardiovascular diseases. Previous studies have highlighted several potential strategies to up-regulate adiponectin secretion and function, including visceral fat reduction through diet therapy and exercise, administration of exogenous adiponectin, treatment with peroxisome proliferator-activating receptor gamma (PPARγ) agonists (e.g., thiazolidinediones (TZDs)) and ligands (e.g., bezafibrate and fenofibrate) or the blocking of the renin-angiotensin system. Likewise, the up-regulation of the expression and stimulation of adiponectin receptors by using adiponectin receptor agonists would be an effective method to treat obesity-related conditions. Notably, adiponectin is an abundantly expressed bioactive protein that also exhibits a wide spectrum of biological properties, such as insulin-sensitizing, anti-diabetic, anti-inflammatory and anti-atherosclerotic activities. Although targeting adiponectin and its receptors has been useful for treating diabetes and other metabolic-related diseases in experimental studies, current drug development based on adiponectin/adiponectin receptors for clinical applications is scarce, and there is a lack of available clinical trial data. This comprehensive review discusses the strategies that are presently being pursued to harness the potential of adiponectin up-regulation. In addition, we examined the current status of drug development and its potential for clinical applications. PMID:25818192

  6. Adiponectin is released via a unique regulated exocytosis pathway from a pre-formed vesicle pool on insulin stimulation.

    PubMed

    Lim, Chun-Yan; Hong, Wanjin; Han, Weiping

    2015-11-01

    Adiponectin, a hormone secreted from adipocytes and released at a high rate into the circulation, plays a pivotal role in maintaining insulin sensitivity at the whole-body level. Despite the importance of this adipokine in metabolic homoeostasis, the mechanism of its secretion from adipocytes remains largely unclear. In the present study, we investigated the subcellular localization of adiponectin, and its secretion regulation in 3T3-L1-differentiated adipocytes, using biochemical methods and fluorescence microscopic imaging. We show that adiponectin is localized in vesicular compartments with no apparent overlap with the Golgi apparatus or endosomes. Moreover, adiponectin-containing vesicles are enriched in two distinct pools: one at the plasma membrane (PM) and the other co-fractionating with endoplasmic reticulum membranes. When viewed under a total internal refection fluorescence microscope, a subset of adiponectin-Venus vesicles is readily observed in proximity to PMs, and could be released in response to insulin. Insulin-stimulated adiponectin release appears to be from a pre-existing pool of vesicles, and is not dependent on new protein synthesis, because adiponectin mRNA levels remain unchanged over a 6-h period of insulin treatment, and inhibition of protein synthesis has no effect on adiponectin release. Disruption of insulin signalling by inhibitors of phosphoinositide 3-kinase and protein kinase B (Akt)-1/2 abrogates the stimulated release of adiponectin. Taken together, our results show that adiponectin is stored in a unique vesicular compartment, and released through a regulated exocytosis pathway that is dependent on insulin signalling. PMID:26330614

  7. A cellular system that degrades misfolded proteins and protects against neurodegeneration.

    PubMed

    Guo, Lili; Giasson, Benoit I; Glavis-Bloom, Alex; Brewer, Michael D; Shorter, James; Gitler, Aaron D; Yang, Xiaolu

    2014-07-01

    Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases. PMID:24882209

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

    SciTech Connect

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

    1989-04-01

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

  9. Mass Spectrometry Analysis of Proteome-wide Proteolytic Post-translational Degradation of Proteins

    SciTech Connect

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

    2008-08-01

    Protein proteolysis is an essential component to proper cell function. Here, we demonstrate a method for studying protein degradation by detection of intermediate intracellular peptides with a high-precision tandem mass spectrometry de novo sequencing-based approach. From a Saccharomyces cerevisiae lysate, we identified >1,200 peptides containing 6-100 amino acids without random false positives and ascribed most identifications as being products of protein degradation. Most protein degradation observed was located in the cytoplasm, and multiple types of cleavage were found to exist in addition to the expected trypsin-like and chymotrypsin-like preferences. The yeast nucleus was found as a proteolysis-inert organelle under the conditions studied and the V-ATPase to be degraded during disassembly. Additionally, matrix associated mitochondrial proteins functioning as transport carriers and gates were found to be commonly degraded. Determining these protein degradation events could eventually aid in understanding of cell biology and detection and treatment of protein degradation-related diseases.

  10. Forage Management Effects on Protein and Fiber Fractions, Protein Degradability, and Dry Matter Yield of Red Clover Conserved as Silage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover (Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein...

  11. Protein degradation to low-molecular compounds after death and during reanimation.

    PubMed

    Konikova, A S; Vinarskaya, A A; Nikulin, V I; Pogossova, A V; Petukhova, L M

    1975-09-11

    The process of protein degradation to amino acids and peptides in rabbits following death and during reanimation in terms of the effects of artificial postmortem cooling on that process has been studied. Protein degradation was judged by increase of low-molecular nitrogenous compounds in serum and in organs by increase in soluble radioactivity with time in animals the proteins of which had been marked in vivo with radioisotopes. It has been found that immediately after death resulting from acute anoxia the processes of protein degradation to amino acids as well as synthesis stops in liver, skeletal and cardiac muscles, spleen, brain and spinal cord. Similar phenomenon takes place in the case of deep hypothermy. During reanimation the process of protein degradation to low-molecular compounds in organs restores. PMID:809921

  12. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    SciTech Connect

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  13. Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy.

    PubMed

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

    1986-11-01

    Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis. PMID:2946236

  14. Chain and pore-blocking effects on matrix degradation in protein-loaded microgels.

    PubMed

    Widenbring, Ronja; Frenning, Göran; Malmsten, Martin

    2014-10-13

    Factors affecting matrix degradation in protein-loaded microgels were investigated for dextran-based microgels, the sugar-binding protein Concanavalin A (ConA), and the dextran-degrading enzyme Dextranase. For this system, effects of enzyme, protein, and glucose concentrations, as well as pH, were considered. Microgel network degradation was monitored by micromanipulator-assisted light microscopy, whereas enzyme and protein distributions were monitored by confocal microscopy. Results show that Dextranase-mediated microgel degradation increased with increasing enzyme concentration, whereas an increased ConA loading in the dextran microgels caused a concentration-dependent decrease in microgel degradation. In the presence of glucose, competitive release of microgel-bound ConA restored the microgel degradation observed in the absence of ConA. To clarify effects of mass transport limitations, microgel degradation was compared to that of non-cross-linked dextran, demonstrating that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. The experimentally observed effects were qualitatively captured by a modified Michaelis-Menten approach for spherical symmetry, in which network blocking by ConA was included. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications. PMID:25144139

  15. Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

    PubMed

    Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

    2015-01-01

    Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. PMID:25240468

  16. The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

    PubMed

    Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R; Dutch, Rebecca E; Wei, Yinan

    2016-04-26

    ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro. PMID:27078234

  17. Redox modulation of cellular metabolism through targeted degradation of signaling proteins by the proteasome

    SciTech Connect

    Squier, Thomas C.

    2006-02-01

    Under conditions of oxidative stress, the 20S proteasome plays a critical role in maintaining cellular homeostasis through the selective degradation of oxidized and damaged proteins. This adaptive stress response is distinct from ubiquitin-dependent pathways in that oxidized proteins are recognized and degraded in an ATP-independent mechanism, which can involve the molecular chaperone Hsp90. Like the regulatory complexes 19S and 11S REG, Hsp90 tightly associates with the 20S proteasome to mediate the recognition of aberrant proteins for degradation. In the case of the calcium signaling protein calmodulin, proteasomal degradation results from the oxidation of a single surface exposed methionine (i.e., Met145); oxidation of the other eight methionines has a minimal effect on the recognition and degradation of calmodulin by the proteasome. Since cellular concentrations of calmodulin are limiting, the targeted degradation of this critical signaling protein under conditions of oxidative stress will result in the downregulation of cellular metabolism, serving as a feedback regulation to diminish the generation of reactive oxygen species. The targeted degradation of critical signaling proteins, such as calmodulin, can function as sensors of oxidative stress to downregulate global rates of metabolism and enhance cellular survival.

  18. Polyphenol, Conditioning, and Conservation Effects on Protein Fractions and Degradability in Forage Legumes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forage legume proteins were fractionated by the Cornell Net Carbohydrate and Protein System or ruminally incubated to assess how conditioning and conservation methods interact with polyphenols (condensed tannins or o-quinones) to alter protein degradability. The presence of polyphenols, conditioning...

  19. Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans.

    PubMed

    Burkewitz, Kristopher; Choe, Keith P; Lee, Elaine Choung-Hee; Deonarine, Andrew; Strange, Kevin

    2012-01-01

    Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50-70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50-80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70-180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought

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

    PubMed

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

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. PMID:26564029

  1. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins

    PubMed Central

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z.; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D.; Sheng, Yong; Crane, Denis I.; Florin, Timothy H.

    2013-01-01

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca2+ or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX’s suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER. PMID:23650437

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

    PubMed

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  3. Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase

    PubMed Central

    Kalveram, Birte

    2013-01-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses—i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus—has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  4. Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Zubenko, George S.; Jones, Elizabeth W.

    1981-01-01

    During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation. PMID:7021321

  5. Degradation-mediated protein quality control at the inner nuclear membrane

    PubMed Central

    Boban, Mirta; Foisner, Roland

    2016-01-01

    abstract An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals. PMID:26760377

  6. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    PubMed

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes. PMID:26521249

  7. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

    PubMed

    Maurer, Matthew J; Spear, Eric D; Yu, Allen T; Lee, Evan J; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  8. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  9. Interaction between resistin and adiponectin in the proliferation of rat vascular smooth muscle cells.

    PubMed

    Hirai, Hiroyuki; Satoh, Hiroaki; Kudoh, Akihiro; Watanabe, Tsuyoshi

    2013-02-01

    We investigated the effect between resistin and adiponectin on the proliferation of vascular smooth muscle cells (VSMCs). We confirmed that resistin significantly increases the number of rat VSMCs as well as thymidine incorporation with them, whereas adiponectin diminishes resistin-induced cell proliferation. Resistin significantly increased p42/44 mitogen-activated protein kinase (MAPK) phosphorylation within rat VSMCs, whereas adiponectin inhibited resistin-induced MAPK phosphorylation. Moreover, resistin significantly increased c-fos expression, whereas adiponectin suppressed resistin-induced c-fos expression. Cell cycle progression is a tightly controlled event that is negatively regulated by cyclin-dependent kinases inhibitors (CDKIs) such as p53, p21, and p27. Resistin significantly decreased the expression of these CDKIs, whereas adiponectin restored the resistin-induced decrease in CDKIs expression. These effects were abolished in the MAPK inhibitors. In conclusion, resistin plays a role in the development of atherosclerosis, whereas adiponectin may be an important in its prevention in insulin-resistant patients. PMID:23267839

  10. Differential Associations between CDH13 Genotypes, Adiponectin Levels, and Circulating Levels of Cellular Adhesive Molecules

    PubMed Central

    Teng, Ming-Sheng; Wu, Semon; Hsu, Lung-An; Chou, Hsin-Hua; Ko, Yu-Lin

    2015-01-01

    CDH13 gene variants with lower adiponectin levels are paradoxically associated with a more favorable metabolic profile. We investigated the statistical association between CDH13 locus variants and adiponectin levels by examining 12 circulating inflammation marker levels and adiposity status in 530 Han Chinese people in Taiwan. After adjustments for clinical covariates, adiponectin levels were positively associated with soluble vascular cell adhesion molecule-1 (sVCAM1) levels and negatively associated with adiposity status and levels of C-reactive protein (CRP), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule-1 (sICAM1). In addition, minor alleles of the CDH13 rs12051272 polymorphism were found to have lower adiponectin levels and higher CRP, sE-selectin, sICAM1, and sVCAM1 levels as well as higher body mass indices and waist circumferences in participants (all P < 0.05). In a subgroup analysis stratified by sex, significant associations between CDH13 genotypes and sE-selectin levels occurred only in men (P = 3.9 × 10−4 and interaction P = 0.005). CDH13 locus variants and adiponectin levels are associated with circulating levels of cellular adhesion molecules and adiposity status in a differential manner that interacts with sex. These results provide further evidence for the crucial role of adiponectin levels and CDH13 gene variants in immune-mediated and inflammatory diseases. PMID:26600672

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

    PubMed

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

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM). PMID:26641055

  12. Dual degradation signals control Gli protein stability and tumor formation

    PubMed Central

    Huntzicker, Erik G.; Estay, Ivette S.; Zhen, Hanson; Lokteva, Ludmila A.; Jackson, Peter K.; Oro, Anthony E.

    2006-01-01

    Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, DN and DC, and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. PMID:16421275

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

    NASA Astrophysics Data System (ADS)

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

    2008-07-01

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

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

    SciTech Connect

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2014-01-24

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

  15. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  16. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    PubMed Central

    Seo, J. K.; Kim, M. H.; Yang, J. Y.; Kim, H. J.; Lee, C. H.; Kim, K. H.; Ha, Jong K.

    2013-01-01

    A series of in vitro studies were carried out to determine i) the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii) the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS) in in vitro experiments. Untreated corn (C) and enzyme-treated corn (EC) were combined with soy bean meal with (ES) and without (S) enzyme treatment or formaldehyde treatment (FS). Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS) with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM) digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS). The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity. PMID:25049798

  17. Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

    PubMed

    Huang, Chun-Yin; Chang, An-Chen; Chen, Hsien-Te; Wang, Shih-Wei; Lo, Yuan-Shun; Tang, Chih-Hsin

    2016-09-01

    Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis. PMID:27252405

  18. ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

    SciTech Connect

    Charlton, Hayley K.; Webster, Julie; Kruger, Sarah; Simpson, Fiona; Richards, Ayanthi A.; Whitehead, Jonathan P.

    2010-02-05

    The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2{beta}, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

  19. Immunolocalization of Tom1 in relation to protein degradation systems in Alzheimer's disease.

    PubMed

    Makioka, Kouki; Yamazaki, Tsuneo; Takatama, Masamitsu; Ikeda, Masaki; Murayama, Shigeo; Okamoto, Koichi; Ikeda, Yoshio

    2016-06-15

    Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Its pathological hallmarks are senile plaques (SPs), which contain extracellular deposits of amyloid β (Aβ) protein fibrils and dystrophic neurites (DNs), and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau. Impairment of protein-degradation systems, including the ubiquitin-proteasome and the autophagy-lysosome systems, has been proposed as one of the causes of the accumulation of these aberrant proteins in AD brains. Tom1 (target of Myb1) was originally identified by the induction of its expression by the v-Myb oncogene and is a part of two major protein-degradation systems. The present study was conducted by immunohistochemical and immunofluorescent stainings to show that Tom1 was localized in DNs, perisomatic granules (PSGs), and NFTs in AD brains. Moreover, in DNs, Tom1 colocalized with ubiquitin, lysosomal proteins, and Tom1-related proteins (Tollip and myosin VI), which act in both protein-degradation systems via Tom1. These results indicate that Tom1 plays important roles in protein-degradation systems in AD pathogenesis. PMID:27206884

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  1. Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

    PubMed

    Tomoshige, Shusuke; Naito, Mikihiko; Hashimoto, Yuichi; Ishikawa, Minoru

    2015-10-14

    We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins. PMID:26338696

  2. Adiponectin Inhibits Nutrient Transporters and Promotes Apoptosis in Human Villous Cytotrophoblasts: Involvement in the Control of Fetal Growth.

    PubMed

    Duval, Fabien; Santos, Esther Dos; Poidatz, Dorothée; Sérazin, Valérie; Gronier, Héloïse; Vialard, François; Dieudonné, Marie-Noëlle

    2016-05-01

    The placenta exchanges nutrients between the mother and the fetus and requires a constant abundant energy supply. Adiponectin (a cytokine produced primarily by adipose tissue) controls glucose and lipid homeostasis. It is well-known that maternal serum adiponectin levels are inversely related to birth weight, suggesting that adiponectin has a negative effect on fetal growth. This effect appears to be related to the control of nutrient transporters in human placenta. However, the underlying molecular mechanisms have not yet been characterized. In the present work, we studied adiponectin's direct effect on human primary cytotrophoblasts from first-trimester placenta. Our result showed that in placental cells, adiponectin 1) inhibits the expression of the major glucose transporters (GLUT1 and GLUT12) and sodium-coupled neutral amino acid transporters (SNAT1, SNAT2, and SNAT4), 2) enhances total ATP production but decreases lactate production, 3) inhibits mitochondrial biogenesis and function, and 4) stimulates cell death by enhancing the expression of the pro-apoptotic B-cell lymphoma-2 (BCL-2)-associated X protein (BAX) and tumor protein P53 (TP53) gene expression and inducing the caspase activity. Small-interfering RNA mediating the down-regulation of adiponectin receptors (ADIPOR1 and ADIPOR2) was used to demonstrate that adiponectin effects on placental nutrient transport and apoptosis seemed to be essentially mediated by these specific receptors. Taken as a whole, these results strongly suggest that adiponectin regulates human placental function by limiting nutrient transporter expression and inducing apoptosis. These findings may help us to better understand adiponectin's role in placental pathologies such as intrauterine growth restriction, which is characterized by fetal weight loss and drastic apoptosis of placental cells. PMID:27030046

  3. Circulating Adiponectin and Risk of Endometrial Cancer

    PubMed Central

    Zheng, Qiaoli; Wu, Haijian; Cao, Jiang

    2015-01-01

    Background Adiponectin is an insulin-sensitizing hormone produced by adipocytes. It has been suggested to be involved in endometrial tumorigenesis. Published data have shown inconsistent results for the association between circulating adiponectin levels and endometrial cancer. In this study, we conducted a meta-analysis to evaluate the predictive value of circulating adiponectin levels on the development of endometrial cancer. Methods PubMed, Embase, ISI web of knowledge, and Cochrane databases were searched for all eligible studies, and the summary relative risk (SRR) was calculated. Additionally, we performed dose-response analysis with eight eligible studies. Results A total of 1,955 cases and 3,458 controls from 12 studies were included. The SRR for the ‘highest’ vs ‘lowest’ adiponectin levels indicated high adiponectin level reduced the risk of endometrial cancer [SRR = 0.40, 95% confidence interval (CI), 0.33–0.66]. Results from the subgroup analyses were consistent with the overall analysis. The SRR for each 1 µg/ml increase of adiponectin indicated a 3% reduction in endometrial cancer risk (95% CI: 2%–4%), and a 14% reduction for each increase of 5 µg/ml (95% CI: 9%–19%). No evidence of publication bias was found. Conclusions This meta-analysis demonstrates that low level of circulating adiponectin is a risk factor for endometrial cancer. PMID:26030130

  4. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed Central

    Carrión, Cristian A.; Martínez, Dana E.; Costa, M. Lorenza; Guiamet, Juan José

    2014-01-01

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves. PMID:27135516

  5. A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

    SciTech Connect

    Ju Donghong; Xie Youming . E-mail: xiey@karmanos.org

    2006-03-10

    The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a 'non-removable' N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting First example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.

  6. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    PubMed Central

    Kaushik, Susmita; Cuervo, Ana Maria

    2015-01-01

    Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LD) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation via CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LD. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs lead to reduced association of ATGL and macrolipophagy-related proteins with LD and the subsequent decrease in lipid oxidation and accumulation of LD. We propose a role of CMA in LD biology and in the maintenance of lipid homeostasis. PMID:25961502

  7. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

    PubMed

    Antunes, Ana T; Goos, Yvonne J; Pereboom, Tamara C; Hermkens, Dorien; Wlodarski, Marcin W; Da Costa, Lydie; MacInnes, Alyson W

    2015-07-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  8. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  9. Adiponectin upregulates ABCA1 expression through liver X receptor alpha signaling pathway in RAW 264.7 macrophages

    PubMed Central

    Liang, Bin; Wang, Xin; Guo, Xiaohong; Yang, Zhiming; Bai, Rui; Liu, Ming; Xiao, Chuanshi; Bian, Yunfei

    2015-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and anti-atherosclerosis. Liver X receptor alpha (LXRα) can stimulate cholesterol efflux through ABCA1. It has been well known that adiponectin has cardiovascular protection. In this study, we attempted to clarify the effect of adiponectin on expression of ABCA1, and explored the role of LXRα in the regulation of ABCA1 in RAW 264.7 macrophages. Our results showed that adiponectin increased ABCA1 expression at both the mRNA and protein levels in a dose-dependent and time-dependent manner. Consequently, adiponectin promoted cholesterol efflux and decreased cholesterol content in RAW 264.7 macrophages. Moreover, adiponectin up-regulated the expression of LXRα in a dose-dependent and time-dependent manner in RAW 264.7 macrophages. LXRα small interfering RNA completely abolished the promotion effects of adiponectin. In summary, adiponectin up-regulates ABCA1 expression via the LXRα pathway in RAW 264.7 macrophages. This novel insight could prove useful for developing new treatment strategies for cardiovascular diseases. PMID:25755733

  10. Adiponectin inhibits leptin-induced oncogenic signalling in oesophageal cancer cells by activation of PTP1B.

    PubMed

    Beales, Ian L P; Garcia-Morales, Carla; Ogunwobi, Olorunseun O; Mutungi, Gabriel

    2014-01-25

    Obesity is characterised by hyperleptinaemia and hypoadiponectinaemia and these metabolic abnormalities may contribute to the progression of several obesity-associated cancers including oesophageal adenocarcinoma (OAC). We have examined the effects of leptin and adiponectin on OE33 OAC cells. Leptin stimulated proliferation, invasion and migration and inhibited apoptosis in a STAT3-dependant manner. Leptin-stimulated MMP-2 secretion in a partly STAT3-dependent manner and MMP-9 secretion via a STAT3-independent pathway. Adiponectin inhibited leptin-induced proliferation, migration, invasion, MMP secretion and reduced the anti-apoptotic effects: these effects of adiponectin were ameliorated by both a non-specific tyrosine phosphatase inhibitor and a specific PTP1B inhibitor. Adiponectin reduced leptin-stimulated JAK2 activation and STAT3 transcriptional activity in a PTP1B-sensitive manner and adiponectin increased both PTP1B protein and activity. We conclude that adiponectin restrains leptin-induced signalling and pro-carcinogenic behaviour by inhibiting the early events in leptin-induced signal transduction by activating PTP1B. Relative adiponectin deficiency in obesity may contribute to the promotion of OAC. PMID:23994026

  11. Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system.

    PubMed

    Sung, Min-Kyung; Reitsma, Justin M; Sweredoski, Michael J; Hess, Sonja; Deshaies, Raymond J

    2016-09-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin-proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

  12. Effects of Adiponectin Including Reduction of Androstenedione Secretion and Ovarian Oxidative Stress Parameters In Vivo

    PubMed Central

    Comim, Fabio V.; Gutierrez, Karina; Bridi, Alessandra; Bochi, Guilherme; Chemeris, Raisa; Rigo, Melânia L.; Dau, Andressa Minussi P.; Cezar, Alfredo S.; Moresco, Rafael Noal; Gonçalves, Paulo Bayard Dias

    2016-01-01

    Adiponectin is the most abundantly produced human adipokine with anti-inflammatory, anti-oxidative, and insulin-sensitizing properties. Evidence from in vitro studies has indicated that adiponectin has a potential role in reproduction because it reduces the production of androstenedione in bovine theca cells in vitro. However, this effect on androgen production has not yet been observed in vivo. The current study evaluated the effect of adiponectin on androstenedione secretion and oxidative stress parameters in a rodent model. Seven-week-old female Balb/c mice (n = 33), previously treated with equine gonadotropin chorionic, were assigned to one of four different treatments: Group 1, control (phosphate-buffered saline); Group 2, adiponectin 0.1 μg/mL; Group 3, adiponectin 1.0 μg/mL; Group 4, adiponectin 5.0 μg/mL. After 24 h, all animals were euthanized and androstenedione levels were measured in the serum while oxidative stress markers were quantified in whole ovary tissue. Female mice treated with adiponectin exhibited a significant reduction (about 60%) in serum androstenedione levels in comparison to controls. Androstenedione levels decreased from 0.78 ± 0.4 ng/mL (mean ± SD) in controls to 0.28 ± 0.06 ng/mL after adiponectin (5 μg/mL) treatment (P = 0.01). This change in androgen secretion after 24 hours of treatment was associated with a significant reduction in the expression of CYP11A1 and STAR (but not CYP17A1). In addition, ovarian AOPP product levels, a direct product of protein oxidation, decreased significantly in adiponectin-treated mice (5 μg/mL); AOPP (mean ± SD) decreased to 4.3 ± 2.1 μmol/L in comparison with that of the controls (11.5 ± 1.7 μmol/L; P = 0.0003). Our results demonstrated for the first time that acute treatment with adiponectin reduced the levels of a direct oxidative stress marker in the ovary as well as decreased androstenedione serum levels in vivo after 24 h. PMID:27158926

  13. AdipoR-increased intracellular ROS promotes cPLA2 and COX-2 expressions via activation of PKC and p300 in adiponectin-stimulated human alveolar type II cells.

    PubMed

    Chen, Hsiao-Mei; Yang, Chuen-Mao; Chang, Jia-Feng; Wu, Chi-Sheng; Sia, Kee-Chin; Lin, Wei-Ning

    2016-08-01

    Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases. PMID:27288489

  14. Adiponectin induces A20 expression in adipose tissue to confer metabolic benefit.

    PubMed

    Hand, Laura E; Usan, Paola; Cooper, Garth J S; Xu, Lance Y; Ammori, Basil; Cunningham, Peter S; Aghamohammadzadeh, Reza; Soran, Handrean; Greenstein, Adam; Loudon, Andrew S I; Bechtold, David A; Ray, David W

    2015-01-01

    Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα(-/-) mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3β activation and induction of A20. Attenuated inflammatory responses in Reverbα(-/-) WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery-induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology. PMID:25190567

  15. Adiponectin Induces A20 Expression in Adipose Tissue To Confer Metabolic Benefit

    PubMed Central

    Hand, Laura E.; Usan, Paola; Cooper, Garth J. S.; Xu, Lance Y.; Ammori, Basil; Cunningham, Peter S.; Aghamohammadzadeh, Reza; Soran, Handrean; Greenstein, Adam; Loudon, Andrew S. I.; Bechtold, David A.; Ray, David W.

    2015-01-01

    Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα−/− mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3β activation and induction of A20. Attenuated inflammatory responses in Reverbα−/− WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery–induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology. PMID:25190567

  16. In vitro ruminal degradation and synthesis of protein on fractions extracted from alfalfa hay and silage.

    PubMed

    Peltekova, V D; Broderick, G A

    1996-04-01

    Net release of degraded N as NH3 and total AA plus microbial protein synthesis, quantified from incorporation of 15NH3 into microbial protein, was used to estimate the rate and extent of in vitro degradation of protein fractions isolated from alfalfa hay and silage. Seven proteins (casein, alfalfa hay, alfalfa silage, extracts from alfalfa hay and silage, and residues from alfalfa hay and silage) were studied. Results from (NH4)2SO4 and SDS-PAGE fractionations suggested that soluble proteins in alfalfa hay and silage differed in susceptibility to proteolytic attack. Although the net release of NH3 plus total AA N from alfalfa silage and alfalfa silage extract was twofold greater than that from alfalfa hay and alfalfa hay extract, net microbial protein synthesis on alfalfa hay and alfalfa hay extract was 33 and 43% greater. Despite greater NPN content in alfalfa silage, protein degradation rate and estimated escape were similar for intact alfalfa hay (0.103/h and 43%) and silage (0.067/h and 43%). This result might be explained by the less efficient microbial utilization of silage NPN, greater protozoal numbers on hay, greater soluble true protein in hay, or differences in molecular mass and stability of soluble proteins in hay versus silage. Use of a two-compartment model, based on water-soluble and insoluble CP fractions assumed to pass with the liquid and solid phases, respectively, yielded RUP estimates for alfalfa hay and silage that were similar to NRC estimates. PMID:8744226

  17. Aphid salivary proteases are capable of degrading sieve-tube proteins.

    PubMed

    Furch, Alexandra C U; van Bel, Aart J E; Will, Torsten

    2015-02-01

    Sieve tubes serve as transport conduits for photo-assimilates and other resources in angiosperms and are profitable targets for piercing-sucking insects such as aphids. Sieve-tube sap also contains significant amounts of proteins with diverse functions, for example in signalling, metabolism, and defence. The identification of salivary proteases in Acyrthosiphon pisum led to the hypothesis that aphids might be able to digest these proteins and by doing so suppress plant defence and access additional nitrogen sources. Here, the scarce knowledge of proteases in aphid saliva is briefly reviewed. In order to provide a better platform for discussion, we conducted a few tests on in vitro protease activity and degradation of sieve-tube sap proteins of Cucurbita maxima by watery saliva. Inhibition of protein degradation by EDTA indicates the presence of different types of proteases (e.g. metalloproteses) in saliva of A. pisum. Proteases in the watery saliva from Macrosiphum euphorbiae and A. pisum were able to degrade the most abundant phloem protein, which is phloem protein 1. Our results provide support for the breakdown of sieve-element proteins by aphid saliva in order to suppress/neutralize the defence responses of the plant and to make proteins of sieve-tube sap accessible as a nitrogen source, as is discussed in detail. Finally, we discuss whether glycosylation of sieve-element proteins and the presence of protease inhibitors may confer partial protection against the proteolytic activity of aphid saliva. PMID:25540441

  18. Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes*

    PubMed Central

    Bhutani, Nidhi; Piccirillo, Rosanna; Hourez, Raphael; Venkatraman, Prasanna; Goldberg, Alfred L.

    2012-01-01

    In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins. PMID:22451661

  19. Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins

    PubMed Central

    Neklesa, Taavi K.; Tae, Hyun Seop; Schneekloth, Ashley R.; Stulberg, Michael J.; Corson, Timothy W.; Sundberg, Thomas B.; Raina, Kanak; Holley, Scott A.; Crews, Craig M.

    2011-01-01

    The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. PMID:21725302

  20. High Molecular Weight Adiponectin and Incident Ischemic Stroke in Postmenopausal Women: A Women’s Health Initiative Study

    PubMed Central

    Ogorodnikova, Alexandra D.; Wassertheil-Smoller, Sylvia; Mancuso, Peter; Sowers, MaryFran R.; Rajpathak, Swapnil N.; Allison, Matthew A.; Baird, Alison E.; Rodriguez, Beatriz; Wildman, Rachel P.

    2010-01-01

    Background and Purpose While low levels of adiponectin are associated with coronary heart disease and cardiovascular disease (CVD) risk factors, it is unclear whether adiponectin levels are related to the risk of developing ischemic stroke. Methods We examined the relationship between baseline high molecular weight (HMW) adiponectin levels and incident ischemic stroke in postmenopausal women, using data and specimens from the Hormones and Biomarkers Predicting Stroke Study, a case-control study nested within the Women’s Health Initiative Observational Study. Included were 855 incident ischemic stroke cases and 855 controls, matched for age, race-ethnicity, date of entry into the cohort, and follow-up time. Odds ratios of incident ischemic stroke associated with baseline HMW adiponectin levels were calculated using conditional logistic regression modeling, adjusting for body mass index (BMI), type 2 diabetes, hypertension, smoking, LDL-C, HDL-C, physical activity, C-reactive protein, and aspirin use. Results Lower levels of HMW adiponectin were significantly associated with type 2 diabetes, hypertension, higher BMI, waist, glucose, and insulin levels, and lower HDL-C levels. The distribution of incident stroke cases by HMW adiponectin quartiles was 49.9%, 50.5%, 50.7%, and 48.9%, respectively (p =0.96). Multivariable-adjusted odds ratios of stroke associated with the top three quartiles of HMW adiponectin versus the first quartile were 0.99 (95%CI 0.71 to 1.37), 1.37 (0.99 to 1.91), and 1.25 (0.88 to 1.79), respectively (p-trend =0.14). Conclusion Despite moderate associations between HMW adiponectin and CVD risk factors, we found no evidence of an association between HMW adiponectin levels and incident ischemic stroke in these postmenopausal women. PMID:20508194

  1. Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

    PubMed Central

    Boname, Jessica M.; Bloor, Stuart; Wandel, Michal P.; Nathan, James A.; Antrobus, Robin; Dingwell, Kevin S.; Thurston, Teresa L.; Smith, Duncan L.; Smith, James C.; Randow, Felix

    2014-01-01

    The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover. PMID:24958774

  2. Adiponectin as a Protective Factor Against the Progression Toward Type 2 Diabetes Mellitus in Postmenopausal Women

    PubMed Central

    Darabi, Hossein; Raeisi, Alireza; Kalantarhormozi, Mohammad Reza; Ostovar, Afshin; Assadi, Majid; Asadipooya, Kamyar; Vahdat, Katayoun; Dobaradaran, Sina; Nabipour, Iraj

    2015-01-01

    Abstract Serum adiponectin levels have been suggested to be predictors of type 2 diabetes mellitus in diverse populations. However, the relationship between circulating adiponectin levels and the risk of development of type 2 diabetes in postmenopausal women has not been investigated. A total of 382 healthy postmenopausal women who participated in a prospective cohort study were followed for 5.8 years. Type 2 diabetes mellitus was defined according to the criteria set out by the American Diabetes Association. Adiponectin, osteoprotegerin (OPG), and high-sensitivity C-reactive protein (hs-CRP) levels were measured using ELISA. Of 195 women who did not have diabetes at baseline and who were reexamined in the second phase of the study for diabetic status, 35 subjects (17.9%) developed type 2 diabetes mellitus during the 5.8 years follow-up period. The women with type 2 diabetes had lower adiponectin levels than the healthy postmenopausal women. Multiple regression analysis showed that, after adjustments were made for age, cardiovascular risk factors, OPG, and hs-CRP levels, higher baseline adiponectin levels were associated with a lower relative risk (RR) of having type 2 (RR = 0.07, confidence interval [CI]: 0.01–0.66, P = 0.021). Higher baseline adiponectin levels functioned as a predictor of a lower risk of developing type 2 diabetes mellitus among postmenopausal women during a 5.8 years follow-up study. Therefore, it is suggested that elevated adiponectin levels may offer protection against the development of type 2 diabetes mellitus after the menopause. PMID:26287420

  3. Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose Tissue Cells: Paracrine Consequences on Vascular Endothelium.

    PubMed

    Fernández-Trasancos, Ángel; Guerola-Segura, Raquel; Paradela-Dobarro, Beatriz; Álvarez, Ezequiel; García-Acuña, José María; Fernández, Ángel Luis; González-Juanatey, José Ramón; Eiras, Sonia

    2016-05-01

    Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin-sensitizer, anti-inflammatory and anti-atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200, or 295 mg/dl glucose, with or without macrophage-conditioned medium (MCM). Expression of adiponectin, GLUT-4 and the insulin receptor was analyzed by real-time PCR. The paracrine effect of stromal cells was determined in co-cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte-conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U-shaped dose-response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis-induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell-conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis-induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression. PMID:26406271

  4. The Adiponectin Variants Contribute to the Genetic Background of Type 2 Diabetes in Turkish Population.

    PubMed

    Arikoglu, Hilal; Ozdemir, Hulya; Kaya, Dudu Erkoc; Ipekci, Suleyman Hilmi; Arslan, Ahmet; Kayis, Seyit Ali; Gonen, Mustafa Sait

    2013-10-26

    Adiponectin, an adipose tissue specific protein encoded by the Adiponectin gene, modulates insulin sensitivity and plays an important role in regulating energy homeostasis. Many studies have shown that single nucleotide polymorphisms (SNPs) in the Adiponectin gene are associated with low plasma adiponectin levels, insulin resistance and an increased risk of type 2 diabetes mellitus. The aim of the present study was to evaluate the contribution of the Adiponectin gene polymorphisms in genetic background of type 2 diabetes in a Turkish population. In total, 169 unrelated and non-obese diabetic patients and 119 age- and BMI-matched non-diabetic individuals with no family history of diabetes were enrolled in this study. We detected a significant association between type 2 diabetes and two SNPs: SNP -11391G>A, which is located in the promoter region of the Adiponectin gene, and SNP +276G>T, which is found in intron 2 of the gene (P<0.05). The silence SNP G15G (+45T>G) in exon 1 and SNP +349A>G in intron 2 also showed a weak association with type 2 diabetes (P=0.06 and P=0.07, respectively), while SNPs -3971A>G in intron 1 and Y111H, R112C and H241P in exon 3 showed no association (P>0.05). In conclusion, these findings suggest that Adiponectin gene polymorphisms might be effective on susceptibility for type 2 diabetes development which emerged from the interactions between multiple genes, variants and environmental factors. PMID:24513330

  5. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  6. E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity.

    PubMed

    Mishra, Amit; Dikshit, Priyanka; Purkayastha, Sudarshana; Sharma, Jaiprakash; Nukina, Nobuyuki; Jana, Nihar Ranjan

    2008-03-21

    The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases. PMID:18201976

  7. Reduction-oxidation state and protein degradation in skeletal muscle of fasted and refed rats

    NASA Technical Reports Server (NTRS)

    Fagan, Julie M.; Tischler, Marc E.

    1986-01-01

    Redox state and protein degradation were measured in isolated muscles of fasted (up to 10 d) and refed (up to 4 d) 7- to 14-wk-old rats. Protein degradation in the extensor digitorum longus muscle, but not in the soleus muscle, was greater in the fasted rats than in weight-matched muscle from fed rats. The NAD couple was more oxidized in incubated and fresh extensor digitorum longus muscles and in some incubated soleus muscles of fasted rats than in weight-matched muscle from fed rats. In the extensor digitorum longus muscle of refed or prolonged fasted rats, protein degradation was slower and the NAD couple was more reduced than in the fed state. Therefore, oxidation of the NAD couple was associated with increased muscle breakdown during fasting, whereas reduction of the NAD couple was associated with muscle conservation and deposition.

  8. Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli.

    PubMed

    Betton, J M; Sassoon, N; Hofnung, M; Laurent, M

    1998-04-10

    The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins. PMID:9535871

  9. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  10. LOW CIRCULATING MATERNAL ADIPONECTIN IN PATIENTS WITH PYELONEPHRITIS: ADIPONECTIN AT THE CROSSROADS OF PREGNANCY AND INFECTION

    PubMed Central

    Mazaki-Tovi, Shali; Romero, Roberto; Vaisbuch, Edi; Chaiworapongsa, Tinnakorn; Erez, Offer; Mittal, Pooja; Kim, Sun Kwon; Gotsch, Francesca; Lamont, Ronald; Ogge, Giovanna; Pacora, Percy; Goncalves, Luis; Kim, Chong Jai; Gomez, Ricardo; Espinoza, Jimmy; Hassan, Sonia S.; Kusanovic, Juan Pedro

    2009-01-01

    Objective An emerging theme in modern biology is that adipose tissue can respond to metabolic stress, and to inflammatory stimuli, by regulating the secretion of a complex network of soluble mediators, termed adipokines. Adiponectin, the most prevalent circulating adipokine in human, has profound insulin-sensitizing and anti-inflammatory properties. Indeed, the notion that adiponectin plays an important role in the interactions between the metabolic and the immune systems has been strongly suggested. Thus, the aim of this study was to determine if pyelonephritis during pregnancy is associated with changes in maternal serum adiponectin concentrations. Study design This cross-sectional study included women in the following groups: 1) normal pregnant women (n=200); and 2) pregnant women with pyelonephritis (n=50). Maternal plasma adiponectin concentrations were determined by ELISA. Non-parametric statistics were used for analyses. Results 1) The median maternal plasma adiponectin concentration was lower in patients with pyelonephritis than in those with a normal pregnancy (p<0.001); 2) among pregnant women with a normal weight, patients with pyelonephritis had a lower median plasma adiponectin concentration than those with a normal pregnancy (p<0.001); 3) similarly, among overweight/obese patients, those with pyelonephritis had a lower median plasma adiponectin concentration than those with a normal pregnancy (p<0.001); and 4) the presence of pyelonephritis was independently associated with maternal plasma adiponectin concentrations after adjustment for maternal age, smoking, gestational age at sampling, and pre-gestational BMI. Conclusion 1) The findings that acute pyelonephritis in pregnancy is characterized by low maternal plasma concentrations of adiponectin in both lean and overweight/obese patients are novel and concur with the anti-inflammatory properties of adiponectin; and 2) the results of this study support the notion that adiponectin may play a role in the

  11. CHIP(-/-)-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications.

    PubMed

    Kim, Sung-Mi; Grenert, James P; Patterson, Cam; Correia, Maria Almira

    2016-01-01

    Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O2 species (ROS) production, triggering oxidative/proteotoxic stress associated with sustained activation of c-Jun NH2-terminal kinase (JNK)-signaling cascades, pro-inflammatory effectors/cytokines, insulin resistance, progressive hepatocellular ballooning and microvesicular steatosis. Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP(-/-)-mice over the first 8-9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5'-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP(-/-)-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models. PMID:27406999

  12. CHIP−/−-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications

    PubMed Central

    Kim, Sung-Mi; Grenert, James P.; Patterson, Cam; Correia, Maria Almira

    2016-01-01

    Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O2 species (ROS) production, triggering oxidative/proteotoxic stress associated with sustained activation of c-Jun NH2-terminal kinase (JNK)-signaling cascades, pro-inflammatory effectors/cytokines, insulin resistance, progressive hepatocellular ballooning and microvesicular steatosis. Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP−/−-mice over the first 8–9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5′-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP−/−-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models. PMID:27406999

  13. Mechanochemical basis of protein degradation by a double-ring AAA+ machine.

    PubMed

    Olivares, Adrian O; Nager, Andrew R; Iosefson, Ohad; Sauer, Robert T; Baker, Tania A

    2014-10-01

    Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates. PMID:25195048

  14. Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster.

    PubMed

    Trost, Martina; Blattner, Ariane C; Lehner, Christian F

    2016-01-01

    The analysis of consequences resulting after experimental elimination of gene function has been and will continue to be an extremely successful strategy in biological research. Mutational elimination of gene function has been widely used in the fly Drosophila melanogaster. RNA interference is used extensively as well. In the fly, exceptionally precise temporal and spatial control over elimination of gene function can be achieved in combination with sophisticated transgenic approaches and clonal analyses. However, the methods that act at the gene and transcript level cannot eliminate protein products which are already present at the time when mutant cells are generated or RNA interference is started. Targeted inducible protein degradation is therefore of considerable interest for controlled rapid elimination of gene function. To this end, a degradation system was developed in yeast exploiting TIR1, a plant F box protein, which can recruit proteins with an auxin-inducible degron to an E3 ubiquitin ligase complex, but only in the presence of the phytohormone auxin. Here we demonstrate that the auxin-inducible degradation system functions efficiently also in Drosophila melanogaster. Neither auxin nor TIR1 expression have obvious toxic effects in this organism, and in combination they result in rapid degradation of a target protein fused to the auxin-inducible degron. PMID:27010248

  15. Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation

    PubMed Central

    Wang, Shiuan; Srivastava, Mayank; Broemer, Meike; Meier, Pascal; Bergmann, Andreas

    2011-01-01

    Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs. PMID:21909282

  16. Degradation of antenna chlrophyll-binding protein CP43 during photoinhibition of photosystem II

    SciTech Connect

    Yamamoto, Yasusi; Akasaka, Tatsuya

    1995-07-18

    When photosystem II (PS II) membranes from spinach were treated with Tris (0.8 M, pH 9.0) and illuminated with white light (5000 {mu}E m{sup -2} s{sup -1}) under aerobic conditions at 25{degrees}C, not only were the reaction center-forming D1 and D2 proteins degraded but the antenna chlorophyll-binding protein CP43 was also degraded. Three products of the degradation of CP43, with molecular masses of 17.0, 15.5, and 14 kDa, respectively, were identified by sodium dodecyl sulfate/urea polyacrylamide gel electrophoresis and Western blotting with a specific antibody. Degradation products of another antenna chlorophyll-binding protein of PS II, CP47, were not detected under the same conditions. Concomitant with the damage to the D1 and D2 proteins and CP43, cross-linked products of the D1 protein, CP43, and CP47 were formed. These products were identified as slow-moving smeared bands in the higher molecular weight range of the gel during electrophoresis. Both the degradation and the cross-linking of these proteins were prevented by the addition of electron donors to PS II, a result that suggests that these processes were caused by the donor-side mechanism of photoinhibition. The photoinduced degradation of CP43 and the cross-linking among the D1 protein, CP43, and CP47 were less obvious in the PS II membranes that had been treated with hydroxylamine rather than Tris and in the membranes that had been treated with Tris and reconstituted by addition of an extrinsic 33-kDa protein (OEC33). These results indicate that removal of OEC33, which is closely associated with CP43, from the PS II complex accelerates the degradation and cross-linking of CP43 during photoinhibition. It is suggested that OEC33 is involved in the stabilization of the antenna chlorophyll-binding proteins in PS II during photoinhibition. 55 refs., 7 figs.

  17. Structural and Functional Similarities between Osmotin from Nicotiana Tabacum Seeds and Human Adiponectin

    PubMed Central

    Colonna, Giovanni

    2011-01-01

    Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm in vivo the molecular modelling data, osmotin has been purified from nicotiana tabacum seeds and its nine residues peptide synthesized. We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico. PMID:21311758

  18. Systematic Protein Level Regulation via Degradation Machinery Induced by Genotoxic Drugs.

    PubMed

    Kume, Kohei; Ishida, Kazushige; Ikeda, Miyuki; Takemoto, Kazuhiro; Shimura, Tsutomu; Young, Lynn; Nishizuka, Satoshi S

    2016-01-01

    In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs. PMID:26625007

  19. Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

    PubMed

    Yoshikawa, Rokusuke; Izumi, Taisuke; Nakano, Yusuke; Yamada, Eri; Moriwaki, Miyu; Misawa, Naoko; Ren, Fengrong; Kobayashi, Tomoko; Koyanagi, Yoshio; Sato, Kei

    2016-06-01

    Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3. PMID:27193350

  20. Hypoglycemic effects of three Iranian edible plants; jujube, barberry and saffron: Correlation with serum adiponectin level.

    PubMed

    Hemmati, Mina; Asghari, Somaye; Zohoori, Elham; Karamian, Mehdi

    2015-11-01

    One of the most common disorders of the endocrine system is diabetes mellitus. This disease is associated with dyslipidemia. Adiponectin is a protein hormone that secreted by adipocytes and has an important role in regulating of glucose and fatty acid metabolic pathways. This study was designed to investigate the changes in serum level of adiponectin in diabetic rats treated with hydroalcoholic extracts of three medicinal plants; jujube (Ziziphus jujuba), barberry (Berberis vulgaris) and saffron (Crocus sativus) in comparison with quercetin. Streptozotocin -induced diabetic male rats were gavaged with specified doses of the extracts (25 and 100mg/kg) for two weeks. At the end of treatment period, fasting blood specimens were collected. The levels of adiponectin, fasting blood sugar (FBS), total Cholesterol, triglycerides, HDL-C and LDL-C were measured. Statistical analysis showed that serum levels of triglyceride and VLDL decreased significantly (P<0.05) in all treated groups. FBS level in all treated groups, decreased significantly and reach to normoglycemic level (P<0.05). Except Jujube, other plant extracts had no effect on cholesterol. Jujube in two doses (25 and 100mg/kg) could increased significantly HDL-C (P<0.05) with no effect on total cholesterol and LDL-C. Serum adiponectin level increased in all treated groups. These beneficial effects of C. sativus, B. vulgaris and Z. jujube extracts and quercetin in diabetic rats may be associated with increase in adiponectin level. PMID:26639503

  1. The regulation of adiponectin receptors in human prostate cancer cell lines

    SciTech Connect

    Mistry, T.; Digby, J.E.; Chen, J.; Desai, K.M.; Randeva, H.S. . E-mail: H.Randeva@warwick.ac.uk

    2006-09-29

    Obesity is a risk factor for prostate cancer, and plasma levels of the adipokine, adiponectin, are low in the former but high in the latter. Adiponectin has been shown to modulate cell proliferation and apoptosis, suggesting that adiponectin and its receptors (Adipo-R1, Adipo-R2) may provide a molecular association between obesity and prostate carcinogenesis. We show for First time, the protein distribution of Adipo-R1 and Adipo-R2 in LNCaP and PC3 cells, and in human prostate tissue. Using real-time RT-PCR we provide novel data demonstrating the differential regulation of Adipo-R1 and Adipo-R2 mRNA expression by testosterone, 5-{alpha} dihydrotestosterone, {beta}-estradiol, tumour necrosis factor-{alpha}, leptin, and adiponectin in LNCaP and PC3 cells. Our findings suggest that adiponectin and its receptors may contribute to the molecular association between obesity and prostate cancer through a complex interaction with other hormones and cytokines that also play important roles in the pathophysiology of obesity and prostate cancer.

  2. Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

    SciTech Connect

    Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi; Hirai, Shizuka; Ohyama, Kana; Kusudo, Tatsuya; Yu, Rina; Yano, Masamichi; Sasaki, Takao; Takahashi, Nobuyuki; Kawada, Teruo

    2008-02-01

    Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-{alpha} and PPAR{gamma}. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPAR{gamma} antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPAR{gamma} activations. The results indicate that auraptene activates PPAR{gamma} in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.

  3. [Considerations about study on the underlying mechanism of acu-moxibustion in the treatment of obesity type polycystic ovary syndrome by regulating adiponectin].

    PubMed

    Liao, Yan-Jun; Shi, Yin; Yu, Li-Qing; Fang, Jian-Qiao

    2012-02-01

    Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease in women of reproductive age, and the obesity and insulin resistance are considered to be the key link in the pathophysiological process of PCOS of obesity type. Adiponectin, a protein hormone, is closely related to insulin resistance and obesity, which has been being researched extensively in recent years. The authors of the present article review the pathogenesis of PCOS of obesity type from the relationship between adiponectin and obesity, and between adiponectin and insulin resistance, separately. In particular, the authors review studies on the underlying mechanism of acupuncture and moxibustion interventions in regulating adiponectin level briefly. The authors think of that acupuncture and moxibustion interventions induced increase of adiponectin level is possibly to improve insulin resistance in obesity and/or PCOS patients, hoping to provide a new target for clinical treatment of PCOS. PMID:22574574

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

    PubMed Central

    Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

    2015-01-01

    Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system. PMID:26090408

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

    PubMed

    Martínez-Castillo, Moisés; Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

    2015-01-01

    Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system. PMID:26090408

  6. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

    PubMed

    Braten, Ori; Livneh, Ido; Ziv, Tamar; Admon, Arie; Kehat, Izhak; Caspi, Lilac H; Gonen, Hedva; Bercovich, Beatrice; Godzik, Adam; Jahandideh, Samad; Jaroszewski, Lukasz; Sommer, Thomas; Kwon, Yong Tae; Guharoy, Mainak; Tompa, Peter; Ciechanover, Aaron

    2016-08-01

    The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes. PMID:27385826

  7. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

    PubMed Central

    Pittman, R C; Carew, T E; Glass, C K; Green, S R; Taylor, C A; Attie, A D

    1983-01-01

    We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples. PMID:6882394

  8. Relation of Adiponectin to All-Cause Mortality, Cardiovascular Mortality, and Major Adverse Cardiovascular Events (from the Dallas Heart Study).

    PubMed

    Witberg, Guy; Ayers, Colby R; Turer, Aslan T; Lev, Eli; Kornowski, Ran; de Lemos, James; Neeland, Ian J

    2016-02-15

    Adiponectin is a key component in multiple metabolic pathways. Studies evaluating associations of adiponectin with clinical outcomes in older adults have reported conflicting results. We investigated the association of adiponectin with mortality and cardiovascular disease (CVD) morbidity in a young, multiethnic adult population. We analyzed data from participants in the Dallas Heart Study without baseline CVD who underwent assessment of total adiponectin from 2000 to 2002. The primary outcome of all-cause mortality was assessed over median 10.4 years of follow-up using multivariable-adjusted Cox proportional hazards models. Secondary outcomes included CVD mortality, major adverse cardiovascular and cerebrovascular events (MACCE), and heart failure (HF). The study cohort included 3,263 participants, mean age 43.4 years, 44% women, and 50% black. There were 184 deaths (63 CVD), 207 MACCE, and 46 HF events. In multivariable models adjusted for age, gender, race, hypertension, diabetes, smoking, high-density lipoprotein cholesterol-C, hyperlipidemia, high-sensitivity C-reactive protein level, estimated glomerular filtration rate, and body mass index, increasing adiponectin quartiles were positively associated with all-cause mortality Q4 versus Q1 (hazard ratio [HR] = 2.27; 95% confidence interval [CI] 1.47, 3.50); CVD mortality Q4 versus Q1 (HR = 2.43; 95% CI 1.15, 5.15); MACCE Q4 versus Q1 (HR = 1.71; 95% CI 1.13, 2.60); and HF Q4 versus Q1 (HR = 2.95; 95% CI 1.14, 7.67). Findings were similar with adiponectin as a continuous variable and consistent across subgroups defined by age, gender, race, obesity, diabetes, metabolic syndrome, or elevated high-sensitivity C-reactive protein. In conclusion, higher adiponectin was associated with increased mortality and CVD morbidity in a young, multiethnic population. These findings may have implications for strategies aimed at lowering adiponectin to prevent adverse outcomes. PMID:26800774

  9. Adiponectin promotes VEGF-A-dependent angiogenesis in human chondrosarcoma through PI3K, Akt, mTOR, and HIF-α pathway

    PubMed Central

    Shih, Jhao-Sheng; Fong, Yi-Chin; Wang, Shih-Wei; Li, Te-Mao; Tang, Chih-Hsin

    2015-01-01

    Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. On the other hand, angiogenesis is a critical step in tumor growth and metastasis. However, the relationship of adiponectin with vascular endothelial growth factor-A (VEGF-A) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study we first demonstrated that the expression of adiponectin was correlated with tumor stage of human chondrosarcoma tissues. In addition, we also found that adiponectin increased VEGF-A expression in human chondrosarcoma cells and subsequently induced migration and tube formation in human endothelial progenitor cells (EPCs). Adiponectin promoted VEGF-A expression through adiponectin receptor (AdipoR), phosphoinositide 3 kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF)-1α signaling cascades. Knockdown of adiponectin decreased VEGF-A expression and also abolished chondrosarcoma conditional medium-mediated tube formation in EPCs in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. Therefore, adiponectin is crucial for tumor angiogenesis and growth, which may represent a novel target for anti-angiogenic therapy in human chondrosarcoma. PMID:26468982

  10. LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and inhibition of migration and invasion of breast cancer cells

    PubMed Central

    Taliaferro-Smith, L; Nagalingam, A; Zhong, D; Zhou, W; Saxena, NK; Sharma, D

    2010-01-01

    Adiponectin is widely known as an adipocytokine with therapeutic potential for its markedly protective function in the pathogenesis of obesity-related disorders, metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. In the present study, we show that adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Further analysis of the underlying molecular mechanisms revealed that adiponectin treatment increased AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase and inhibition of p70S6 kinase (S6K). Intriguingly, we discovered that adiponectin treatment increases the expression of tumor suppressor gene LKB1 in breast cancer cells. Overexpression of LKB1 in breast cancer cells further increased adiponectin-mediated phosphorylation of AMPK. Using isogenic LKB1 knockdown cell line pair, we found that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and more importantly, inhibition of adhesion, migration and invasion of breast cancer cells. Taken together these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Our findings indicate the possibility of using adiponectin analogues to inhibit invasion and migration of breast cancer cells. PMID:19483724

  11. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease

    PubMed Central

    Shin, Yongdae; Davis, Joseph H.; Brau, Ricardo R.; Martin, Andreas; Kenniston, Jon A.; Baker, Tania A.; Sauer, Robert T.; Lang, Matthew J.

    2009-01-01

    ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5′-[γ-thio]triphosphate (ATPγS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function. PMID:19892734

  12. Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance.

    PubMed

    Yang, Lihua; Harding, Jason D; Ivanov, Alexander V; Ramasubramanyan, Natarajan; Dong, Diane D

    2015-03-13

    Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS-PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions. PMID:25680549

  13. Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

    PubMed

    Armenti, Stephen T; Lohmer, Lauren L; Sherwood, David R; Nance, Jeremy

    2014-12-01

    The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events. PMID:25377555

  14. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

    PubMed Central

    Wagner, I; Arlt, H; van Dyck, L; Langer, T; Neupert, W

    1994-01-01

    ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria. Images PMID:7957078

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

    PubMed

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

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group. PMID:17869110

  16. Carbohydrate Source and Protein Degradability Alter Lactation, Ruminal, and Blood Measures.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effects of nonfiber carbohydrate source (NFC) and protein degradability (RDP) in the diets of lactating dairy cattle on intake, production, efficiency, and ruminal measures were evaluated in a three period (21 d) partially balanced incomplete latin square design with a 3x2 factorial arrangement of t...

  17. EFFECTS OF FEEDING DAIRY COWS PROTEIN SUPPLEMENTS OF VARYING RUMINAL DEGRADABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-five (10 ruminally cannulated) Holstein cows averaging 82 ± 34 days in milk were assigned to 5x5 Latin squares (21-d periods) and fed diets supplemented with one of four different proteins to assess effects on production, ruminal metabolism, omasal flow of N fractions, and degradation rates o...

  18. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    PubMed

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

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  19. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    ERIC Educational Resources Information Center

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  20. Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components*

    PubMed Central

    Lee, Kyoung-Hee; Biswas, Amlan; Liu, Yuen-Joyce; Kobayashi, Koichi S.

    2012-01-01

    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. PMID:23019338

  1. Developing an in vitro method for determining feed soluble protein degradation rate by mixed ruminal microorganisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For the purposes of ration balancing using predictive computer models, more accurate content and rumen soluble protein degradability values are needed, especially for highly processed feeds. Consequently, a standardized method of determination is needed. Hence, a novel ruminal in vitro method is d...

  2. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. Rates of protein degradation were determined in response to minimal essential media (MEM) of various amino acid (AA)...

  3. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  4. Serum Adiponectin Levels, Neuroimaging, and Cognition in the Mayo Clinic Study of Aging

    PubMed Central

    Wennberg, Alexandra M. V.; Gustafson, Deborah; Hagen, Clinton E.; Roberts, Rosebud O.; Knopman, David; Jack, Clifford; Petersen, Ronald C.; Mielke, Michelle M.

    2016-01-01

    BACKGROUND Adiponectin, a protein involved in inflammatory pathways, may impact the development and progression of Alzheimer’s disease (AD). Adiponectin levels have been associated with mild cognitive impairment (MCI) and AD; however, its association with Alzheimer-associated neuroimaging and cognitive outcomes is unknown. OBJECTIVE Determine the cross-sectional association between plasma adiponectin and neuroimaging and cognitive outcomes in an older population-based sample. METHODS Multivariable adjusted regression models were used to investigate the association between plasma adiponectin and hippocampal volume (HVa), PiB-PET, FDG PET, cortical thickness, MCI diagnosis, and neuropsychological test performance. Analyses included 535 non-demented participants aged 70 and older enrolled in the Mayo Clinic Study of Aging. RESULTS Women had higher adiponectin than men (12,631 ng/mL vs. 8,908 ng/mL, P < .001). Among women, higher adiponectin was associated with smaller HVa (B=−0.595; 95% CI −1.19, −0.005), poorer performance in language (B−0.676; 95% CI −1.23, −0.121) and global cognition (B=−0.459; 95% CI −0.915, −0.002), and greater odds of a MCI diagnosis (OR=6.23; 95% CI 1.20, 32.43). In analyses stratified by sex and elevated amyloid (PiB-PET SUVR>1.4), among women with elevated amyloid, higher adiponectin was associated with smaller HVa (B=−0.723; 95% CI −1.43, −0.014), poorer performance in memory (B=−1.02; 95% CI −1.73, −0.312), language (B=−0.896; 95% CI −1.58, −0.212), and global (B=−0.650; 95% CI −1.18, −0.116) cognition, and greater odds of MCI (OR=19.34; 95% CI 2.72, 137.34). CONCLUSION Higher plasma adiponectin was associated with neuroimaging and cognitive outcomes among women. Longitudinal analyses are necessary to determine whether higher adiponectin predicts neurodegeneration and cognitive decline. PMID:27163809

  5. Osmotin: a plant sentinel and a possible agonist of mammalian adiponectin

    PubMed Central

    Anil Kumar, S.; Hima Kumari, P.; Shravan Kumar, G.; Mohanalatha, C.; Kavi Kishor, P. B.

    2015-01-01

    Osmotin is a stress responsive antifungal protein belonging to the pathogenesis-related (PR)-5 family that confers tolerance to both biotic and abiotic stresses in plants. Protective efforts of osmotin in plants range from high temperature to cold and salt to drought. It lyses the plasma membrane of the pathogens. It is widely distributed in fruits and vegetables. It is a differentially expressed and developmentally regulated protein that protects the cells from osmotic stress and invading pathogens as well, by structural or metabolic alterations. During stress conditions, osmotin helps in the accumulation of the osmolyte proline, which quenches reactive oxygen species and free radicals. Osmotin expression results in the accumulation of storage reserves and increases the shelf-life of fruits. It binds to a seven-transmembrane-domain receptor-like protein and induces programmed cell death in Saccharomyces cerevisiae through RAS2/cAMP signaling pathway. Adiponectin, produced in adipose tissues of mammals, is an insulin-sensitizing hormone. Strangely, osmotin acts like the mammalian hormone adiponectin in various in vitro and in vivo models. Adiponectin and osmotin, the two receptor binding proteins do not share sequence similarity at the amino acid level, but interestingly they have a similar structural and functional properties. In experimental mice, adiponectin inhibits endothelial cell proliferation and migration, primary tumor growth, and reduces atherosclerosis. This retrospective work examines the vital role of osmotin in plant defense and as a potential targeted therapeutic drug for humans. PMID:25852715

  6. Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

    PubMed

    Stapels, D A C; Kuipers, A; von Köckritz-Blickwede, M; Ruyken, M; Tromp, A T; Horsburgh, M J; de Haas, C J C; van Strijp, J A G; van Kessel, K P M; Rooijakkers, S H M

    2016-04-01

    Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus. PMID:26418545

  7. Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif.

    PubMed

    Belizario, José E; Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João Marcelo

    2008-06-01

    The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif. PMID:18537676

  8. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems

    PubMed Central

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals. PMID:25483043

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

    USGS Publications Warehouse

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

    2009-01-01

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

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

    SciTech Connect

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

    2008-03-30

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

  11. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  12. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    PubMed Central

    Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-01-01

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARa degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARa fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  13. Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de Novo-Protein Unique Sequence Tags Approach

    SciTech Connect

    Shen, Yufeng; Tolic, Nikola; Purvine, Samuel O.; Smith, Richard D.

    2010-08-01

    Disulfide bonds are a form of posttranslational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, and specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags) we unambiguously correlated the spectra to specific database proteins. Examination of the UStags’ prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to ~10 kDa.

  14. Identification of disulfide bonds in protein proteolytic degradation products using de novo-protein unique sequence tags approach.

    PubMed

    Shen, Yufeng; Tolić, Nikola; Purvine, Samuel O; Smith, Richard D

    2010-08-01

    Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa. PMID:20590115

  15. Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

    PubMed

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

    2015-04-14

    The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. PMID:25843722

  16. Determinants of rodent longevity in the chaperone-protein degradation network.

    PubMed

    Rodriguez, Karl A; Valentine, Joseph M; Kramer, David A; Gelfond, Jonathan A; Kristan, Deborah M; Nevo, Eviatar; Buffenstein, Rochelle

    2016-05-01

    Proteostasis is an integral component of healthy aging, ensuring maintenance of protein structural and functional integrity with concomitant impact upon health span and longevity. In most metazoans, increasing age is accompanied by a decline in protein quality control resulting in the accrual of damaged, self-aggregating cytotoxic proteins. A notable exception to this trend is observed in the longest-lived rodent, the naked mole-rat (NMR, Heterocephalus glaber) which maintains proteostasis and proteasome-mediated degradation and autophagy during aging. We hypothesized that high levels of the proteolytic degradation may enable better maintenance of proteostasis during aging contributing to enhanced species maximum lifespan potential (MLSP). We test this by examining proteasome activity, proteasome-related HSPs, the heat-shock factor 1 (HSF1) transcription factor, and several markers of autophagy in the liver and quadriceps muscles of eight rodent species with divergent MLSP. All subterranean-dwelling species had higher levels of proteasome activity and autophagy, possibly linked to having to dig in soils rich in heavy metals and where underground atmospheres have reduced oxygen availability. Even after correcting for phylogenetic relatedness, a significant (p < 0.02) positive correlation between MLSP, HSP25, HSF1, proteasome activity, and autophagy-related protein 12 (ATG12) was observed, suggesting that the proteolytic degradation machinery and maintenance of protein quality play a pivotal role in species longevity among rodents. PMID:26894765

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

    ERIC Educational Resources Information Center

    Felsenberg, Johannes; Dombrowski, Vincent; Eisenhardt, Dorothea

    2012-01-01

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

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

    USGS Publications Warehouse

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

    2011-01-01

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

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

    USGS Publications Warehouse

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

    2011-01-01

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

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

    USGS Publications Warehouse

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

    2011-01-01

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

  1. Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens

    PubMed Central

    Johnson, Christopher J.; Bennett, James P.; Biro, Steven M.; Duque-Velasquez, Juan Camilo; Rodriguez, Cynthia M.; Bessen, Richard A.; Rocke, Tonie E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted. PMID:21589935

  2. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  3. Effects of feeding dairy cows protein supplements of varying ruminal degradability.

    PubMed

    Reynal, S M; Broderick, G A

    2003-03-01

    Twenty-five (10 ruminally cannulated) Holstein cows averaging 82 +/- 34 d in milk were assigned to 5 x 5 Latin squares (21-d periods) and fed diets supplemented with one of four different proteins to assess effects on production, ruminal metabolism, omasal flow of N fractions, and degradation rates of protein supplements. Total mixed diets contained (dry matter basis) 44% corn silage, 22% alfalfa silage, 2% urea, and 31% concentrate. Five concentrate mixes were fed: 31% high-moisture shelled corn (HMSC; basal); 9% solvent soybean meal (SSBM), 22% HMSC; 10% expeller soybean meal (ESBM), 21% HMSC; 5.5% blood meal (BM), 25.5% HMSC; and 7% corn gluten meal (CGM), 24% HMSC. Diets averaged, respectively, 15.8, 19.1, 19.7, 20.3, and 19.3% crude protein. Feeding the basal diet reduced intake and yield of milk, fat-corrected milk (FCM), and all milk components compared to the protein-supplemented diets. Milk yield was higher for cows fed ESBM and CGM, fat yield was higher for cows fed SSBM and CGM, but FCM and protein yields were not different among cows fed supplemental protein. Based on omasal sampling, mean in vivo estimates of ruminal degradation rate for the crude protein in SSBM, ESBM, BM, and CGM was, respectively, 0.417, 0.179, 0.098, and 0.051/h (computed using passage rates observed for the small particle phase; mean = 0.14/h), and 0.179, 0.077, 0.042, and 0.026/h (computed using a passage rate of 0.06/h). The in vivo degradation rate computed for SSBM at a passage rate = 0.06/h was similar to that estimated using the inhibitor in vitro method. However, in vivo degradation rates computed at passage rate = 0.06/h for ESBM, BM, and CGM were about two, four, and three times more rapid than those estimated by inhibitor in vitro. Experimental proteins fed in this trial will be used as standards for developing in vitro methods for predicting rates of ruminal protein degradation. PMID:12703620

  4. Adiponectin stimulates autophagy and reduces oxidative stress to enhance insulin sensitivity during high-fat diet feeding in mice.

    PubMed

    Liu, Ying; Palanivel, Rengasamy; Rai, Esther; Park, Min; Gabor, Tim V; Scheid, Michael P; Xu, Aimin; Sweeney, Gary

    2015-01-01

    Numerous studies have characterized the antidiabetic effects of adiponectin, yet the precise cellular mechanisms in skeletal muscle, in particular, changes in autophagy, require further clarification. In the current study, we used a high-fat diet (HFD) to induce obesity and insulin resistance in wild-type (WT) or adiponectin knockout (Ad-KO) mice with and without adiponectin replenishment. Temporal analysis of glucose tolerance and insulin sensitivity using hyperinsulinemic-euglycemic clamp and muscle insulin receptor substrate and Akt phosphorylation demonstrated exaggerated and more rapid HFD-induced insulin resistance in skeletal muscle of Ad-KO mice. Superoxide dismutase activity, the reduced glutathione-to-glutathione disulfide ratio, and lipid peroxidation indicated that HFD-induced oxidative stress was corrected by adiponectin. Gene array analysis implicated several antioxidant enzymes, including Gpxs, Prdx, Sod, and Nox4, in mediating this effect. Adiponectin also attenuated palmitate-induced reactive oxygen species production in cultured myotubes and improved insulin-stimulated glucose uptake in primary muscle cells. Increased LC3-II and decreased p62 expression suggested that HFD induced autophagy in muscle of WT mice; however, these changes were not observed in Ad-KO mice. Replenishing adiponectin in Ad-KO mice increased LC3-II and Beclin1 and decreased p62 protein levels, induced fibroblast growth factor-21 expression, and corrected HFD-induced decreases in LC3, Beclin1, and ULK1 gene expression. In vitro studies examining changes in phospho-ULK1 (Ser555), LC3-II, and lysosomal enzyme activity confirmed that adiponectin directly induced autophagic flux in cultured muscle cells in an AMPK-dependent manner. We overexpressed an inactive mutant of Atg5 to create an autophagy-deficient cell model, and together with pharmacological inhibition of autophagy, demonstrated reduced insulin sensitivity under these conditions. In summary, adiponectin stimulated

  5. Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2

    PubMed Central

    Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Halbsguth, Tobias; Bröker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf

    2012-01-01

    The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search. PMID:22327575

  6. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    PubMed Central

    Cazanave, S C; Wang, X; Zhou, H; Rahmani, M; Grant, S; Durrant, D E; Klaassen, C D; Yamamoto, M; Sanyal, A J

    2014-01-01

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis. PMID:24769730

  7. Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

    PubMed Central

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

    2013-01-01

    Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation. PMID:23472144

  8. The effect of adiponectin on osteonectin gene expression by oxidized low density lipoprotein-treated vascular smooth muscle cells.

    PubMed

    Niknam, Sara; Ghatreh-Samani, Keihan; Farrokhi, Effat

    2015-01-01

    Osteonectin is a bone- associated protein involved in vascular calcification. Adiponectin may protect against cardiovascular disease but possible effects on vascular calcification have been poorly studied. The aim of this study was to investigate the modulatory effect of adiponectin on oxidized low density lipoprotein (oxLDL)- induced expression of osteonectin in human aorta vascular smooth muscle cells (HA/VSMCs). HA/VSMCs were cultured in F12K media and then treated with oxLDL (100 µg/mL) in the presence or absence of adoponectin (5 µg/mL) for 24 and 48 hours. mRNA expression and protein level of osteonectin were determined by quantitative real-time PCR and western blot analysis, respectively. After exposure to oxLDL, osteonectin expression increased 1.62 ± 0.23- and 6.62 ± 0.48-fold after 24 and 48 hours respectively compared to the control. Adiponectin increased oxLDL- induced osteonectin expression in a time-dependent manner after 24 and 48 hours (3.24 ± 0.39- and 24.93 ± 2.15-fold, respectively). Western blotting confirmed that osteonectin protein was upregulated by adiponectin.Our data suggest that OxLDL might cause the increase of osteonectin expression both at mRNA and protein level. This upregulation is intensified by adiponectin. PMID:25815284

  9. The Effect of Adiponectin on Osteonectin Gene Expression by Oxidized Low Density Lipoprotein-Treated Vascular Smooth Muscle Cells

    PubMed Central

    Niknam, Sara; Ghatreh-Samani, Keihan; Farrokhi, Effat

    2015-01-01

    Osteonectin is a bone- associated protein involved in vascular calcification. Adiponectin may protect against cardiovascular disease but possible effects on vascular calcification have been poorly studied. The aim of this study was to investigate the modulatory effect of adiponectin on oxidized low density lipoprotein (oxLDL)- induced expression of osteonectin in human aorta vascular smooth muscle cells (HA/VSMCs). HA/VSMCs were cultured in F12K media and then treated with oxLDL (100 µg/mL) in the presence or absence of adoponectin (5 µg/mL) for 24 and 48 hours. mRNA expression and protein level of osteonectin were determined by quantitative real-time PCR and western blot analysis, respectively. After exposure to oxLDL, osteonectin expression increased 1.62 ± 0.23- and 6.62 ± 0.48-fold after 24 and 48 hours respectively compared to the control. Adiponectin increased oxLDL- induced osteonectin expression in a time-dependent manner after 24 and 48 hours (3.24 ± 0.39- and 24.93 ± 2.15-fold, respectively). Western blotting confirmed that osteonectin protein was upregulated by adiponectin.Our data suggest that OxLDL might cause the increase of osteonectin expression both at mRNA and protein level. This upregulation is intensified by adiponectin. PMID:25815284

  10. PI3K-resistant GSK3 controls adiponectin formation and protects from metabolic syndrome.

    PubMed

    Chen, Hong; Fajol, Abul; Hoene, Miriam; Zhang, Bingbing; Schleicher, Erwin D; Lin, Yun; Calaminus, Carsten; Pichler, Bernd J; Weigert, Cora; Häring, Hans U; Lang, Florian; Föller, Michael

    2016-05-17

    Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome. PMID:27140617

  11. Myocardial Mitochondrial and Contractile Function Are Preserved in Mice Lacking Adiponectin

    PubMed Central

    Braun, Martin; Hettinger, Niko; Koentges, Christoph; Pfeil, Katharina; Cimolai, Maria C.; Hoffmann, Michael M.; Osterholt, Moritz; Doenst, Torsten; Bode, Christoph; Bugger, Heiko

    2015-01-01

    Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ-/-) mice and wildtypes (WT). In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24%) in ADQ-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions. PMID:25785965

  12. A structural overview of GH61 proteins - fungal cellulose degrading polysaccharide monooxygenases.

    PubMed

    Lo Leggio, Leila; Welner, Ditte; De Maria, Leonardo

    2012-01-01

    Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full. PMID:24688660

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

    PubMed Central

    Leggio, Leila Lo; Welner, Ditte; De Maria, Leonardo

    2012-01-01

    Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full. PMID:24688660

  14. Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

    PubMed Central

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R.; Liu, Yu; Powers, David L.; Kelly, Jeffery W.; Gierasch, Lila M.; Powers, Evan T.

    2015-01-01

    SUMMARY The folding fate of a protein in vivo is determined by the interplay between a protein’s folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding vs. aggregation vs. degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. PMID:25843722

  15. Mineralocorticoid Receptor Blockade Reverses Obesity-Related Changes in Expression of Adiponectin, PPARγ and Pro-inflammatory Adipokines

    PubMed Central

    Guo, Christine; Ricchiuti, Vincent; Lian, Bill Q.; Yao, Tham M.; Coutinho, Patricia; Romero, José R.; Li, Jianmin; Williams, Gordon H.; Adler, Gail K.

    2009-01-01

    Background In obesity, decreases in adiponectin and increases in pro-inflammatory adipokines are associated with heart disease. Since adipocytes express mineralocorticoid receptor (MR) and MR blockade reduces cardiovascular inflammation and injury, we tested the hypothesis that MR blockade reduces inflammation and expression of pro-inflammatory cytokines in adipose tissue and increases adiponectin expression in adipose tissue and hearts of obese mice. Methods and Results We determined the effect of MR blockade (eplerenone, 100 mg/kg/day for 16 weeks) on gene expression in retroperitoneal adipose and heart tissue from obese, diabetic db/db mice (n=8) as compared with untreated obese, diabetic db/db mice (n=10) and lean, non-diabetic db/+ littermates (n=11). There was increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor type-1 (PAI-1) and macrophage protein CD68 and decreased expression of adiponectin and peroxisome proliferator-activated receptor-γ (PPARγ) in retroperitoneal adipose tissue from obese versus lean mice. Also, adiponectin expression in heart was reduced in obese versus lean mice. MR blockade prevented these obesity-related changes in gene expression. Further, treatment of undifferentiated preadipocytes with aldosterone (10−8 M for 24 h) increased mRNA levels of TNF-α and MCP-1, and reduced mRNA and protein levels of PPARγ and adiponectin, supporting a direct aldosterone effect on gene expression. Conclusions MR blockade reduced expression of pro-inflammatory and pro-thrombotic factors in adipose tissue and increased expression of adiponectin in heart and adipose tissue of obese, diabetic mice. These effects on adiponectin and adipokine gene expression may represent a novel mechanism for the cardioprotective effects of MR blockade. PMID:18427128

  16. Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

    PubMed

    Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

    2016-09-01

    HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. PMID:27460547

  17. Inhibitors of cholesterol biosynthesis increase hepatic low-density lipoprotein receptor protein degradation.

    PubMed

    Ness, G C; Zhao, Z; Lopez, D

    1996-01-15

    Inhibitors of cholesterol biosynthesis are believed to lower serum cholesterol levels by enhancing the removal of serum low-density lipoprotein (LDL) by increasing hepatic LDL receptor function. Thus, the effects of several different inhibitors of cholesterol biosynthesis were examined for their effects on the expression of the hepatic LDL receptor in rats. We found that administration of inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase such as lovastatin, pravastatin, fluvastatin, and rivastatin resulted in increased hepatic LDL receptor mRNA levels. Surprisingly, these agents failed to increase levels of immunoreactive LDL receptor protein in rat liver even when the dose and length of treatment were increased. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, caused even greater increases in hepatic LDL receptor mRNA levels, but did not increase levels of immunoreactive protein. Further investigation revealed that the rate of degradation of the hepatic LDL receptor was increased in rats given inhibitors of cholesterol biosynthesis. The greatest increase in the rate of degradation was seen in animals treated with zaragozic acid A which caused the largest increase in hepatic LDL receptor mRNA levels. In contrast, hepatic LDL receptor protein was stabilized in cholesterol-fed rats. It appears that increased potential for LDL receptor protein synthesis, reflected in increased mRNA levels, is offset by a corresponding increase in the rate of receptor protein degradation resulting in constant steady-state levels of hepatic LDL receptor protein. These findings are suggestive of increased cycling of the hepatic LDL receptor. This postulated mechanism can provide for enhanced hepatic uptake of lipoproteins without increasing steady-state levels of LDL receptor protein. PMID:8561503

  18. c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock.

    PubMed Central

    Lüscher, B; Eisenman, R N

    1988-01-01

    The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells. Images PMID:3043180

  19. From start to finish: amino-terminal protein modifications as degradation signals in plants.

    PubMed

    Gibbs, Daniel J; Bailey, Mark; Tedds, Hannah M; Holdsworth, Michael J

    2016-09-01

    Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the in vivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants. PMID:27439310

  20. TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

    PubMed Central

    Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

    2016-01-01

    Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

  1. TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis.

    PubMed

    Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

    2016-01-01

    Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin-proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

  2. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

    PubMed

    Peth, Andreas; Nathan, James A; Goldberg, Alfred L

    2013-10-01

    The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost. PMID:23965995

  3. Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

    PubMed

    Falkevall, Annelie; Alikhani, Nyosha; Bhushan, Shashi; Pavlov, Pavel F; Busch, Katrin; Johnson, Kenneth A; Eneqvist, Therese; Tjernberg, Lars; Ankarcrona, Maria; Glaser, Elzbieta

    2006-09-29

    Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease. PMID:16849325

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

    PubMed Central

    2014-01-01

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

  5. Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation.

    PubMed

    Jung, Eun Sun; Hong, HyunSeok; Kim, Chaeyoung; Mook-Jung, Inhee

    2015-01-01

    Beta-amyloid (Aβ), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway. PMID:25740315

  6. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  7. Multiple Sclerosis Autoantigen Myelin Basic Protein Escapes Control by Ubiquitination during Proteasomal Degradation*

    PubMed Central

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-01-01

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome. PMID:24739384

  8. Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation

    PubMed Central

    Li, Jun; Chen, Cancan; Ma, Xiancai; Geng, Guannan; Liu, Bingfeng; Zhang, Yijun; Zhang, Shaoyang; Zhong, Fudi; Liu, Chao; Yin, Yue; Cai, Weiping; Zhang, Hui

    2016-01-01

    Long noncoding RNAs (lncRNAs) play multiple key regulatory roles in various cellular pathways. However, their functions in HIV-1 latent infection remain largely unknown. Here we show that a lncRNA named NRON, which is highly expressed in resting CD4+ T lymphocytes, could be involved in HIV-1 latency by specifically inducing Tat protein degradation. Our results suggest that NRON lncRNA potently suppresses the viral transcription by decreasing the cellular abundance of viral transactivator protein Tat. NRON directly links Tat to the ubiquitin/proteasome components including CUL4B and PSMD11, thus facilitating Tat degradation. Depletion of NRON, especially in combination with a histone deacetylase (HDAC) inhibitor, significantly reactivates the viral production from the HIV-1-latently infected primary CD4+ T lymphocytes. Our data indicate that lncRNAs play a role in HIV-1 latency and their manipulation could be a novel approach for developing latency-reversing agents. PMID:27291871

  9. Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids.

    PubMed

    Sobolesky, Philip M; Harrell, Tyler S; Parry, Celeste; Venn-Watson, Stephanie; Janech, Michael G

    2016-01-01

    Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however, insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21)/Adiponectin/Ceramide (Cer), could provide further support to this hypothesis. We measured 2-h post-prandial serum FGF21, total adiponectin, percent unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0) over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129-1599 pg/ml, but did not significantly change over the 24-week study period. Total adiponectin levels (mean ± SD) significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The percent unmodified adiponectin levels (mean ± SD) decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001). We quantified the abundances of serum ceramides and sphingosines (SPH) because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor-mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, and Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion

  10. Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids

    PubMed Central

    Sobolesky, Philip M.; Harrell, Tyler S.; Parry, Celeste; Venn-Watson, Stephanie; Janech, Michael G.

    2016-01-01

    Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however, insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21)/Adiponectin/Ceramide (Cer), could provide further support to this hypothesis. We measured 2-h post-prandial serum FGF21, total adiponectin, percent unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0) over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129–1599 pg/ml, but did not significantly change over the 24-week study period. Total adiponectin levels (mean ± SD) significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The percent unmodified adiponectin levels (mean ± SD) decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001). We quantified the abundances of serum ceramides and sphingosines (SPH) because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor-mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, and Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion

  11. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.

    PubMed

    Zhang, Liangyu; Ward, Jordan D; Cheng, Ze; Dernburg, Abby F

    2015-12-15

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3' UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  12. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

    PubMed Central

    Zhang, Liangyu; Ward, Jordan D.; Cheng, Ze; Dernburg, Abby F.

    2015-01-01

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  13. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  14. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    PubMed Central

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2013-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits HIV-1 infection of myeloid-lineage cells 1,2 as well as resting CD4+ T cells 3,4 by reducing the cellular dNTP concentration to a level where the viral reverse transcriptase cannot function 5,6. In other lentiviruses, including HIV-2 and related SIVs, SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation 7,8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. We present here the crystal structure of a ternary complex of Vpx with the host cell’s E3 ligase substrate adaptor DCAF1 and the C-terminal region of SAMHD1. Vpx is made up of a three-helical bundle, stabilised by a zinc finger motif and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C-terminus making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure provides the first description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system. PMID:24336198

  15. Adiponectin and the systemic inflammatory response in weight-losing patients with non-small cell lung cancer.

    PubMed

    Jamieson, Nigel B; Brown, Duncan J F; Michael Wallace, A; McMillan, Donald C

    The aim of the present study was to examine the relationship between adiponectin and the systemic inflammatory response in weight-losing patients with non-small cell lung cancer (NSCLC). Measurement of anthropometry, acute phase proteins, interleukin-6, leptin (total and free) and adiponectin were carried out on healthy subjects (n = 13) and non-small cell lung cancer patients with weight loss (n = 20). The groups were age and sex matched. Compared with the controls the cancer group had a lower BMI (p < 0.01), mid-upper arm circumference (p < 0.001), triceps skinfold thickness (p < 0.05) and circulating concentrations of albumin (p < 0.001), haemoglobin (p < 0.05), free and total leptin (p < 0.05) and adiponectin (p < 0.01). In contrast, the cancer group had elevated circulating concentrations of interleukin-6 and C-reactive protein concentrations (p < 0.001). In the cancer group circulating adiponectin concentrations were significantly inversely correlated with both free (rs = -0.675, p = 0.001) and total leptin concentrations (rs = -0.690, p = 0.001). However, neither weight loss, interleukin-6 or C-reactive protein concentrations were correlated with either adiponectin, free or total leptin concentrations in the cancer group. These results suggest that adipokine production is normal and is unlikely to play a major role in the abnormal fat metabolism in weight-losing cancer patients. PMID:15242698

  16. Globular adiponectin ameliorates metabolic insulin resistance via AMPK-mediated restoration of microvascular insulin responses.

    PubMed

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W; Barrett, Eugene J; Cao, Wenhong; Liu, Zhenqi

    2015-09-01

    Adiponectin is an adipokine with anti-inflammatory and anti-diabetic properties. Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance in obesity and diabetes. Insulin resistance is present in muscle microvasculature and this may contribute to decreased insulin delivery to, and action in, muscle. In this study we examined whether adiponectin ameliorates metabolic insulin resistance by affecting muscle microvascular recruitment. We demonstrated that a high-fat diet induces vascular adiponectin and insulin resistance but globular adiponectin administration can restore vascular insulin responses and improve insulin's metabolic action via an AMPK- and nitric oxide-dependent mechanism. This suggests that globular adiponectin might have a therapeutic potential for improving insulin resistance and preventing cardiovascular complications in patients with diabetes via modulation of microvascular insulin responses. Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance, and microvasculature plays a critical role in the regulation of insulin action in muscle. Here we tested whether adiponectin replenishment could improve metabolic insulin sensitivity in male rats fed a high-fat diet (HFD) via the modulation of microvascular insulin responses. Male Sprague-Dawley rats were fed either a HFD or low-fat diet (LFD) for 4 weeks. Small resistance artery myograph changes in tension, muscle microvascular recruitment and metabolic response to insulin were determined. Compared with rats fed a LFD, HFD feeding abolished the vasodilatory actions of globular adiponectin (gAd) and insulin on pre-constricted distal saphenous arteries. Pretreatment with gAd improved insulin responses in arterioles isolated from HFD rats, which was blocked by AMP-activated protein kinase (AMPK) inhibition. Similarly, HFD abolished microvascular responses to either gAd or insulin and decreased insulin-stimulated glucose disposal by

  17. Degradation of the 32 kD herbicide binding protein in far red light. [Spirodella oligorrhiza

    SciTech Connect

    Gaba, V.; Marder, J.B.; Greenberg, B.M.; Mattoo, A.K.; Edelman, M.

    1987-06-01

    White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (greater than or equal to700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths > 730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals.

  18. ClpXP, an ATP-powered unfolding and protein-degradation machine

    PubMed Central

    Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies. PMID:21736903

  19. PBRM1 (BAF180) protein is functionally regulated by p53-induced protein degradation in renal cell carcinomas.

    PubMed

    Macher-Goeppinger, Stephan; Keith, Martina; Tagscherer, Katrin E; Singer, Stephan; Winkler, Juliane; Hofmann, Thomas G; Pahernik, Sascha; Duensing, Stefan; Hohenfellner, Markus; Kopitz, Juergen; Schirmacher, Peter; Roth, Wilfried

    2015-12-01

    About 40% of clear-cell renal cell carcinomas (ccRCC) harbour mutations in Polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex. This qualifies PBRM1 as a major cancer gene in ccRCC. The PBRM1 protein alters chromatin structure and its known functions include transcriptional regulation by controlling the accessibility of DNA and influencing p53 transcriptional activity. Since little is known about the regulation of PBRM1, we studied possible mechanisms and interaction partners involved in the regulation of PBRM1 expression. Activation of p53 in RCC cells resulted in a marked decrease of PBRM1 protein levels. This effect was abolished by siRNA-mediated down-regulation of p53, and transcriptional activity was not crucial for p53-dependent PBRM1 regulation. Pulse-chase experiments determined post-translational protein degradation to be the underlying mechanism for p53-dependent PBRM1 regulation, which was accordingly inhibited by proteasome inhibitors. The effects of p53 activation on PBRM1 expression were confirmed in RCC tissue ex vivo. Our results demonstrate that PBRM1 is a target of p53-induced proteasomal protein degradation and provide further evidence for the influence of PBRM1 on p53 function in RCC tumour cells. Considering the paramount role of p53 in carcinogenesis and the presumptive impact of PBRM1 in RCC development, this novel regulation mechanism might be therapeutically exploited in the future. PMID:26178300

  20. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice. PMID:27471256

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

    SciTech Connect

    Radulescu, Razvan T.; Duckworth, William C.; Levy, Jennifer L.; Fawcett, Janet

    2010-04-30

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

  2. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.

    PubMed Central

    Sanz-Ezquerro, J J; de la Luna, S; Ortín, J; Nieto, A

    1995-01-01

    In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit. PMID:7884889

  3. Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation

    NASA Technical Reports Server (NTRS)

    Tischler, M. E.; Satarug, S.; Aannestad, A.; Munoz, K. A.; Henriksen, E. J.

    1997-01-01

    Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.

  4. In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays.

    PubMed

    Lorenz, Martin M; Hayot Carbonero, Christine; Smith, Lydia; Udén, Peter

    2012-05-23

    This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding. PMID:22494200

  5. Quorum sensing controls hyphal initiation in Candida albicans through Ubr1-mediated protein degradation

    PubMed Central

    Lu, Yang; Su, Chang; Unoje, Ohimai; Liu, Haoping

    2014-01-01

    Candida albicans is the most common cause of invasive fungal infections in humans. Its ability to undergo the morphological transition from yeast to hyphal growth forms is critical for its pathogenesis. Hyphal initiation requires the activation of the cAMP-PKA pathway, which down-regulates the expression of NRG1, the major repressor of hyphal development. Hyphal initiation also requires inoculation of a small amount of C. albicans cells from overnight culture to fresh medium. This inoculation releases the inhibition from farnesol, a quorum-sensing molecule of C. albicans, that accumulated in the spent medium. Here, we show that farnesol inhibits hyphal initiation mainly through blocking the protein degradation of Nrg1. Through screening a kinase mutant library, we identified Sok1 as the kinase required for Nrg1 degradation during inoculation. SOK1 expression is transiently activated on inoculation during hyphal initiation, and overexpression of SOK1 overcomes the farnesol-mediated inhibition of hyphal initiation. Screening a collection of transcription factor mutants, the homeodomain-containing transcription repressor Cup9 is found to be responsible for the repression of SOK1 expression in response to farnesol inhibition. Interestingly, farnesol inhibits Cup9 degradation mediated by the N-end rule E3 ubiquitin ligase, Ubr1. Therefore, hyphal initiation requires both the cAMP-PKA pathway-dependent transcriptional down-regulation of NRG1 and Sok1-mediated degradation of Nrg1 protein. The latter is triggered by the release from farnesol inhibition of Cup9 degradation and consequently, derepression of SOK1 transcription. Neither pathway alone is sufficient for hyphal initiation. PMID:24449897

  6. Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

    PubMed

    Kemp, C M; Wheeler, T L

    2011-10-01

    Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs. PMID:21622882

  7. Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.

    PubMed Central

    Pagès, S; Gal, L; Bélaïch, A; Gaudin, C; Tardif, C; Bélaïch, J P

    1997-01-01

    The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation. PMID:9139893

  8. Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage.

    PubMed

    El-Zahar, Khaled; Chobert, Jean-Marc; Sitohy, Mahmoud; Dalgalarrondo, Michèle; Haertlé, Thomas

    2003-06-01

    Yoghurts are mostly produced from cow milk and to a very limited extent from ewe milk. The evolution of caseins and whey proteins in ovine milk submitted to different thermal treatments (63 degrees C/30 min; 73 degrees C/15 min; 85 degrees C/10 min or 96 degrees C/5 min) was followed during fermentation of yoghurts and during their storage up to 14 days, using two different sets of starters. One set of starter LAB was a "ropy" culture (YC-191), which is a well-defined mixed strain culture containing Streptococcus thermophilus ST-143 and Lactobacillus delbrueckii subsp. bulgaricus (LB-18 and LB-CH2). The other set of starter bacteria (YC-460) was a standard yoghurt culture("non-ropy") containing mixed strain culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Contents of free amino groups in produced yoghurts increased gradually during the fermentation, up to a maximal value obtained after 4 h fermentation, then they did not change significantly during storage of yoghurt produced with YC-191 starter. In contrary, a large drop in the amount of free amino groups was observed in the first 24 h of storage in the case of yoghurt made with YC-460 indicating that microorganisms continue still to grow in low temperatures. During fermentation and storage of both yoghurt types, alpha-lactalbumin was hydrolyzed to a slightly bigger extent than beta-lactoglobulin. During fermentation, beta-casein was slightly more degraded than alpha(s)-caseins; however, the opposite was observed during storage up to 14 days. Generally, a more intense heat pretreatment led to a higher degradation of whey proteins and caseins during fermentation and storage. Differences in proteolytic activity between the two starters used (whey proteins more degraded by YC-191; caseins more degraded by YC-460) may lead to improvement in production and formulation of yoghurts differing in their physicochemical and rheological properties. PMID:12866624

  9. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis

    PubMed Central

    Briz, Victor; Hsu, Yu-Tien; Li, Yi; Lee, Erin; Bi, Xiaoning; Baudry, Michel

    2013-01-01

    Memory consolidation has been suggested to be protein synthesis-dependent. Recent data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2 but not calpain-1 treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knock-down of calpain-2 but not calpain-1 by siRNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity. PMID:23467348

  10. Effect of feed deprivation and insulin-like growth hormone on indices of protein degradation in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin-like growth factor-I (IGF-I) is a hormone that promotes growth by both increasing protein synthesis and decreasing protein degradation. This study utilizes a comparative slaughter approach to determine the effect of feed deprivation and IGF-I treatment on weight loss and indices of protein ...

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

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

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

    PubMed

    Chakrabarti, Sanjukta; Barrow, Colin J; Kanwar, Rupinder K; Ramana, Venkata; Kanwar, Jagat R

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  13. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  14. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

    PubMed Central

    Ito, N; Li, Y; Suzuki, T; Stechschulte, D J; Dileepan, K N

    1998-01-01

    The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression. PMID:9927232

  15. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  16. Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

    PubMed

    Fingerhut, R; van der Horst, G T; Verheijen, F W; Conzelmann, E

    1992-09-15

    Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates. PMID:1396669

  17. Adiponectin inhibits oxidized low density lipoprotein-induced increase in matrix metalloproteinase 9 expression in vascular smooth muscle cells

    PubMed Central

    Saneipour, Maryam; Ghatreh-Samani, Keihan; Heydarian, Esfandiar; Farrokhi, Effat; Abdian, Narges

    2015-01-01

    BACKGROUND High expression of matrix metalloproteinase 9 (MMP9) during vascular injury and inflammation plays an important role in atherosclerotic plaque formation and rupture. In the process of atherosclerosis, oxidized low-density lipoprotein (oxLDL) upregulates MMP9 in human aortic vascular smooth muscle cells (HA/VSMCs). Adiponectin is an adipose tissue-derived hormone that has been shown to exert anti-atherogenic and anti-inflammatory effects. The aim of this study was to investigate the effect of adiponectin on MMP9 expression under pathogenic condition created by oxLDL in HA/VSMCs. METHODS In this experimental study, HA/VSMC were stimulated with oxLDL alone and in the presence of adiponectin for 24 and 48 h. The expression of MMP9 gene was determined by real-time polymerase chain reaction method. The protein level of this gene was investigated by western blotting technique. RESULTS An oxLDL increased MMP9 expression 2.16 ± 0.24- and 3.32 ± 0.25-fold after 24 and 48 h, respectively and adiponectin decreased oxLDL-induced MMP9 expression in a time-dependent manner. CONCLUSION These results show that adiponectin changes extracellular matrix by reducing MMP9 mRNA and protein, therefore, may stabilize lesions and reduce atheroma rupture. PMID:26405452

  18. β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes*

    PubMed Central

    Jakhria, Toral; Hellewell, Andrew L.; Porter, Morwenna Y.; Jackson, Matthew P.; Tipping, Kevin W.; Xue, Wei-Feng; Radford, Sheena E.; Hewitt, Eric W.

    2014-01-01

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. PMID:25378395

  19. Mathematical aspects of the kinetics of formation and degradation of linear peptide or protein aggregates.

    PubMed

    Zhdanov, Vladimir P

    2016-08-01

    In cells, peptides and proteins are sometimes prone to aggregation. In neurons, for example, amyloid β peptides form plaques related to Alzheimer's disease (AD). The corresponding kinetic models either ignore or do not pay attention to degradation of these species. Here, the author proposes a generic kinetic model describing formation and degradation of linear aggregates. The process is assumed to occur via reversible association of monomers and attachment of monomers to or detachment from terminal parts of aggregates. Degradation of monomers is described as a first-order process. Degradation of aggregates is considered to occur at their terminal and internal parts with different rates and these steps are described by first-order equations as well. Irrespective of the choice of the values of the rate constants, the model predicts that eventually the system reaches a stable steady state with the aggregate populations rapidly decreasing with increasing size at large sizes. The corresponding steady-state size distributions of aggregates are illustrated in detail. The transient kinetics are also shown. The observation of AD appears, however, to indicate that the peptide production becomes eventually unstable, i.e., the growth of the peptide population is not properly limited. This is expected to be related to the specifics of the genetic networks controlling the peptide production. Following this line, two likely general networks with, respectively, global negative and positive feedbacks in the peptide production are briefly discussed. PMID:27132946

  20. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

    PubMed Central

    Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Takatsuki, Hanae; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent. PMID:26368533

  1. Protein binding mediation of biomaterial-dependent monocyte activation on a degradable polar hydrophobic ionic polyurethane.

    PubMed

    Battiston, Kyle G; Labow, Rosalind S; Santerre, J Paul

    2012-11-01

    Protein adsorption is an important phenomenon influencing the cellular response to biomaterials. Previous studies comparing monocyte activation on a degradable polar hydrophobic ionic polyurethane (D-PHI) indicated a reduced pro-inflammatory monocyte response relative to tissue culture polystyrene (TCPS) and poly(lactide-co-glycolide) (PLGA) substrates. The present study investigated the influence of protein binding in order to gain further insight into the observed differential monocyte activation. Several proteins, identified in different relative amounts within the bound protein layers on D-PHI vs. PLGA and TCPS, were evaluated for their effect on monocyte activation. It was found that, in general, both non-coated and protein pre-adsorbed D-PHI supported a reduced pro-inflammatory response relative to PLGA, as indicated by lower levels of tumor necrosis factor-α (TNF-α) release. An initial increase in TNF-α release occurred when α(2)-macroglobulin (A2M) was pre-adsorbed to D-PHI, which was shown to involve the α(2)-macroglobulin receptor and was active on D-PHI but not on the two other biomaterials. This response was not observed during competitive protein binding in the presence of fetal bovine serum (FBS), suggesting that a more complex arrangement of the bound proteins and their interactions with one another, as well as with the surface chemistry of the individual biomaterials, resulted in the low-activating character of D-PHI when interacting with human monocytes. PMID:22940217

  2. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.

    PubMed

    van der Goot, Annemieke T; Zhu, Wentao; Vázquez-Manrique, Rafael P; Seinstra, Renée I; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C; Ruiz Silva, Mariana; Thijssen, Karen L; Kema, Ido P; Neri, Christian; Oefner, Peter J; Nollen, Ellen A A

    2012-09-11

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  3. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    PubMed Central

    van der Goot, Annemieke T.; Zhu, Wentao; Vázquez-Manrique, Rafael P.; Seinstra, Renée I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Ruiz Silva, Mariana; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  4. The TSG101 protein binds to connexins and is involved in connexin degradation

    SciTech Connect

    Auth, Tanja Schlueter, Sharazad; Urschel, Stephanie; Kussmann, Petra; Sonntag, Stephan; Hoeher, Thorsten; Kreuzberg, Maria M.; Dobrowolski, Radoslaw; Willecke, Klaus

    2009-04-01

    Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

  5. Maternal Overweight Programs Insulin and Adiponectin Signaling in the Offspring

    PubMed Central

    Shankar, Kartik; Kang, Ping; Harrell, Amanda; Zhong, Ying; Marecki, John C.; Ronis, Martin J. J.; Badger, Thomas M.

    2010-01-01

    Gestational exposure to maternal overweight (OW) influences the risk of obesity in adult life. Male offspring from OW dams gain greater body weight and fat mass and develop insulin resistance when fed high-fat diets (45% fat). In this report, we identify molecular targets of maternal OW-induced programming at postnatal d 21 before challenge with the high-fat diet. We conducted global transcriptome profiling, gene/protein expression analyses, and characterization of downstream signaling of insulin and adiponectin pathways in conjunction with endocrine and biochemical characterization. Offspring born to OW dams displayed increased serum insulin, leptin, and resistin levels (P < 0.05) at postnatal d 21 preceding changes in body composition. A lipogenic transcriptome signature in the liver, before development of obesity, was evident in OW-dam offspring. A coordinated locus of 20 sterol regulatory element-binding protein-1-regulated target genes was induced by maternal OW. Increased nuclear levels of sterol regulatory element-binding protein-1 and recruitment to the fatty acid synthase promoter were confirmed via ELISA and chromatin immunoprecipitation analyses, respectively. Higher fatty acid synthase and acetyl coenzyme A carboxylase protein and pAKT (Thr308) and phospho-insulin receptor-β were confirmed via immunoblotting. Maternal OW also attenuated AMP kinase/peroxisome proliferator-activated receptor-α signaling in the offspring liver, including transcriptional down-regulation of several peroxisome proliferator-activated receptor-α-regulated genes. Hepatic mRNA and circulating fibroblast growth factor-21 levels were significantly lower in OW-dam offspring. Furthermore, serum levels of high-molecular-weight adiponectin (P < 0.05) were decreased in OW-dam offspring. Phosphorylation of hepatic AMP-kinase (Thr172) was significantly decreased in OW-dam offspring, along with lower AdipoR1 mRNA. Our results strongly suggest that gestational exposure to maternal

  6. Maternal adiponectin controls milk composition to prevent neonatal inflammation.

    PubMed

    Jin, Zixue; Du, Yang; Schwaid, Adam G; Asterholm, Ingrid W; Scherer, Philipp E; Saghatelian, Alan; Wan, Yihong

    2015-04-01

    Adiponectin is an important adipokine. Increasing evidence suggests that altered adiponectin levels are linked with metabolic and inflammatory disorders. Here we report an important yet previously unrecognized function of adiponectin in lactation by which maternal adiponectin determines the inflammatory status in the nursing neonates. Surprisingly, both maternal adiponectin overexpression in the transgenic mice and maternal adiponectin deletion in the knockout mice lead to systemic inflammation in the pups, manifested as transient hair loss. However, distinct mechanisms are involved. Adiponectin deficiency triggers leukocyte infiltration and production of inflammatory cytokines in the lactating mammary gland. In contrast, adiponectin overabundance increases lipid accumulation in the lactating mammary gland, resulting in excessive long-chain saturated fatty acids in milk. Interestingly, in both cases, the inflammation and alopecia in the pups can be rescued by Toll-like receptor (TLR)-2/4 deletion because TLR2/4 double-knockout pups are resistant. Mechanistically, long-chain saturated fatty acid activation of inflammatory genes is TLR2/4 dependent and can be potentiated by proinflammatory cytokines, indicating that the inflammatory stimuli in both scenarios functionally converge by activating the TLR2/4 signaling. Therefore, our findings reveal adiponectin as a dosage-dependent regulator of lactation homeostasis and milk quality that critically controls inflammation in the nursing neonates. Furthermore, these results suggest that inflammatory infantile disorders may result from maternal adiponectin dysregulation that can be treated by TLR2/4 inhibition. PMID:25590242

  7. New Insight into Adiponectin Role in Obesity and Obesity-Related Diseases

    PubMed Central

    Nigro, Ersilia; Scudiero, Olga; Monaco, Maria Ludovica; Palmieri, Alessia; Mazzarella, Gennaro; Costagliola, Ciro; Bianco, Andrea; Daniele, Aurora

    2014-01-01

    Obesity is a major health problem strongly increasing the risk for various severe related complications such as metabolic syndrome, cardiovascular diseases, respiratory disorders, diabetic retinopathy, and cancer. Adipose tissue is an endocrine organ that produces biologically active molecules defined “adipocytokines,” protein hormones with pleiotropic functions involved in the regulation of energy metabolism as well as in appetite, insulin sensitivity, inflammation, atherosclerosis, cell proliferation, and so forth. In obesity, fat accumulation causes dysregulation of adipokine production that strongly contributes to the onset of obesity-related diseases. Several advances have been made in the treatment and prevention of obesity but current medical therapies are often unsuccessful even in compliant patients. Among the adipokines, adiponectin shows protective activity in various processes such as energy metabolism, inflammation, and cell proliferation. In this review, we will focus on the current knowledge regarding the protective properties of adiponectin and its receptors, AdipoRs (“adiponectin system”), on metabolic complications in obesity and obesity-related diseases. Adiponectin, exhibiting antihyperglycemic, antiatherogenic, and anti-inflammatory properties, could have important clinical benefits in terms of development of therapies for the prevention and/or for the treatment of obesity and obesity-related diseases. PMID:25110685

  8. New insight into adiponectin role in obesity and obesity-related diseases.

    PubMed

    Nigro, Ersilia; Scudiero, Olga; Monaco, Maria Ludovica; Palmieri, Alessia; Mazzarella, Gennaro; Costagliola, Ciro; Bianco, Andrea; Daniele, Aurora

    2014-01-01

    Obesity is a major health problem strongly increasing the risk for various severe related complications such as metabolic syndrome, cardiovascular diseases, respiratory disorders, diabetic retinopathy, and cancer. Adipose tissue is an endocrine organ that produces biologically active molecules defined "adipocytokines," protein hormones with pleiotropic functions involved in the regulation of energy metabolism as well as in appetite, insulin sensitivity, inflammation, atherosclerosis, cell proliferation, and so forth. In obesity, fat accumulation causes dysregulation of adipokine production that strongly contributes to the onset of obesity-related diseases. Several advances have been made in the treatment and prevention of obesity but current medical therapies are often unsuccessful even in compliant patients. Among the adipokines, adiponectin shows protective activity in various processes such as energy metabolism, inflammation, and cell proliferation. In this review, we will focus on the current knowledge regarding the protective properties of adiponectin and its receptors, AdipoRs ("adiponectin system"), on metabolic complications in obesity and obesity-related diseases. Adiponectin, exhibiting antihyperglycemic, antiatherogenic, and anti-inflammatory properties, could have important clinical benefits in terms of development of therapies for the prevention and/or for the treatment of obesity and obesity-related diseases. PMID:25110685

  9. Cross-Talk between Adiponectin and IGF-IR in Breast Cancer

    PubMed Central

    Mauro, Loredana; Naimo, Giuseppina Daniela; Ricchio, Emilia; Panno, Maria Luisa; Andò, Sebastiano

    2015-01-01

    Obesity is a chronic and multifactorial disorder that is reaching epidemic proportions. It is characterized by an enlarged mass of adipose tissue caused by a combination of size increase of preexisting adipocytes (hypertrophy) and de novo adipocyte differentiation (hyperplasia). Obesity is related to many metabolic disorders like hypertension, type 2 diabetes, metabolic syndrome, and cardiovascular disease, and it is associated with an increased risk of cancer development in different tissues including breast. Adipose tissue is now regarded as not just a storage reservoir for excess energy, but rather as an endocrine organ, secreting a large number of bioactive molecules called adipokines. Among these, adiponectin represents the most abundant adipose tissue-excreted protein, which exhibits insulin sensitizing, anti-inflammatory, and antiatherogenic properties. The serum concentrations of adiponectin are inversely correlated with body mass index. Recently, low levels of plasma adiponectin have been associated with an increased risk for obesity-related cancers and development of more aggressive phenotype, concomitantly with alterations in the bioavailability of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) signaling pathways. In this review, we discuss the cross-talk between adiponectin/AdipoR1 and IGF-I/IGF-IR in breast cancer. PMID:26236690

  10. Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation

    PubMed Central

    Di Costanzo, Antonella; Festa, Luisa; Duverger, Olivier; Vivo, Maria; Guerrlal, Luisa; La Mantia, Girolama; Morasso, Marla I.; Calabro, Viola

    2009-01-01

    The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant ΔNp63α in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of ΔNp63α are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raft phosphorylation. Dlx3 is unable to promote p63 degradation in Raft depleted MEF cells or upon pharmacological knockdown of Raft. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of ΔNp63α protein level, a mechanism that may contribute to reduce the abundance of ΔNp63α during differentiation of stratified epithelia. PMID:19282665

  11. Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.

    PubMed

    Di Costanzo, Antonella; Festa, Luisa; Duverger, Olivier; Vivo, Maria; Guerrini, Luisa; La Mantia, Girolama; Morasso, Maria I; Calabrò, Viola

    2009-04-15

    The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant DeltaNp63alpha in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of DeltaNp63alpha are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raf1 phosphorylation. Dlx3 is unable to promote p63 degradation in Raf1 depleted MEF cells or upon pharmacological knockdown of Raf1. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of DeltaNp63alpha protein level, a mechanism that may contribute to reduce the abundance of DeltaNp63alpha during differentiation of stratified epithelia. PMID:19282665

  12. Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

    PubMed

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

    2016-03-01

    Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies. PMID:26041514

  13. Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication.

    PubMed

    Murphy, Christopher M; Xu, Yanping; Li, Feng; Nio, Kouki; Reszka-Blanco, Natalia; Li, Xiaodong; Wu, Yaxu; Yu, Yanbao; Xiong, Yue; Su, Lishan

    2016-09-13

    The hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4(HBx) substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4(HBx) E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression. PMID:27626656

  14. Diversity in growth and protein degradation by dairy relevant lactic acid bacteria species in reconstituted whey.

    PubMed

    Pescuma, Micaela; Hébert, Elvira M; Bru, Elena; Font de Valdez, Graciela; Mozzi, Fernanda

    2012-05-01

    The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37 °C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, β-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey. PMID:22559062

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

    PubMed

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

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. PMID:26505494

  16. Phenotypes on demand via switchable target protein degradation in multicellular organisms

    PubMed Central

    Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S.; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R. Jürgen; Schnittger, Arp; Dissmeyer, Nico

    2016-01-01

    Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

  17. Phenotypes on demand via switchable target protein degradation in multicellular organisms.

    PubMed

    Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R Jürgen; Schnittger, Arp; Dissmeyer, Nico

    2016-01-01

    Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

  18. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    PubMed

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P < 0.05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin. PMID:17445338

  19. A molecular nanodevice for targeted degradation of mRNA during protein synthesis

    PubMed Central

    Lee, Kyung-Ho; Min, Seung-Eui; Kim, Haseong; Lee, Seung-Goo; Kim, Dong-Myung

    2016-01-01

    RNase H is an endonuclease that catalyzes the cleavage of RNA. Because it only acts on RNA in RNA:DNA hybrids, RNase H can be used for targeted degradation of RNA when used in combination with antisense oligodeoxyribonucleotides (ASODNs) designed against a specific sequence of the target RNA. In this study, ASODN and RNase H were co-conjugated on magnetic nanoparticles. The resulting nanoparticles, having integrated functions of probing and processing target RNA, were able to remove target mRNA sequences more effectively than free ASODNs. The paramagnetic property of the nanoparticles also enabled timed engagement and disengagement of the RNA-degrading components in a given system, and these nanoparticles were able to be used for ON/OFF control of gene expression during cell-free protein synthesis reactions. PMID:26857021

  20. The role of local protein synthesis and degradation in axon regeneration

    PubMed Central

    Gumy, Laura F.; Tan, Chin Lik; Fawcett, James W.

    2010-01-01

    In axotomised regenerating axons, the first step toward successful regeneration is the formation of a growth cone. This requires a variety of dynamic morphological and biochemical changes in the axon, including the appearance of many new cytoskeletal, cell surface and signalling molecules. These changes suggest the activation of coordinated complex cellular processes. A recent development has been the demonstration that the regenerative ability of some axons depends on their capacity to locally synthesise new proteins and degrade others at the injury site autonomously from the cell body. There are also events involving the degradation of cytoskeletal and other molecules, and activation of signalling pathways, with axotomy-induced calcium changes probably being an initiating event. A future challenge will be to understand how this complex network of processes interacts in order to find therapeutic ways of promoting the regeneration of CNS axons. PMID:19520073

  1. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

    PubMed Central

    Zhao, Jinghui; Zhai, Bo; Gygi, Steven P.; Goldberg, Alfred Lewis

    2015-01-01

    Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth. PMID:26669439

  2. Stress-Regulated Translational Attenuation Adapts Mitochondrial Protein Import Through Tim17A Degradation

    PubMed Central

    Rainbolt, T. Kelly; Atanassova, Neli; Genereux, Joseph C.; Wiseman, R. Luke

    2014-01-01

    SUMMARY Stress-regulated signaling pathways protect mitochondrial proteostasis, and thus mitochondrial function, from pathologic insults. Despite the importance of stress-regulated signaling pathways in mitochondrial proteome maintenance, the molecular mechanisms by which these pathways maintain mitochondrial proteostasis remain largely unknown. Here, we identify Tim17A as a stress-regulated subunit of the Translocase of the Inner Membrane 23 (TIM23) mitochondrial protein import complex. We show that Tim17A protein levels are decreased downstream of stress-regulated translational attenuation induced by eIF2α phosphorylation through a mechanism dependent on the mitochondrial protease YME1L. Furthermore, we demonstrate that decreasing Tim17A protein levels attenuates TIM23-dependent protein import, promotes the induction of mitochondrial Unfolded Protein Response-associated proteostasis genes, and confers stress-resistance in C. elegans and mammalian cells. Thus, our results indicate that Tim17A degradation is a stress-responsive mechanism by which cells adapt mitochondrial protein import efficiency and promote mitochondrial proteostasis in response to the numerous pathologic insults that induce stress-regulated translation attenuation. PMID:24315374

  3. Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction

    PubMed Central

    Iwamoto, Takeo; Takada, Koji; Okuda, Ken-ichi; Tajima, Akiko; Iwase, Tadayuki

    2013-01-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction. PMID:23316041

  4. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements

    PubMed Central

    Wang, Y.; Jin, L.; Wen, Q. N.; Kopparapu, N. K.; Liu, J.; Liu, X. L.; Zhang, Y. G.

    2016-01-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  5. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements.

    PubMed

    Wang, Y; Jin, L; Wen, Q N; Kopparapu, N K; Liu, J; Liu, X L; Zhang, Y G

    2016-02-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

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

    PubMed

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

    2014-10-01

    A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio <1) the concentrations of His, Lys, and Trp, and increased (ratio >1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality. PMID:25108871

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

    PubMed Central

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

    2012-01-01

    Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein. PMID:22347431

  8. Recycling of a regulatory protein by degradation of the RNA to which it binds.

    PubMed

    Deikus, Gintaras; Babitzke, Paul; Bechhofer, David H

    2004-03-01

    When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription. PMID:14976255

  9. Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability.

    PubMed

    Rineau, Francois; Stas, Jelle; Nguyen, Nhu H; Kuyper, Thomas W; Carleer, Robert; Vangronsveld, Jaco; Colpaert, Jan V; Kennedy, Peter G

    2016-03-01

    In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4 (+) and NO3 (-)) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability. PMID:26682855

  10. Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

    SciTech Connect

    Ezaki, Hisao; Yoshida, Yuichi; Saji, Yukiko; Takemura, Takayo; Fukushima, Juichi; Matsumoto, Hitoshi; Kamada, Yoshihiro; Wada, Akira; Igura, Takumi; Kihara, Shinji; Funahashi, Tohru; Shimomura, Iichiro; Tamura, Shinji; Kiso, Shinichi Hayashi, Norio

    2009-01-02

    We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) {alpha} and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

  11. Requirement for alanine in the amino acid control of deprivation-induced protein degradation in liver.

    PubMed Central

    Pösö, A R; Mortimore, G E

    1984-01-01

    Protein degradation in liver is actively controlled by a small group of inhibitory amino acids--leucine, tyrosine (or phenylalanine), glutamine, proline, histidine, tryptophan, and methionine. Other evidence, however, suggests that one or more of the remaining 12 noninhibitory amino acids is also required for suppression of proteolysis at normal concentrations. This question was investigated in livers of fed rats perfused in the single-pass mode. The deletion of alanine at normal (1x), but not at 4x or 10x normal, plasma amino acid concentrations evoked a near-maximal acceleration of protein degradation. No other noninhibitory amino acid was effective. Because alanine alone was not directly inhibitory and its omission was not associated with a decrease in inhibitory amino acid pools, alanine was presumed to act as a coregulator in the expression of inhibitory activity. When tested alone, the inhibitory group was as effective as the complete mixture at 0.5x and 4x levels, but it lost its suppressive ability within a narrow zone of concentration centered slightly above 1x. The addition of 1x (0.48 mM) alanine completely restored the inhibition. Pyruvate and lactate could be effectively substituted, but only at concentrations 10-20 times greater than that of alanine. These, together with earlier findings, indicate the existence of a regulatory complex that recognizes specific amino acids and transmits positive and negative signals to proteolytic sites. The results also suggest that alanine can provide an important regulatory link between energy demands and protein degradation. PMID:6589593

  12. Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice*

    PubMed Central

    Garyali, Punitee; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2014-01-01

    Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a−/−, Epm2b−/−, and Epm2a−/− Epm2b−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b−/− and Epm2a−/− Epm2b−/− cells) but not laforin (Epm2a−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. PMID:24914213

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

    PubMed

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

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. PMID:25687693

  14. Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

    PubMed

    Ichimura, Mayuko; Kato, Shigeko; Tsuneyama, Koichi; Matsutake, Sachiko; Kamogawa, Mai; Hirao, Eri; Miyata, Ayako; Mori, Sawako; Yamaguchi, Noriaki; Suruga, Kazuhito; Omagari, Katsuhisa

    2013-05-01

    Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein α in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome. PMID:23684441

  15. Objectively Measured Physical Activity and Its Association With Adiponectin and Other Novel Metabolic Markers

    PubMed Central

    Metcalf, Brad S.; Jeffery, Alison N.; Hosking, Joanne; Voss, Linda D.; Sattar, Naveed; Wilkin, Terence J.

    2009-01-01

    OBJECTIVE—Recent evidence suggests that, in children, traditional markers of metabolic disturbance are related only weakly to physical activity. We therefore sought to establish the corresponding relationships with newer metabolic markers. RESEARCH DESIGN AND METHODS—This was a nonintervention longitudinal study of 213 healthy children recruited from 54 schools in Plymouth, U.K. MTI accelerometers were used to make objective 7-day recordings of physical activity at ages 5 ± 0.3 (mean ± SD), 6, 7, and 8 years. Overall physical activity was taken as the average of the four annual time points. The metabolic markers at 8 years were adiponectin, leptin, high-sensitivity C-reactive protein (hsCRP), and insulin resistance (homeostasis model assessment). Potential confounders included percent body fat measured by dual-energy X-ray absorptiometry and diet measured by food frequency questionnaire. RESULTS—Whereas physical activity did not correlate with insulin resistance (r = −0.01), leptin (r = +0.04), or hsCRP (r = +0.01) independently of percent body fat, it did correlate with adiponectin, but inversely (r = −0.18, P = 0.02). This unexpected inverse relationship was strongest among the less active children (physical activity < median: r = −0.30, P = 0.01) but negligible in the more active children (physical activity > median: r = +0.04, P = 0.76). Adiponectin was significantly higher (0.52 SD, P < 0.01) in the least active tertile compared with the other two tertiles. Insulin resistance, however, did not differ across the physical activity tertiles (P = 0.62). CONCLUSIONS—Adiponectin levels in children are highest among those who are least active, but their insulin resistance is no different. Adiponectin has a known insulin-sensitizing effect, and our findings are consistent with a selective effect at low levels of physical activity. PMID:19033408

  16. Association of Bio-energy Processing-Induced Protein Molecular Structure Changes with CNCPS-Based Protein Degradation and Digestion of Co-products in Dairy Cows.

    PubMed

    Li, Xinxin; Zhang, Yonggen; Yu, Peiqiang

    2016-05-25

    The primary objective of this study was to develop a model to predict Cornell Net Carbohydrate Protein System (CNCPS) protein degradation and digestion based on protein molecular structure changes induced by bio-energy processing in different types of co-products (CoPR, CoPC, CoPS = co-products from bioprocessing of rapeseed, canola seed, and soybean, respectively). The results showed that the inherent structure changes induced by the processing had a close relationship with CNCPS predicted protein degradable, undegradable, and digestible contents. The amide I to II ratio and α-helix to β-sheet ratio could be used to predict total degradable protein (R(2) = 0.99, RSD = 0.84, P < 0.001). Total CNCPS intestinal digestible protein could be predicted by protein structure α-helix to β-sheet ratio (R(2) = 0.93, RSD = 0.33, P < 0.001). In conclusion, the processing-induced protein molecular structure changes were highly linked to protein nutritive value of the co-products and could be used as predictors for CNCPS protein degradation and digestion in dairy cattle. PMID:27112731

  17. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

    NASA Astrophysics Data System (ADS)

    Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai P.; Wu, Jun; Li, Ren-Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef M.; Brumell, John H.; Liu, Peter P.

    2014-03-01

    The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1-/- mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

  18. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus

    PubMed Central

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-01-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates. PMID:23902518

  19. The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes

    PubMed Central

    Cornfine, Susanne; Himmel, Mirko; Kopp, Petra; el Azzouzi, Karim; Wiesner, Christiane; Krüger, Marcus; Rudel, Thomas; Linder, Stefan

    2011-01-01

    Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA–induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures. PMID:21119006

  20. ORMDL orosomucoid-like proteins are degraded by free-cholesterol-loading–induced autophagy

    PubMed Central

    Wang, Shuhui; Robinet, Peggy; Smith, Jonathan D.; Gulshan, Kailash

    2015-01-01

    Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol–induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol. PMID:25775599

  1. Distinct protein degradation profiles are induced by different disuse models of skeletal muscle atrophy

    PubMed Central

    Bialek, Peter; Morris, Carl; Parkington, Jascha; St. Andre, Michael; Owens, Jane; Yaworsky, Paul; Seeherman, Howard

    2011-01-01

    Skeletal muscle atrophy can be a consequence of many diseases, environmental insults, inactivity, age, and injury. Atrophy is characterized by active degradation, removal of contractile proteins, and a reduction in muscle fiber size. Animal models have been extensively used to identify pathways that lead to atrophic conditions. We used genome-wide expression profiling analyses and quantitative PCR to identify the molecular changes that occur in two clinically relevant mouse models of muscle atrophy: hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7, and 14 days after casting or injury. The total amount of muscle loss, as measured by wet weight and muscle fiber size, was equivalent between models on day 14, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tenotomy resulted in the regulation of significantly more mRNA transcripts then did casting. Analysis of the regulated genes and pathways suggest that the mechanisms of atrophy are distinct between these models. The degradation following casting was ubiquitin-proteasome mediated, while degradation following tenotomy was lysosomal and matrix-metalloproteinase mediated, suggesting a possible role for autophagy. These data suggest that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat atrophy resulting from different conditions. PMID:21791639

  2. Aldehyde oxidase 1 is highly abundant in hepatic steatosis and is downregulated by adiponectin and fenofibric acid in hepatocytes in vitro

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas S.; Schmidl, Christian; Buettner, Roland; Bollheimer, Cornelius; Aslanidis, Charalampos; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2006-11-24

    Adiponectin protects the liver from steatosis caused by obesity or alcohol and therefore the influence of adiponectin on human hepatocytes was analyzed. GeneChip experiments indicated that recombinant adiponectin downregulates aldehyde oxidase 1 (AOX1) expression and this was confirmed by real-time RT-PCR and immunoblot. AOX1 is a xenobiotic metabolizing protein and produces reactive oxygen species (ROS), that promote cell damage and fibrogenesis. Adiponectin and fenofibric acid activate peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) and both suppress AOX1 protein and this is blocked by the PPAR-{alpha} antagonist RU486. Obesity is associated with low adiponectin, reduced hepatic PPAR-{alpha} activity and fatty liver, and AOX1 was found induced in the liver of rats on a high-fat diet when compared to controls. Free fatty acids and leptin, that are elevated in obesity, failed to upregulate AOX1 in vitro. The current data indicate that adiponectin reduces AOX1 by activating PPAR-{alpha} whereas fatty liver disease is associated with elevated hepatic AOX1. High AOX1 may be associated with higher ROS well described to induce fibrogenesis in liver tissue but may also influence drug metabolism and activity.

  3. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham.

    PubMed

    Gallego, Marta; Mora, Leticia; Fraser, Paul D; Aristoy, María-Concepción; Toldrá, Fidel

    2014-04-15

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered. PMID:24295685

  4. Protein Transmission, Seeding and Degradation: Key Steps for α-Synuclein Prion-Like Propagation

    PubMed Central

    Ximerakis, Methodios; Vekrellis, Kostas

    2014-01-01

    Converging lines of evidence suggest that cell-to-cell transmission and the self-propagation of pathogenic amyloidogenic proteins play a central role in the initiation and the progression of several neurodegenerative disorders. This "prion-like" hypothesis has been recently reported for α-synuclein, a presynaptic protein implicated in the pathogenesis of Parkinson's disease (PD) and related disorders. This review summarizes recent findings on α-synuclein prion-like propagation, focusing on its transmission, seeding and degradation and discusses some key questions that remain to be explored. Understanding how α-synuclein exits cells and propagates from one brain region to another will lead to the development of new therapeutic strategies for the treatment of PD, aiming at slowing or stopping the disease progression. PMID:25548532

  5. Protein degradation by rumen microbes of heat-treated whole cottonseed.

    PubMed

    Tagari, H; Pena, F; Satter, L D

    1986-06-01

    In vitro and in situ rumen digestion trials were conducted to measure the effect of heat treatment on protein degradation by rumen microbes of cottonseed autoclaved and dry-heated (120, 140, 160 and 180 C) for different lengths of time (20, 40, 60, 90 and 120 min). Autoclaving (120 C, 1 kg/cm2 of pressure for 60 min) was effective in reducing ammonia nitrogen concentration in vitro, but dry heat at this temperature was without effect. Dry heating caused a linear decrease of in vitro ammonia concentration as temperature increased from 140 to 180 C and as exposure time increased from 20 to 120 min. The in situ results corroborated the in vitro findings. Crude protein disappearance from dacron bags decreased from 87 to 48% as temperature increased from 140 to 180 C after 20 min of heating. PMID:3755430

  6. Quercetin downregulates Mcl-1 by acting on mRNA stability and protein degradation

    PubMed Central

    Spagnuolo, C; Cerella, C; Russo, M; Chateauvieux, S; Diederich, M; Russo, G L

    2011-01-01

    Background: We recently demonstrated that quercetin, a flavonoid naturally present in food and beverages belonging to the large class of phytochemicals, was able to sensitise leukaemic cells isolated from patients with chronic lymphocytic leukaemia (CLL) when associated with recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or anti-CD95. We also showed that quercetin potentiated the effect of fludarabine on resistant B cells from CLL patients. Resistance to therapy in CLL depends on the expression and activity of anti-apoptotic proteins of the Bcl-2 family. Among these, myeloid cell leukaemia-1 (Mcl-1) has been associated with apoptotic resistance in CLL. Therefore, we investigate here whether the sensitising activity of this flavonoid, which leads to increased apoptosis in both cell lines and CLL, could be related to Mcl-1 expression and stability. Results: B cells isolated from CLL patients showed different levels of Mcl-1 protein expression, resulting, in several cases, in increased sensitivity to fludarabine. Quercetin significantly enhanced the downregulation of Mcl-1 in B cells isolated from selected patients expressing detectable levels of Mcl-1. In U-937 cells, quercetin increased Mcl-1 mRNA instability in the presence of actinomycin D. When cells were treated with MG-132, a proteasome inhibitor, Mcl-1 protein level increased. However, quercetin, in the presence of Z-Vad-FMK, continued to lower Mcl-1 protein expression, indicating its independence from caspase-mediated degradation. In contrast, co-treatment of quercetin and MG-132 did not revert the effect of MG-132 mono-treatment, thus suggesting a possible interference of quercetin in regulating the proteasome-dependent degradation of Mcl-1. Gossypol, a small-molecule inhibitor of Bcl-2 family members, mimics the activity of quercetin by lowering Mcl-1 expression and sensitising U-937 cells to apoptosis induced by recombinant TRAIL and the Fas-ligand. Conclusion: This study

  7. Association of polymorphism in adiponectin (+45 T/G) and leptin (–2548 G/A) genes with type 2 diabetes mellitus in male Egyptians

    PubMed Central

    Motawi, Tarek; Salman, Tarek; Shaker, Olfat

    2015-01-01

    Introduction Adiponectin is an adipose tissue-specific protein with insulin-sensitizing properties. Many investigators have explored the association between adiponectin single nucleotide polymorphisms (SNPs) and type 2 diabetes mellitus (T2DM) in different ethnic populations from different regions. Leptin is a protein hormone constituting an important signal in the regulation of adipose tissue mass and body weight. The aim of this study was to explore potential associations between SNP +45 T>G of the adiponectin gene and SNP 2548G/A of leptin with T2DM and the effect of SNPs on serum adiponectin and leptin levels. Material and methods From the Egyptian population, we enrolled 110 T2DM patients and 90 non-diabetic controls. Serum lipid profile, blood glucose, serum adiponectin, and leptin were measured. Genotyping for two common SNPs of the adiponectin and leptin genes was performed by polymerase chain reaction–restriction fragment length polymorphism. Results The G allele and TG/GG genotype of SNP 45 occurred more frequently than the T allele and TT genotype in T2DM patients compares to the controls. Subjects with the GG + TG genotype of SNP 45 were at increased risk for T2DM (OR = 6.476; 95% CI: 3.401–12.33) and associated with a low serum adiponectin level compared with the TT genotype. The serum leptin concentration of GA + AA genotype carriers was not significantly different from that of the GG genotype in the diabetic group. Conclusions The G allele carriers who have reduced plasma concentrations of adiponectin may have an association with T2DM, while leptin SNP 2548 G/A is not associated with the risk of development of T2DM in the Egyptian population. PMID:26528333

  8. Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

    2012-01-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636

  9. Amyloid-beta protein clearance and degradation (ABCD) pathways and their role in Alzheimer's disease.

    PubMed

    Baranello, Robert J; Bharani, Krishna L; Padmaraju, Vasudevaraju; Chopra, Nipun; Lahiri, Debomoy K; Greig, Nigel H; Pappolla, Miguel A; Sambamurti, Kumar

    2015-01-01

    Amyloid-β proteins (Aβ) of 42 (Aβ42) and 40 aa (Aβ40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aβ precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aβ and also induce the entire spectrum of pathology associated with the disease. Aβ accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by β-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aβ. Although Aβ accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aβ production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aβ42 or both Aβ40 and Aβ42. However, the vast majority of AD patients accumulate Aβ without these known mutations. This led to proposals that impairment of Aβ degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aβ and we have recently established that the mechanism is by skirting Aβ degradation. This review outlines major cellular pathways of Aβ degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for A

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  11. Estrogen inhibits phorbol ester-induced I kappa B alpha transcription and protein degradation.

    PubMed

    Sun, W H; Keller, E T; Stebler, B S; Ershler, W B

    1998-03-27

    Estrogen (E2) is known to prevent bone loss and the mechanism is, at least in part, mediated by inhibition of expression of cytokines such as interleukin-6 (IL-6). Expression of IL-6 is tightly regulated and the transcription factor NF kappa B can upregulate IL-6 gene expression by binding to its promoter region. NF kappa B is kept in an inactive state by associating with its cytoplasmic inhibitor I kappa B alpha. Upon mitogenic stimulation, I kappa B alpha becomes phosphorylated, followed by a rapid protein degradation. As a result, NF kappa B is released and translocate to the nucleus where DNA binding occurs. It has been shown that E2 treatment downregulates mitogen-induced IL-6 expression by inhibiting NF kappa B activity. Here, we sought to determine whether E2 regulates IL-6 gene expression by modulating the levels of I kappa B alpha. Our results show that E2 treatment almost completely inhibits phorbol ester-induced I kappa B alpha protein degradation. In addition, E2 inhibits phorbol ester-stimulated I kappa B alpha gene expression. Taken together, our results suggest that E2 maintains steady state levels of I kappa B alpha upon mitogen stimulation, resulting in inhibition of NF kappa B activation and IL-6 gene expression. This may explain the protective effect of E2 on bone loss. PMID:9535726

  12. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin

    PubMed Central

    Svitil, A. L.; Chadhain, S.; Moore, J. A.; Kirchman, D. L.

    1997-01-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products. PMID:16535505

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

    PubMed Central

    de Laat, Rian; Meabon, James S.; Wiley, Jesse C.; Hudson, Mark P.; Montine, Thomas J.; Bothwell, Mark

    2015-01-01

    Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP) by β-secretase and γ-secretase generate amyloid β (Aβ) peptides, which are thought to contribute to Alzheimer's disease (AD). Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD. PMID:25758563

  14. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

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

    PubMed

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

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. PMID:27084451

  16. An Armadillo Motif in Ufd3 Interacts with Cdc48 and is Involved in Ubiquitin Homeostasis and Protein Degradation

    SciTech Connect

    Zhao, G.; Li, G; Schindelin, H; Lennarz, W

    2009-01-01

    The yeast AAA-ATPase Cdc48 and the ubiquitin fusion degradation (UFD) proteins play important, evolutionarily conserved roles in ubiquitin dependent protein degradation. The N-terminal domain of Cdc48 interacts with substrate-recruiting cofactors, whereas the C terminus of Cdc48 binds to proteins such as Ufd3 that process substrates. Ufd3 is essential for efficient protein degradation and for maintaining cellular ubiquitin levels. This protein contains an N-terminal WD40 domain, a central ubiquitin-binding domain, and a C-terminal Cdc48-binding PUL domain. The crystal structure of the PUL domain reveals an Armadillo repeat with high structural similarity to importin-a, and the Cdc48-binding site could be mapped to the concave surface of the PUL domain by biochemical studies. Alterations of the Cdc48 binding site of Ufd3 by site-directed mutagenesis resulted in a depletion of cellular ubiquitin pools and reduced activity of the ubiquitin fusion degradation pathway. Therefore, our data provide direct evidence that the functions of Ufd3 in ubiquitin homeostasis and protein degradation depend on its interaction with the C terminus of Cdc48.

  17. Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

    SciTech Connect

    Chen Baoying; Lam, Karen S.L.; Wang Yu; Wu Donghai; Lam, Michael C.; Shen Jiangang; Wong Laiching; Hoo, Ruby L.C.; Zhang Jialiang; Xu Aimin . E-mail: amxu@hkucc.hku.hk

    2006-03-10

    Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H{sub 2}O{sub 2})-induced dysregulation of adiponectin and PAI-1 production. H{sub 2}O{sub 2} treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and CCAAT/enhancer binding protein (C/EBP{alpha}), but had no effect on HIF-1{alpha}, whereas hypoxia stabilized HIF-1{alpha} and decreased expression of C/EBP{alpha}, but not PPAR{gamma}. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.

  18. Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation

    PubMed Central

    Lee, Jae Ho; Lee, Gha Young; Jang, Hagoon; Choe, Sung Sik; Koo, Seung-Hoi; Kim, Jae Bum

    2014-01-01

    Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. Conclusion: RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation

  19. Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics

    SciTech Connect

    Manes, Nathan P.; Gustin, Jean K.; Rue, Joanne; Mottaz, Heather M.; Purvine, Samuel O.; Norbeck, Angela D.; Monroe, Matthew E.; Zimmer, Jennifer S.; Metz, Thomas O.; Adkins, Joshua N.; Smith, Richard D.; Heffron, Fred

    2007-04-01

    The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

  20. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  1. Regulation of Endoplasmic Reticulum-Associated Protein Degradation (ERAD) by Ubiquitin

    PubMed Central

    Lemus, Leticia; Goder, Veit

    2014-01-01

    Quality control of protein folding inside the endoplasmic reticulum (ER) includes chaperone-mediated assistance in folding and the selective targeting of terminally misfolded species to a pathway called ER-associated protein degradation, or simply ERAD. Once selected for ERAD, substrates will be transported (back) into the cytosol, a step called retrotranslocation. Although still ill defined, retrotranslocation likely involves a protein conducting channel that is in part formed by specific membrane-embedded E3 ubiquitin ligases. Early during retrotranslocation, reversible self-ubiquitination of these ligases is thought to aid in initiation of substrate transfer across the membrane. Once being at least partially exposed to the cytosol, substrates will become ubiquitinated on the cytosolic side of the ER membrane by the same E3 ubiquitin ligases. Ubiquitin on substrates was originally thought to be a permanent modification that (1) promotes late steps of retrotranslocation by recruiting the energy-providing ATPase Cdc48p/p97 via binding to its associated adaptor proteins and that (2) serves to target substrates to the proteasome. Recently it became evident, however, that the poly-ubiquitin chains (PUCs) on ERAD substrates are often subject to extensive remodeling, or processing, at several stages during ERAD. This review recapitulates the current knowledge and recent findings about PUC processing on ERAD substrates and ubiquitination of ERAD machinery components and discusses their functional consequences. PMID:25100021

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

    PubMed Central

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

    2014-01-01

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

  3. Comparison of sludge digestion under aerobic and anaerobic conditions with a focus on the degradation of proteins at mesophilic temperature.

    PubMed

    Shao, Liming; Wang, Tianfeng; Li, Tianshui; Lü, Fan; He, Pinjing

    2013-07-01

    Aerobic and anaerobic digestion are popular methods for the treatment of waste activated sludge. However, the differences in degradation of sludge during aerobic and anaerobic digestion remain unclear. In this study, the sludge degradation during aerobic and anaerobic digestion was investigated at mesophilic temperature, focused on protein based on the degradation efficiency and degree of humification. The duration of aerobic and anaerobic digestion was about 90 days. The final degradation efficiency of volatile solid was 66.1 ± 1.6% and 66.4 ± 2.4% under aerobic and anaerobic conditions, respectively. The final degradation efficiency of protein was 67.5 ± 1.4% and 65.1 ± 2.6% under aerobic and anaerobic conditions, respectively. The degradation models of volatile solids were consistent with those of protein under both aerobic and anaerobic conditions. The solubility of protein under aerobic digestion was greater than that under anaerobic digestion. Moreover, the humification index of dissolved organic matter of aerobic digestion was greater than that during anaerobic digestion. PMID:23685650

  4. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  5. Evaluation of Plasma Fibrinogen Degradation Products and Total Serum Protein Concentration in Oral Submucous Fibrosis

    PubMed Central

    B.N.V.S., Satish; B., Maharudrappa; K.M., Prashant; Hugar, Deepa; Allad, Umesh; Prabhu, Prasanth S.

    2014-01-01

    Background: Oral submucous fibrosis (OSMF) is a potentially malignant disorder with a multifactorial etiology. Malnutrition is a major problem for the inhabitants of most countries where OSMF is prevalent. Recently, a new direction in the etiopathogenesis was provided by the identification of fibrinogen degradation products (FDP) in the plasma of OSMF patients. Aims and Objectives: To assess the role of FDP in the etiology of OSMF and to correlate with the nutritional status by evaluating the total serum protein level. The study also determines to evaluate the correlation between the levels of plasma FDP with respect to the staging and grading of OSMF. Correlation between the levels of Total Serum Protein (TSP) with respect to the staging and grading of OSMF was also evaluated. Materials and Methods: The study included 30 cases clinically and histopathologically diagnosed as oral submucous fibrosis. The FDP levels were assessed using both qualitative and semi quantitative method as supplied by ‘Tulip Diagnostics (P) Ltd. Total Serum Protein (TSP) estimation was done by Biuret method using Liquixx Protein kit by Erba, Manheim. Results: The study indicates that in qualitative assessment of FDP only 14 subjects showed the presence of FDP levels>200ng/ml. In semiquantitative assessment there is no significant association between varying clinical stages and histopathological grades and FDP levels. Total serum Protein level showed a marginal increase in all subjects. The study revealed a positive correlation between FDP and TSP in all OSMF subjects. Conclusion: A larger sample size which would be a better representation of the population and the use of different methods which have higher sensitivities and specificities to evaluate FDP level and detailed fractional analysis of protein along with immunoglobulin profiling would facilitate in attaining more conclusive results. PMID:24995245

  6. NITRIC OXIDE-DEPENDENT PROTEASOMAL DEGRADATION OF CYTOCHROME P450 2B PROTEINS*

    PubMed Central

    Lee, Choon-Myung; Kim, Bong-Yoon; Li, Lian; Morgan, Edward T.

    2007-01-01

    Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes following exposure to bacterial endotoxin (LPS) in a nitric oxide (NO)-dependent manner. In the present study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed more than 60% after 6h treatment with interleukin-1β (IL-1). This effect was NO-dependent, and treatment of cells with the NO-donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione (GSNO), and S-nitroso, N-acetylpenicillamine (SNAP) also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH4Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with GSNO caused S-nitrosylation of CYP2B protein, and enhanced the ubiquitination pattern of CYP2B compared to unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species. PMID:17993647

  7. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    PubMed

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. PMID:26231209

  8. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    PubMed Central

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro

    2014-01-01

    The ubiquitin–proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  9. Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

    PubMed

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-09-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  10. Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

    PubMed Central

    Gao, Caiji; Zhuang, Xiaohong; Cui, Yong; Fu, Xi; He, Yilin; Zhao, Qiong; Zeng, Yonglun; Shen, Jinbo; Luo, Ming; Jiang, Liwen

    2015-01-01

    Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process. PMID:25624505

  11. Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor {gamma}, controls hepatitis B virus replication

    SciTech Connect

    Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2011-01-20

    In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein {alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), adiponectin, liver X receptor {alpha} (LXR{alpha}), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPAR{gamma} and C/EBP{alpha}. Conversely, HBV replication was upregulated by adiponectin and PPAR{gamma} agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

  12. Association of adiponectin and metabolic syndrome in women

    PubMed Central

    Sanjari, Mojgan; Khodashahi, Mandana; Gholamhoseinian, Ahmad; Shokoohi, Mostafa

    2011-01-01

    BACKGROUND: An inverse association between serum adiponectin level and metabolic syndrome was seen in few studies. The aim of this study was to assess the association between serum adiponectin levels and metabolic syndrome in a sample of Iranian women from Kerman. METHODS: In a cross-sectional study 946 subjects were studied to determine the prevalence of metabolic syndrome and in a case control study (170 subjects for each group) the association between serum adiponectin levels and metabolic syndrome were investigated. Metabolic syndrome was defined using International Diabetes Federation (IDF) criteria. Socio-demographics factors and measures of waist circumference, blood pressure and lipid profiles were collected. Serum adiponectin level was measured by ELISA method. RESULTS: The prevalence of the metabolic syndrome was 36.7%. Mean of serum adiponectin level in individuals with metabolic syndrome was lower than individuals without it (10.5 ± 4.1 and 13.45 ± 5.6 μg/ml, respectively, p < 0.001). Low level of adiponectin was a good predictor for metabolic syndrome (a range of β coefficients out of -2.03 to -2.85 according to five models). Systolic blood pressure, body mass index (BMI) and diastolic blood pressure were independent predictors of serum adiponectin (p values were 0.001, 0.009 and 0.034, respectively). CONCLUSIONS: We found that adiponectin is negatively associated with metabolic syndrome. Systolic and diastolic blood pressure and BMI were identified as independent predictors. PMID:22973360

  13. Linkage analysis of circulating levels of adiponectin in hispanic children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adiponectin, a hormone produced exclusively by adipose tissue, is inversely associated with insulin resistance and pro-inflammatory conditions. The aim of this study was to find quantitative trait loci (QTLs) that affect circulating levels of adiponectin in Hispanic children participating in the VVA...

  14. Linkage analysis of circulating levels of adiponectin in Hispanic children

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adiponectin is produced exclusively by adipose tissue and is inversely associated with insulin resistance and pro-inflammatory conditions. The aim of this study was to conduct a genome scan of circulating levels of adiponectin in Hispanic children. The present study included extended families with ...

  15. Intracerebroventricular injection of adiponectin regulates locomotor activity in rats.

    PubMed

    Miyatake, Yumiko; Shiuchi, Tetsuya; Ueta, Tomoyo; Taniguchi, Yasuko; Futami, Akari; Sato, Fukiko; Kitamura, Tadahiro; Tsutsumi, Rie; Harada, Nagakatsu; Nakaya, Yutaka; Sakaue, Hiroshi

    2015-01-01

    Enhancing exercise motivation is the best way to prevent obesity and diabetes. In this study, we examined whether adiponectin affects locomotion activity in Wister and Spontaneously-Running Tokushima-Shikoku (SPORTS) rats using two types of behavioral assays: home cage and wheel running activity. SPORTS rats were established from an original line from Wister strain that had shown high level of wheel running activity in our laboratory. Injection of adiponectin into the lateral ventricle of Wister rats and SPORTS rats decreased home cage activity, but no change was observed in the food intake and oxygen consumption. This result indicates the possibility that adiponectin can reduce non-exercise activity thermogenesis (NEAT) and physical activity via the central nervous system. In contrast, injection of adiponectin did not change wheel running activity in SPORTS rats. We produced hypothalamus-destructed model rat using monosodium glutamate (MSG) to elucidate the regulation site of adiponectin. Injection of adiponectin into MSG-treated SPORTS rats did not change amount of home cage activity and food intake, suggesting that adiponectin action on home cage activity was in the hypothalamic area. These results suggest that adiponectin regulates locomotion activity through mediobasal hypothalamus. PMID:26399348

  16. Adiponectin Inhibits LPS-Induced HMGB1 Release through an AMP Kinase and Heme Oxygenase-1-Dependent Pathway in RAW 264 Macrophage Cells

    PubMed Central

    Kaede, Ryuji; Okamatsu-Ogura, Yuko

    2016-01-01

    High mobility group protein B1 (HMGB1) is a late inflammatory mediator that exaggerates septic symptoms. Adiponectin, an adipokine, has potent anti-inflammatory properties. However, possible effects of adiponectin on lipopolysaccharide- (LPS-) induced HMGB1 release are unknown. The aim of this study was to investigate effects of full length adiponectin on HMGB1 release in LPS-stimulated RAW 264 macrophage cells. Treatment of the cells with LPS alone significantly induced HMGB1 release associated with HMGB1 translocation from the nucleus to the cytosol. However, prior treatment with adiponectin suppressed LPS-induced HMGB1 release and translocation. The anti-inflammatory cytokine interleukin- (IL-) 10 similarly suppressed LPS-induced HMGB1 release. Adiponectin treatment decreased toll-like receptor 4 (TLR4) mRNA expression and increased heme oxygenase- (HO-) 1 mRNA expression without inducing IL-10 mRNA, while IL-10 treatment decreased TLR2 and HMGB1 mRNA expression and increased the expression of IL-10 and HO-1 mRNA. Treatment with the HO-1 inhibitor ZnPP completely prevented the suppression of HMGB1 release by adiponectin but only partially inhibited that induced by IL-10. Treatment with compound C, an AMP kinase (AMPK) inhibitor, abolished the increase in HO-1 expression and the suppression of HMGB1 release mediated by adiponectin. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through an AMPK-mediated and HO-1-dependent IL-10-independent pathway. PMID:27313399

  17. Degradation of the interferon-induced 68,000-M(r) protein kinase by poliovirus requires RNA.

    PubMed Central

    Black, T L; Barber, G N; Katze, M G

    1993-01-01

    Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells. Images PMID:7678306

  18. Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

    SciTech Connect

    Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Dan; Hammel, Ken

    2008-10-02

    Fungal lignin-degrading systems must include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, secretion of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Despite their importance, little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of them by shotgun liquid chromatography/tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein I.D. 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were up-regulated under ligninolytic conditions. Real time reverse transcription polymerase chain reaction assays showed that RNA transcripts encoding all of these proteins were also up-regulated in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium, and are therefore most likely associated with the outer envelope of the fungus.

  19. Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

    PubMed Central

    Golisz, Anna; Sikorski, Pawel J.; Kruszka, Katarzyna; Kufel, Joanna

    2013-01-01

    Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5′–3′ mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5′–3′ exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants. PMID:23620288

  20. Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

    PubMed

    Hwang, J-Y; Lee, J; Oh, C-K; Kang, H W; Hwang, I-Y; Um, J W; Park, H C; Kim, S; Shin, J-H; Park, W-Y; Darnell, R B; Um, H-D; Chung, K C; Kim, K; Oh, Y J

    2016-01-01

    Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP(+)) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP(+) reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP(+)-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP(+)-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP(+)-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration. PMID:27253404

  1. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

    PubMed Central

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community. PMID:26441929

  2. Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2.

    PubMed

    Kano, Satoshi; Miyajima, Naoto; Fukuda, Satoshi; Hatakeyama, Shigetsugu

    2008-07-15

    Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs. PMID:18632609

  3. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer.

    PubMed

    Raina, Kanak; Lu, Jing; Qian, Yimin; Altieri, Martha; Gordon, Deborah; Rossi, Ann Marie K; Wang, Jing; Chen, Xin; Dong, Hanqing; Siu, Kam; Winkler, James D; Crew, Andrew P; Crews, Craig M; Coleman, Kevin G

    2016-06-28

    Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC. PMID:27274052

  4. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

    PubMed

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community. PMID:26441929

  5. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

    PubMed Central

    Raina, Kanak; Lu, Jing; Qian, Yimin; Altieri, Martha; Gordon, Deborah; Rossi, Ann Marie K.; Wang, Jing; Chen, Xin; Dong, Hanqing; Siu, Kam; Winkler, James D.; Crew, Andrew P.; Crews, Craig M.; Coleman, Kevin G.

    2016-01-01

    Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC. PMID:27274052

  6. Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins.

    PubMed Central

    Sodeinde, O A; Sample, A K; Brubaker, R R; Goguen, J D

    1988-01-01

    The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid. Images PMID:2843471

  7. Chlorogenic Acid Improves Late Diabetes through Adiponectin Receptor Signaling Pathways in db/db Mice

    PubMed Central

    Jin, Shasha; Chang, Cuiqing; Zhang, Lantao; Liu, Yang; Huang, Xianren; Chen, Zhimin

    2015-01-01

    The aim of this study was to examine the effects of chlorogenic acid (CGA) on glucose and lipid metabolism in late diabetic db/db mice, as well as on adiponectin receptors and their signaling molecules, to provide evidence for CGA in the prevention of type 2 diabetes. We randomly divided 16 female db/db mice into db/db-CGA and db/db-control (CON) groups equally; db/m mice were used as control mice. The mice in both the db/db-CGA and db/m-CGA groups were administered 80 mg/kg/d CGA by lavage for 12 weeks, whereas the mice in both CON groups were given equal volumes of phosphate-buffered saline (PBS) by lavage. At the end of the intervention, we assessed body fat and the parameters of glucose and lipid metabolism in the plasma, liver and skeletal muscle tissues as well as the levels of aldose reductase (AR) and transforming growth factor-β1 (TGF-β1) in the kidneys and measured adiponectin receptors and the protein expression of their signaling molecules in liver and muscle tissues. After 12 weeks of intervention, compared with the db/db-CON group, the percentage of body fat, fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) in the db/db-CGA group were all significantly decreased; TGF-β1 protein expression and AR activity in the kidney were both decreased; and the adiponectin level in visceral adipose was increased. The protein expression of adiponectin receptors (ADPNRs), the phosphorylation of AMP-activated protein kinase (AMPK) in the liver and muscle, and the mRNA and protein levels of peroxisome proliferator-activated receptor alpha (PPAR-α) in the liver were all significantly greater. CGA could lower the levels of fasting plasma glucose and HbA1c during late diabetes and improve kidney fibrosis to some extent through the modulation of adiponectin receptor signaling pathways in db/db mice. PMID:25849026

  8. Association of adiponectin with hepatic steatosis: a study of 1,349 subjects in a random population sample

    PubMed Central

    2014-01-01

    Background Objective of the present study was to examine the association between adiponectin and hepatic steatosis, and other biochemical and anthropometric parameters in healthy subjects. Results A total of 1349 subjects (age 18–65 years) underwent ultrasound examination of the liver. Mean adiponectin concentration for the study collective was 11.35 ± 6.28 μg/mL. The following parameters were assessed for their association with adiponectin: body-mass index (BMI); age; sex; arterial blood pressure; nicotine use; alcohol consumption; physical activity; metabolic syndrome; total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol; triglycerides; aspartate aminotransferase (AST); alanine aminotransferase (ALT); γ-glutamyltransferase (GGT); alkaline phosphatase (AP); C-reactive protein (CRP); insulin sensitivity according to the Homeostasis Model Assessment (HOMA); random blood glucose; and the degree of steatosis of the liver. The numerical differences in the variables influencing adiponectin returned in the descriptive analysis were confirmed at bivariate analysis for BMI, ALT, AST, GGT, AP, total and HDL cholesterol, triglycerides, CRP, arterial blood pressure, metabolic syndrome, nicotine use and alcohol consumption. The logistic regression of the multivariate analysis showed that male sex, hepatic steatosis, BMI, metabolic syndrome, tobacco smoking and CRP correlate negatively with adiponectin, while age, moderate alcohol consumption and HDL cholesterol exhibit a positive association. Conclusions The results of the present study confirm the findings of previous research. Adiponectin correlates negatively with cardiometabolic risk factors and is an independent indicator for non-alcoholic fatty liver disease (NAFLD). PMID:24693952

  9. Stimulated release of a hyperpolarizing factor (ADHF) from mesenteric artery perivascular adipose tissue: involvement of myocyte BKCa channels and adiponectin

    PubMed Central

    Weston, A H; Egner, I; Dong, Y; Porter, E L; Heagerty, A M; Edwards, G

    2013-01-01

    Background and Purpose Perivascular adipose tissue (PVAT) releases adipocyte-derived hyperpolarizing factors (ADHFs) that may partly act by opening myocyte K+ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K+ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT. Experimental Approach Myocyte membrane potential was recorded from de-endothelialized, non-contracted rat and mouse mesenteric arteries in the presence and absence of PVAT. Key Results The β3-adrenoceptor agonist, CL-316,243 (10 μM), generated PVAT-dependent, iberiotoxin-sensitive myocyte hyperpolarizations resulting from BKCa channel opening and which were partially blocked by L-NMMA (100 μM). Adiponectin (5 μg·mL−1) also produced iberiotoxin-sensitive hyperpolarizations in PVAT-denuded arterioles. Activation of myocyte AMP-activated protein kinase (AMPK) using 5 μM A-769662 also induced BKCa-mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL-316,243, adiponectin and A-769662. In vessels from Adipo−/− mice, hyperpolarizations to CL-316,243 were absent whereas those to A-769662 and adiponectin were normal. In rat vessels, adipocyte-dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM) were unaltered. Conclusions and Implications Under basal, non-contracted conditions, β3-adrenoceptor stimulation of PVAT releases an ADHF, which is probably adiponectin. This activates AMPK to open myocyte BKCa channels indirectly and additionally liberates NO, which also contributes to the observed PVAT-dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A-769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF-induced myocyte electrical changes mediated via the opening of BKCa channels. PMID:23488724

  10. Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.

    PubMed

    Urbán, Noelia; van den Berg, Debbie L C; Forget, Antoine; Andersen, Jimena; Demmers, Jeroen A A; Hunt, Charles; Ayrault, Olivier; Guillemot, François

    2016-07-15

    Quiescence is essential for long-term maintenance of adult stem cells. Niche signals regulate the transit of stem cells from dormant to activated states. Here, we show that the E3-ubiquitin ligase Huwe1 (HECT, UBA, and WWE domain-containing 1) is required for proliferating stem cells of the adult mouse hippocampus to return to quiescence. Huwe1 destabilizes proactivation protein Ascl1 (achaete-scute family bHLH transcription factor 1) in proliferating hippocampal stem cells, which prevents accumulation of cyclin Ds and promotes the return to a resting state. When stem cells fail to return to quiescence, the proliferative stem cell pool becomes depleted. Thus, long-term maintenance of hippocampal neurogenesis depends on the return of stem cells to a transient quiescent state through the rapid degradation of a key proactivation factor. PMID:27418510

  11. Enhanced adiponectin actions by overexpression of adiponectin receptor 1 in macrophages

    PubMed Central

    Luo, Nanlan; Chung, B Hong; Wang, Xiangdong; Klein, Richard L.; Tang, Chao-Ke; Garvey, W. Timothy; Fu, Yuchang

    2013-01-01

    Objective Adiponectin is one of several important, metabolically active cytokines secreted from adipose tissue. Epidemiologic studies have associated low circulating levels of this adipokine with multiple metabolic disorders including obesity, insulin resistance, type II diabetes, and cardiovascular disease. To investigate how enhanced adiponectin-mediated changes in metabolism in vivo, we generated transgenic mice which specifically overexpress the gene coding for adiponectin receptor 1 (AdipoR1) in mouse macrophages using the human scavenger receptor A-I gene (SR-AI) enhancer/promoter. We found that macrophage-specific AdipoR1 transgenic mice (AdR1-TG) presented reduced whole body weight, fat accumulation and liver steatosis when these transgenic mice were fed with a high fat diet. Moreover, these macrophage AdR1-TG mice exhibited enhanced whole-body glucose tolerance and insulin sensitivity with reduced proinflammatory cytokines, MCP-1 and TNF-α, both in the serum and in the insulin target metabolic tissues. Additional studies demonstrated that these macrophage AdR1-TG animals exhibited reduced macrophage foam cell formation in the arterial wall when these transgenic mice were crossed with a low-density lipoprotein receptor (Ldlr) deficient mouse model. Conclusions These results suggest that AdipoR1 overexpressed in macrophages can physiologically modulate metabolic activities in vivo by enhancing adiponectin actions in distal metabolically active tissues. The AdipoR1 modified macrophages provide unique interactions with the residented tissues/cells, suggesting a novel role of macrophage adiponectin receptor in improving metabolic disorders in vivo. PMID:23510830

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

    SciTech Connect

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-05-03

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

  13. Multiple sequence signals direct recognition and degradation of protein substrates by the AAA+ protease HslUV.

    PubMed

    Sundar, Shankar; McGinness, Kathleen E; Baker, Tania A; Sauer, Robert T

    2010-10-29

    Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V(max). By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease. PMID:20837023

  14. Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

    PubMed

    Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

    2015-11-17

    Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation. PMID:26488608

  15. Establishment of a concept of visceral fat syndrome and discovery of adiponectin

    PubMed Central

    Matsuzawa, Yuji

    2010-01-01

    Although obesity is a major background of life style-related diseases such as diabetes mellitus, lipid disorder, hypertension and cardiovascular disease, the extent of whole body fat accumulation does not necessarily the determinant for the occurrence of these diseases. We developed the method for body fat analysis using CT scan and established the concept of visceral fat obesity, in other word metabolic syndrome in which intra-abdominal visceral fat accumulation has an important role in the development of diabetes, lipid disorder, hypertension and atherosclerosis. In order to clarify the mechanism that visceral fat accumulation causes metabolic and cardiovascular diseases, we have analyzed gene expression profile in subcutaneous adipose tissue and visceral adipose tissue. From the analysis, we found that adipose tissue, especially visceral adipose tissue expressed abundantly the genes encoding bioactive substances such as cytokines, growth factors and complements. In addition to known bioactive substances, we found a novel collagen-like protein which we named adiponectin. Adiponectin is present in plasma at a very high concentration and is inversely associated with visceral fat accumulation. Adiponectin has anti-diabetic, anti-hypertensive and anti-atherogenic properties and recent studies revealed that this protein has an anti-inflammatory and anti-oncogenic function. Therefore hypoadiponectinemia induced by visceral fat accumulation should become a strong risk factor for metabolic and cardiovascular diseases and also some kinds of cancers. In this review article, I would like to discuss the mechanism of life style-related diseases by focusing on the dysregulation of adiponectin related to obesity, especially visceral obesity. PMID:20154470

  16. Taxol and tau overexpression induced calpain-dependent degradation of the microtubule-destabilizing protein SCG10.

    PubMed

    Vega, Irving E; Hamano, Tadanori; Propost, Josh A; Grenningloh, Gabriele; Yen, Shu-Hui

    2006-11-01

    Microtubule-stabilizing and -destabilizing proteins play a crucial role in regulating the dynamic instability of microtubules during neuronal development and synaptic transmission. The microtubule-destabilizing protein SCG10 is a neuron-specific protein implicated in neurite outgrowth. The SCG10 protein is significantly reduced in mature neurons, suggesting that its expression is developmentally regulated. In contrast, the microtubule-stabilizing protein tau is expressed in mature neurons and its function is essential for the maintenance of neuronal polarity and neuronal survival. Thus, the establishment and maintenance of neuronal polarity may down-regulate the protein level/function of SCG10. In this report, we show that treatment of PC12 cells and neuroblastoma cells with the microtubule-stabilizing drug Taxol induced a rapid degradation of the SCG10 protein. Consistently, overexpression of tau protein in neuroblastoma cells also induced a reduction in SCG10 protein levels. Calpain inhibitor MDL-28170, but not caspase inhibitors, blocked a significant decrease in SCG10 protein levels. Collectively, these results indicate that tau overexpression and Taxol treatment induced a calpain-dependent degradation of the microtubule-destabilizing protein SCG10. The results provide evidence for the existence of an intracellular mechanism involved in the regulation of SCG10 upon microtubule stabilization. PMID:16822511

  17. Network analysis and cross species comparison of protein-protein interaction networks of human, mouse and rat cytochrome P450 proteins that degrade xenobiotics.

    PubMed

    Karthikeyan, Bagavathy Shanmugam; Akbarsha, Mohammad Abdulkader; Parthasarathy, Subbiah

    2016-06-21

    Cytochrome P450 (CYP) enzymes that degrade xenobiotics play a critical role in the metabolism and biotransformation of drugs and xenobiotics in humans as well as experimental animal models such as mouse and rat. These proteins function as a network collectively as well as independently. Though there are several reports on the organization, regulation and functionality of various CYP enzymes at the molecular level, the understanding of organization and functionality of these proteins at the holistic level remain unclear. The objective of this study is to understand the organization and functionality of xenobiotic degrading CYP enzymes of human, mouse and rat using network theory approaches and to study species differences that exist among them at the holistic level. For our analysis, a protein-protein interaction (PPI) network for CYP enzymes of human, mouse and rat was constructed using the STRING database. Topology, centrality, modularity and robustness analyses were performed for our predicted CYP PPI networks that were then validated by comparison with randomly generated network models. Network centrality analyses of CYP PPI networks reveal the central/hub proteins in the network. Modular analysis of the CYP PPI networks of human, mouse and rat resulted in functional clusters. These clusters were subjected to ontology and pathway enrichment analysis. The analyses show that the cluster of the human CYP PPI network is enriched with pathways principally related to xenobiotic/drug metabolism. Endo-xenobiotic crosstalk dominated in mouse and rat CYP PPI networks, and they were highly enriched with endogenous metabolic and signaling pathways. Thus, cross-species comparisons and analyses of human, mouse and rat CYP PPI networks gave insights about species differences that existed at the holistic level. More investigations from both reductionist and holistic perspectives can help understand CYP metabolism and species extrapolation in a much better way. PMID:27194593

  18. Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells.

    PubMed

    Wang, J F; Becks, G P; Hill, D J

    1997-02-01

    In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44-46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44-46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44-46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5. To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A-Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A-Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells. Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 degrees C for 16 h. Both 44-46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells

  19. Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

    PubMed Central

    von Nandelstadh, Pernilla; Gucciardo, Erika; Lohi, Jouko; Li, Rui; Sugiyama, Nami; Carpen, Olli; Lehti, Kaisa

    2014-01-01

    Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion. PMID:24989798

  20. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    PubMed

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway. PMID:27130438

  1. Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2005-01-01

    Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family. PMID:16212488

  2. The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation

    NASA Technical Reports Server (NTRS)

    Seider, M. J.; Kapp, R.; Chen, C.-P.; Booth, F. W.

    1980-01-01

    Skeletal muscle preparations using cut muscle fibers have often been used in studies of protein metabolism. The present paper reports an investigation of the effect of muscle cutting or stretching in vitro on the rates of protein synthesis and/or degradation. Protein synthesis and content, and ATP and phosphocreatine levels were monitored in soleus and extensor digitorum longus muscles from the rat with various extents of muscle fiber cuts and following stretching to about 120% the resting length. Rates of protein synthesis are found to be significantly lower and protein degradation higher in the cut muscles than in uncut controls, while ATP and phosphocreatine concentrations decreased. Stretched intact muscles, on the other hand, are observed to have higher concentrations of high-energy phosphates than unstretched muscles, while rates of protein degradation were not affected. Results thus demonstrate that the cutting of skeletal muscle fibers alters many aspects of muscle metabolism, and that moderate decreases in ATP concentration do not alter rates of protein concentration in intact muscles in vitro.

  3. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  4. Proteomic and SAGE profiling of murine melanoma progression indicates the reduction of proteins responsible for ROS degradation.

    PubMed

    de Souza, Gustavo A; Godoy, Lyris M F; Teixeira, Veronica R; Otake, Andreia H; Sabino, Adão; Rosa, José C; Dinarte, Anemari R; Pinheiro, Daniel G; Silva, Wilson A; Eberlin, Marcos N; Chammas, Roger; Greene, Lewis J

    2006-03-01

    Using 2-DE of total cell protein extracts, we compared soluble proteins from murine melanoma lines Tm1 and Tm5 with proteins from the nontumoral cell melan-a from which they were derived. Seventy-one of the 452 spots (average) detected with CBB were differentially accumulated, i.e., increased or decreased twofold. Forty-four spots were identified by PMF/MALDI-TOF, 15 with increased and 29 with decreased protein levels. SAGE showed that 17/34 (50%) of the differentially accumulated proteins, pI range 4-7, presented similar differences at the mRNA level. Major reductions in protein were observed in tumor cells of proteins that degrade reactive oxygen species (ROS). Decreases of > or = twofold in GST, superoxide dismutase, aldehyde dehydrogenase, thioredoxin, peroxiredoxin 2, and peroxiredoxin 6 protein were observed. SAGE indicated the reduction of other proteins involved in ROS degradation. As expected, the accumulation of exogenous peroxides was significantly higher in the tumor cells while the levels of glutathionylation were two times lower in the tumor cells compared to melan-a. The differential accumulation of proteins involved in oncogene/tumor suppressor pathways was observed. Melanoma cells can favor survival pathways activated by ROS by inhibiting p53 pathways and activation of Ras and c-myc pathways. PMID:16429458

  5. Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Evert, Bernd O; Rubinsztein, David C; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-12-01

    Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyl transferases may affect chromatin structure and transcription and thus contribute to neurodegeneration. PMID:16456924

  6. Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Evert, Bernd O; Rubinsztein, David C; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-09-01

    Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyltransferases may affect chromatin structure and transcription and thus contribute to neurodegeneration. PMID:15994095

  7. Effects of feed intake and protein degradability on ruminal characteristics and site of digestion in steers.

    PubMed

    Firkins, J L; Berger, L L; Merchen, N R; Fahey, G C; Nelson, D R

    1986-08-01

    Four multiple-fistulated Hereford steers were used in a 4 X 4 Latin square design with a 2 X 2 factorial arrangement of treatments [two intakes (9.1 and 6.1 kg dry matter/d) and two protein sources differing in ruminal degradability (dry distillers grains and dry corn gluten feed)]. Steers fed at the high intake had faster fluid dilution rates (7.63 versus 6.52%/h), higher ruminal fluid outflows (120.2 versus 91.7 L/d), lower apparent ruminal digestibilities of organic matter (41.3 versus 44.3%) and neutral detergent fiber (56.0 versus 60.2%), and lower total tract digestibilities of neutral detergent fiber (64.3 versus 68.7%) than when they were fed at the low intake. Steers fed dry corn gluten feed had higher apparent ruminal digestibilities of organic matter (45.5 versus 40.1%) and neutral detergent fiber (60.2 versus 56.0%) and lower duodenal flows of nonammonia-nonbacterial N (40.1 versus 52.2% of N intake) than when they were fed dry distillers grains. Efficiency of ruminal bacterial growth was higher when steers were fed at the high versus low intakes. Efficiency of ruminal bacterial growth and site and extent of fiber digestion, especially hemicellulose, but not ruminal escape of protein, can be readily altered by manipulation of feed intake of moderately high forage diets. PMID:3020102

  8. Application of Universal Stress Proteins in Probing the Dynamics of Potent Degraders in Complex Terephthalate Metagenome

    PubMed Central

    Mbah, Andreas N.; Isokpehi, Raphael D.

    2013-01-01

    The culture-independent strategies to study microbial diversity and function have led to a revolution in environmental genomics, enabling fundamental questions about the distribution of microbes and their influence on bioremediation to be addressed. In this research we used the expression of universal stress proteins as a probe to determine the changes in degrading microbial population from a highly toxic terephthalate wastewater to a less toxic activated sludge bioreactor. The impact of relative toxicities was significantly elaborated at the levels of genus and species. The results indicated that 23 similar prokaryotic phyla were represented in both metagenomes irrespective of their relative abundance. Furthermore, the following bacteria taxa Micromonosporaceae, Streptomyces, Cyanothece sp. PCC 7822, Alicyclobacillus acidocaldarius, Bacillus halodurans, Leuconostoc mesenteroides, Lactococcus garvieae, Brucellaceae, Ralstonia solanacearum, Verminephrobacter eiseniae, Azoarcus, Acidithiobacillus ferrooxidans, Francisella tularensis, Methanothermus fervidus, and Methanocorpusculum labreanum were represented only in the activated sludge bioreactor. These highly dynamic microbes could serve as taxonomic biomarkers for toxic thresholds related to terephthalate and its derivatives. This paper, highlights the application of universal stress proteins in metagenomics analysis. Dynamics of microbial consortium of this nature can have future in biotechnological applications in bioremediation of toxic chemicals and radionuclides. PMID:24151583

  9. Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

    PubMed

    Gruosso, Tina; Mieulet, Virginie; Cardon, Melissa; Bourachot, Brigitte; Kieffer, Yann; Devun, Flavien; Dubois, Thierry; Dutreix, Marie; Vincent-Salomon, Anne; Miller, Kyle Malcolm; Mechta-Grigoriou, Fatima

    2016-01-01

    Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients. PMID:27006338

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

    PubMed Central

    Mao, Yanwei; Zhang, Yimin; Liang, Rongrong; Ren, Lulu; Zhu, He; Li, Ke; Zhu, Lixian; Luo, Xin

    2012-01-01

    The objective of this study was to investigate the effect of rapid chilling (RC) on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC) were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES) treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM). The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness. PMID:25049681

  11. Inhibition of ABCA1 protein degradation promotes HDL cholesterol efflux capacity and RCT and reduces atherosclerosis in mice.

    PubMed

    Huang, LinZhang; Fan, BaoYan; Ma, Ang; Shaul, Philip W; Zhu, HaiBo

    2015-05-01

    ABCA1 plays a key role in the initial lipidation of apoA-I, which generates circulating HDL cholesterol. Whereas it is known that the transcriptional upregulation of ABCA1 promotes HDL formation and reverse cholesterol transport (RCT), it is not known how the inhibition of ABCA1 protein degradation impacts HDL function. Employing the small molecule triacetyl-3-hydroxyphenyladenosine (IMM-H007), we determined how the attenuation of ABCA1 protein degradation affects HDL cholesterol efflux capacity, RCT, and atherosclerotic lesion formation. Pulse-chase analysis revealed that IMM-H007 inhibits ABCA1 degradation and facilitates its cell-surface localization in macrophages, and additional studies in macrophages showed that IMM-H007 thereby promotes cholesterol efflux. IMM-H007 treatment of Paigen diet-fed mice caused an increase in circulating HDL level, it increased the cholesterol efflux capacity of HDL, and it enhanced in vivo RCT from macrophages to the plasma, liver, and feces. Furthermore, ABCA1 degradation suppression by IMM-H007 reduced atherosclerotic plaque formation in apoE(-/-) mice. Thus, via effects on both ABCA1-expressing cells and circulating HDL function, the inhibition of ABCA1 protein degradation by IMM-H007 promotes HDL cholesterol efflux capacity and RCT and attenuates atherogenesis. IMM-H007 potentially represents a lead compound for the development of agents to augment HDL function. PMID:25761370

  12. A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

    PubMed Central

    Doherty, F J; Wassell, J A; Mayer, R J

    1987-01-01

    Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Images Fig. 1. Fig. 5. PMID:3593223

  13. Adiponectin signaling and function in insulin target tissues

    PubMed Central

    Ruan, Hong; Dong, Lily Q.

    2016-01-01

    Obesity-linked type 2 diabetes is one of the paramount causes of morbidity and mortality worldwide, posing a major threat on human health, productivity, and quality of life. Despite great progress made towards a better understanding of the molecular basis of diabetes, the available clinical counter-measures against insulin resistance, a defect that is central to obesity-linked type 2 diabetes, remain inadequate. Adiponectin, an abundant adipocyte-secreted factor with a wide-range of biological activities, improves insulin sensitivity in major insulin target tissues, modulates inflammatory responses, and plays a crucial role in the regulation of energy metabolism. However, adiponectin as a promising therapeutic approach has not been thoroughly explored in the context of pharmacological intervention, and extensive efforts are being devoted to gain mechanistic understanding of adiponectin signaling and its regulation, and reveal therapeutic targets. Here, we discuss tissue- and cell-specific functions of adiponectin, with an emphasis on the regulation of adiponectin signaling pathways, and the potential crosstalk between the adiponectin and other signaling pathways involved in metabolic regulation. Understanding better just why and how adiponectin and its downstream effector molecules work will be essential, together with empirical trials, to guide us to therapies that target the root cause(s) of type 2 diabetes and insulin resistance. PMID:26993044

  14. Role of Adiponectin in Coronary Heart Disease Risk

    PubMed Central

    Lawlor, Debbie A.; de Oliveira, Cesar; White, Jon; Horta, Bernardo Lessa; Barros, Aluísio J.D.

    2016-01-01

    Rationale: Hypoadiponectinemia correlates with several coronary heart disease (CHD) risk factors. However, it is unknown whether adiponectin is causally implicated in CHD pathogenesis. Objective: We aimed to investigate the causal effect of adiponectin on CHD risk. Methods and Results: We undertook a Mendelian randomization study using data from genome-wide association studies consortia. We used the ADIPOGen consortium to identify genetic variants that could be used as instrumental variables for the effect of adiponectin. Data on the association of these genetic variants with CHD risk were obtained from CARDIoGRAM (22 233 CHD cases and 64 762 controls of European ancestry) and from CARDIoGRAMplusC4D Metabochip (63 746 cases and 130 681 controls; ≈ 91% of European ancestry) consortia. Data on the association of genetic variants with adiponectin levels and with CHD were combined to estimate the influence of blood adiponectin on CHD risk. In the conservative approach (restricted to using variants within the adiponectin gene as instrumental variables), each 1 U increase in log blood adiponectin concentration was associated with an odds ratio for CHD of 0.83 (95% confidence interval, 0.68–1.01) in CARDIoGRAM and 0.97 (95% confidence interval, 0.84–1.12) in CARDIoGRAMplusC4D Metabochip. Findings from the liberal approach (including variants in any locus across the genome) indicated a protective effect of adiponectin that was attenuated to the null after adjustment for known CHD predictors. Conclusions: Overall, our findings do not support a causal role of adiponectin levels in CHD pathogenesis. PMID:27252388

  15. Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8.

    PubMed

    Kichise, Tomoyasu; Hisano, Tamao; Takeda, Kazuki; Miki, Kunio

    2009-09-01

    Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non-oxygenolytic ring opening, and beta-oxidation-like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non-oxygenolytic ring-opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 A. PaaG consists of three identical subunits related by local three-fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4-chlorobenzoyl-CoA dehalogenase and peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase, indicating that PaaG may undergo isomerization or a ring-opening reaction via a Delta(3),Delta(2)-enoyl-CoA isomerase-like mechanism. PMID:19452559

  16. Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut.

    PubMed

    Fescemyer, Howard W; Sandoya, Germán V; Gill, Torrence A; Ozkan, Seval; Marden, James H; Luthe, Dawn S

    2013-03-01

    Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function. PMID:23306018

  17. Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks.

    PubMed

    Shimamoto, Saki; Ijiri, Daichi; Nakashima, Kazuki; Kawaguchi, Mana; Ishimaru, Yoshitaka; Furukawa, Airi; Ohtsuka, Akira

    2016-08-01

    To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases. PMID:27055887

  18. Effect of Source of Rumen-Degraded Protein on Production and Ruminal Metabolism in Lactating Dairy Cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-eight (8 with ruminal cannulas) lactating Holstein cows were assigned to a 16-wk, 4 x 4 Latin squares study to examine the effect on production and ruminal metabolism of feeding differing proportions of rumen-degraded protein (RDP) from soybean meal and urea. Diets contained dry matter [(DM) ...

  19. The interaction of harvesting time of day of switchgrass hay and ruminal degradability of supplemental protein to beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to interact AM ( 0600 ) vs PM ( 1800 ) harvest with ruminal degradability of a protein supplement (HI or LO ) to change voluntary intake, apparent digestibility or N retention by steers fed Alamo switchgrass (Panicum virgatum L.) hay. Black steers (255 ± 14kg BW) we...

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

    PubMed Central

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-01-01

    Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in-vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis. PMID:23578663

  1. Estimating Rumen Degradable Protein in Forage Legume Hays and Silages by In Situ Disappearance Kinetics vs. Alternative Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently, the in situ kinetic method is considered the “gold standard” for estimating rumen degradable protein (RDP) in forages. When such estimates are impractical (e.g. numerous samples, limited sample quantities, or lack of ruminally fistulated cattle) alternative RDP methods are used, but their...

  2. Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    PubMed Central

    Kim, Nari; Kim, Min-Jung; Sung, Pil Soo; Bae, Yong Chul; Shin, Eui-Cheol; Yoo, Joo-Yeon

    2016-01-01

    Hepatitis C virus (HCV) utilizes autophagy to promote its propagation. Here we show the autophagy-mediated suppression of HCV replication via the endoplasmic reticulum (ER) protein SCOTIN. SCOTIN overexpression inhibits HCV replication and infectious virion production in cells infected with cell culture-derived HCV. HCV nonstructural 5A (NS5A) protein, which is a critical factor for HCV RNA replication, interacts with the IFN-β-inducible protein SCOTIN, which transports NS5A to autophagosomes for degradation. Furthermore, the suppressive effect of SCOTIN on HCV replication is impaired in both ATG7-silenced cells and cells treated with autophagy or lysosomal inhibitors. SCOTIN does not affect the overall flow of autophagy; however, it is a substrate for autophagic degradation. The physical association between the transmembrane/proline-rich domain (TMPRD) of SCOTIN and Domain-II of NS5A is essential for autophagosomal trafficking and NS5A degradation. Altogether, our findings suggest that IFN-β-induced SCOTIN recruits the HCV NS5A protein to autophagosomes for degradation, thereby restricting HCV replication. PMID:26868272

  3. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators. PMID:27155164

  4. Positive feedback regulation between adiponectin and T-cadherin impacts adiponectin levels in tissue and plasma of male mice.

    PubMed

    Matsuda, Keisuke; Fujishima, Yuya; Maeda, Norikazu; Mori, Takuya; Hirata, Ayumu; Sekimoto, Ryohei; Tsushima, Yu; Masuda, Shigeki; Yamaoka, Masaya; Inoue, Kana; Nishizawa, Hitoshi; Kita, Shunbun; Ranscht, Barbara; Funahashi, Tohru; Shimomura, Iichiro

    2015-03-01

    Adiponectin (Adipo), a multimeric adipocyte-secreted protein abundant in the circulation, is implicated in cardiovascular protective functions. Recent work documented that Adipo locally associates with responsive tissues through interactions with T-cadherin (Tcad), an atypical, glycosylphosphatidylinositol (GPI)-anchored cadherin cell surface glycoprotein. Mice deficient for Tcad lack tissue-associated Adipo, accumulate Adipo in the circulation, and mimic the Adipo knockout (KO) cardiovascular phenotype. In reverse, Tcad protein is visibly reduced from cardiac tissue in Adipo-KO mice, suggesting interdependent regulation of the 2 proteins. Here, we evaluate the effect of Adipo on Tcad protein expression. Adipo and Tcad proteins were colocalized in aorta, heart, and skeletal muscle. Adipo positively regulated levels of Tcad protein in vivo and in endothelial cell (EC) cultures. In Tcad-KO mice, binding of endogenous and exogenously administered Adipo to cardiovascular tissues was dramatically reduced. Consistently, knockdown of Tcad in cultured murine vascular ECs significantly diminished Adipo binding. In search for a possible mechanism, we found that enzymatic cleavage of Tcad with phosphatidylinositol-specific phospholipase C increases plasma Adipo while decreasing tissue-bound levels. Similarly, pretreatment of cultured ECs with serum containing Adipo attenuated phosphatidylinositol-specific phospholipase C-mediated Tcad cleavage. In vivo administration of adenovirus producing Adipo suppressed plasma levels of GPI phospholipase D, the endogenous cleavage enzyme for GPI-anchored proteins. In conclusion, our data show that both circulating and tissue-bound Adipo levels are dependent on Tcad and, in reverse, regulate tissue Tcad levels through a positive feedback loop that operates by suppressing phospholipase-mediated Tcad release from the cell surface. PMID:25514086

  5. Heat shock transcription factor δ³² is targeted for degradation via an ubiquitin-like protein ThiS in Escherichia coli.

    PubMed

    Xu, Xibing; Niu, Yulong; Liang, Ke; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2015-04-01

    The posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins (UBLs) plays an important role in eukaryote biology, through which substrate proteins are targeted for degradation by the proteasome. Prokaryotes have been thought to degrade proteins by an ubiquitin independent pathway. Here, we show that ThiS, an ubiquitin-like protein, is covalently attached to δ(32) and at least 27 other proteins, leading to their subsequent degradation by proteases, in a similar manner to the ubiquitin-proteasome system (UPS) in eukaryotes. Molecular biology and biochemical studies confirm that specific lysine sites in δ(32) can be modified by ThiS. The results presented here establish a new model for δ(32) degradation and show that Escherichia coli uses a small-protein modifier to control protein stability. PMID:25721662

  6. Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

    PubMed

    St John, A C; Goldberg, A L

    1980-09-01

    Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA c