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Sample records for adiponectin protein degradation

  1. Biomarkers of Adiponectin: Plasma Protein Variation and Genomic DNA Polymorphisms

    PubMed Central

    Gu, Harvest F.

    2009-01-01

    Adiponectin is secreted by white adipose tissue and exists as the most abundant adipokine in the human plasma. Recent research has indicated that plasma adiponectin levels are inversely correlated with body mass index (BMI) and insulin resistance. Reduction of plasma adiponectin levels is commonly observed in the patients with type 2 diabetes (T2D) and/or in those who are obese in comparison with healthy control individuals. The adiponectin (AdipoQ) gene has a moderate linkage disequilibrium (LD), but two small LD blocks are observed, respectively, in the promoter region and the boundary of exon 2-intron 2. Genetic association studies have demonstrated that single nucleotide polymorphisms (SNPs) +45G15G(T/G) in exon 2 and +276G/T in intron 2 of the AdipoQ gene confer the risk susceptibility to the development of T2D, obesity and diabetic nephropathy (DN). The SNPs in the promoter region, including −11426A/G, −11377C/G and −11391G/A, are found to be associated with T2D and DN. Recent research has indicated that the promoter polymorphisms interfere with the AdipoQ promoter activity. The haplotypes constructed by the promoter polymorphisms and SNP +276G/T in intron 2 are associated with circulating adiponectin levels. This review summarises genetic and pathophysiological relevancies of adiponectin and discusses about the biomarkers of adiponectin plasma protein variation and genomic DNA polymorphisms. PMID:20029651

  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. 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

  4. The Effects of Aerobic Exercise on Plasma Adiponectin Level and Adiponectin-related Protein Expression in Myocardial Tissue of ApoE(-/-) Mice.

    PubMed

    Zhu, Xiao-Juan; Chen, Li-Hui; Li, Jiang-Hua

    2015-12-01

    Numerous reports have confirmed the effect of ApoE knockout in the induction of cardiovascular diseases and the protective effect of adiponectin against the progression of cardiovascular diseases. The aim of this study was to reveal the roles of adiponectin signaling in the progression of cardiovascular diseases induced by ApoE knockout and to analyze the healthy effects of aerobic exercise on ApoE knockout mice (ApoE(-/-) mice) through observing the changes of adiponectin signaling caused by ApoE knockout and aerobic exercise. A twelve-week aerobic exercise program was carried out on the male ApoE(-/-) mice and the C57BL / 6J mice (C57 mice) of the same strain. Results show that the body weights, blood lipid level, plasma adiponectin level and adiponectin-related proteins in myocardial tissue were all significantly changed by ApoE knockout. A twelve-week aerobic exercise program exerted only minimal effects on the body weights, blood lipid levels, and plasma adiponectin levels of ApoE(-/-) mice, but increased the expressions of four adiponectin-related proteins, AdipoR1, PPARα, AMPK and P-AMPK, in the myocardial tissue of the ApoE(-/-) mice. In summary, adiponectin signaling may play an import role in the progression of cardiovascular diseases induced by ApoE knockout, and the beneficial health effects of aerobic exercise on ApoE(-/-) mice may be mainly from the increased adiponectin-related protein expression in myocardial tissue. Key pointsA twelve-week aerobic exercise program exerted only limited effects on the body weights and the plasma adiponectin levels of both the normal mice and the ApoE(-/-) mice but did effectively regulate the blood lipid levels of the normal mice (but not the ApoE(-/-) mice).After 12 weeks of aerobic exercise, expression of the adiponectin-related proteins in the myocardial tissue of the ApoE(-/-) and normal mice was increased, but the increased amplitudes of these proteins in the ApoE(-/-) mice were much larger in the Apo

  5. Secretion of the Adipocyte-Specific Secretory Protein Adiponectin Critically Depends on Thiol-Mediated Protein Retention▿ †

    PubMed Central

    Wang, Zhao V.; Schraw, Todd D.; Kim, Ja-Young; Khan, Tayeba; Rajala, Michael W.; Follenzi, Antonia; Scherer, Philipp E.

    2007-01-01

    Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lα, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones. PMID:17353260

  6. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    PubMed Central

    Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

  7. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.

    PubMed

    Frizzell, Norma; Rajesh, Mathur; Jepson, Matthew J; Nagai, Ryoji; Carson, James A; Thorpe, Suzanne R; Baynes, John W

    2009-09-18

    S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219-34228). We show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for approximately 7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.

  8. Effects of sugar-sweetened beverages on plasma acylation stimulating protein, leptin, and adiponectin: Relationships with metabolic outcomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE: The effects of fructose and glucose consumption on plasma acylation stimulating protein (ASP), adiponectin, and leptin concentrations relative to energy intake, body weight, adiposity, circulating triglycerides, and insulin sensitivity were determined. DESIGN AND METHODS: Thirty two over...

  9. Rat myocardial protein degradation.

    PubMed

    Steer, J H; Hopkins, B E

    1981-07-01

    1. Myocardial protein degradation rates were determined by following tyrosine release from rat isolated left hemi-atria in vitro. 2. After two 20 min preincubations the rate of tyrosine release from hemi-atria was constant for 4 h. 3. Skeletal muscle protein degradation was determined by following tyrosine release from rat isolated hemi-diaphragm (Fulks, Li & Goldberg, 1975). 4. Insulin (10(-7) M) inhibited tyrosine release from hemi-atria and hemi-diaphragm to a similar extent. A 48 h fast increased tyrosine release rate from hemi-diaphragm and decreased tyrosine release rate from hemi-atria. Hemi-diaphragm tyrosine release was inhibited by 15 mmol/l D-glucose but a variety of concentrations of D-glucose (0, 5, 15 mmol/l) had no effect on tyrosine release from hemi-atria. Five times the normal plasma levels of the branched-chain amino acids leucine, isoleucine and valine had no effect on tyrosine release from either hemi-atria or hemi-diaphragm.

  10. Adiponectin in chronic kidney disease has an opposite impact on protein-energy wasting and cardiovascular risk: two sides of the same coin.

    PubMed

    Park, S-H; Carrero, J J; Lindholm, B; Stenvinkel, P

    2009-08-01

    Adiponectin, an anti-inflammatory, anti-atherogenic and insulin sensitizing adipokine exists in several isoforms in the circulation. In patients with chronic kidney disease (CKD), circulating levels of total as well as high-molecular-weight adiponectin are elevated. In contrast to initial studies, several recent and larger studies on outcomes do not support a protective effect of high adiponectin on cardiovascular disease (CVD) and overall mortality in CKD patients. Paradoxically, high adiponectin predicts increased overall and cardiovascular mortality in CKD patients. This effect seems unrelated to a direct effect of adiponectin, but rather due to a process of protein-energy (PEW) wasting. This review summarizes recent conflicting findings on adiponectin in relation to outcomes and discusses the pathophysiologic roles of adiponectin in PEW, insulin resistance and vascular injuries of CKD patients.

  11. 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

  12. 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

  13. 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

  14. C-reactive protein inhibits high-molecular-weight adiponectin expression in 3T3-L1 adipocytes via PI3K/Akt pathway.

    PubMed

    Liu, Yuanxin; Liu, Cuiping; Jiang, Chao; Wang, Su; Yang, Qichao; Jiang, Dan; Yuan, Guoyue

    2016-03-25

    Adiponectin, an adipose-specific protein hormone, is secreted from white adipose tissue and involved in glucose and lipid metabolism. It is assembled into low-molecular-weight trimer (LMW), middle-molecular-weight hexameric (MMW) and high-molecular-weight (HMW), among which HMW exhibits higher activity. In this study, we proved that C-reactive protein (CRP), an inflammatory marker, inhibited adiponectin expression, especially HMW in time-and dose-dependent manners. Furthermore, CRP decreased the HMW/total adiponectin ration and reduced adiponectin assembly by increasing ERp44, and decreasing Ero1-α and DsbA-L. CRP activated pAkt, the downstream of PI3K. Inhibition of PI3K or pAkt abolished the effect of CRP. Our study suggested that CRP decreased adiponectin expression and multimerization, while CRP-induced decline in adiponectin might be mediated through the PI3K/Akt pathway.

  15. 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

  16. The Effect of Vegan Protein-Based Diets on Metabolic Parameters, Expressions of Adiponectin and Its Receptors in Wistar Rats

    PubMed Central

    Chen, Jie-Hua; Song, Jia; Chen, Yan; Ding, Qiang; Peng, Anfang; Mao, Limei

    2016-01-01

    Vegan protein-based diet has attracted increasing interest in the prevention of metabolic syndrome (MetS). Meanwhile, adiponectin has become a highly potential molecular target in the prevention of MetS. Our study will identify a potential vegan protein diet for the prevention of MetS using rat models. Thirty-six Wistar rats were randomly assigned into three groups and given diets containing one of the following proteins for 12 weeks: casein (CAS, control diet), soy protein (SOY), and gluten-soy mixed protein (GSM). Changes in metabolic parameters as well as the expressions of adiponectin and its receptors were identified. Compared to CAS diet, both SOY and GSM diets led to decreases in blood total cholesterol and triglycerides, but only GSM diet led to an increase in HDL-cholesterol; no marked difference was observed in blood glucose in all three groups; HOMA-IR was found lower only in SOY group. Among groups, the order of serum adiponectin level was found as GSM > SOY > CAS. Similar order pattern was also observed in expression of adiponectin in adipose tissue and AdipoR1 mRNA in skeletal muscle. Our results suggested for the first time that, besides SOY diet, GSM diet could also be a possible substitute of animal protein to prevent MetS. PMID:27763537

  17. (-)-Catechin suppresses expression of Kruppel-like factor 7 and increases expression and secretion of adiponectin protein in 3T3-L1 cells.

    PubMed

    Cho, Si Young; Park, Pil Joon; Shin, Hyun Jung; Kim, Young-Kyung; Shin, Dong Wook; Shin, Eui Seok; Lee, Hyoung Ho; Lee, Byeong Gon; Baik, Joo-Hyun; Lee, Tae Ryong

    2007-04-01

    Adiponectin is an adipocyte-specific secretory hormone that can increase insulin sensitivity and promote adipocyte differentiation. Administration of adiponectin to obese or diabetic mice reduces plasma glucose and free fatty acid levels. Green tea polyphenols possess many pharmacological activities such as antioxidant, anti-inflammatory, antiobesity, and antidiabetic activities. To investigate whether green tea polyphenols have an effect on the regulation of adiponectin, we measured expression and secretion levels of adiponectin protein after treatment of each green tea polyphenols in 3T3-L1 adipocytes. We found that (-)-catechin enhanced the expression and secretion of adiponectin protein in a dose- and time-dependent manner. Furthermore, treatment of (-)-catechin increased insulin-dependent glucose uptake in differentiated adipocytes and augmented the expression of adipogenic marker genes, including PPARgamma, CEBPalpha, FAS, and SCD-1, when (-)-catechin was treated during adipocyte differentiation. In search of the molecular mechanism responsible for inducible effect of (-)-catechin on adiponectin expression, we found that (-)-catechin markedly suppresses the expression of Kruppel-like factor 7 (KLF7) protein, which has recently been reported to inhibit the expression of adiponectin and other adipogenesis related genes, including leptin, PPARgamma, C/EBPalpha, and aP2 in adipocytes. KLF7 is a transcription factor in adipocyte and plays an important role in the pathogenesis of type 2 diabetes. Taken together, these data suggest that the upregulation of adiponectin protein by (-)-catechin may involve, at least in part, suppression of KLF7 in 3T3-L1 cells.

  18. 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.

  19. Trans-Cinnamic Acid Increases Adiponectin and the Phosphorylation of AMP-Activated Protein Kinase through G-Protein-Coupled Receptor Signaling in 3T3-L1 Adipocytes

    PubMed Central

    Kopp, Christina; Singh, Shiva P.; Regenhard, Petra; Müller, Ute; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    Adiponectin and intracellular 5′adenosine monophosphate-activated protein kinase (AMPK) are important modulators of glucose and fat metabolism. Cinnamon exerts beneficial effects by improving insulin sensitivity and blood lipids, e.g., through increasing adiponectin concentrations and AMPK activation. The underlying mechanism is unknown. The Gi/Go-protein-coupled receptor (GPR) 109A stimulates adiponectin secretion after binding its ligand niacin. Trans-cinnamic acid (tCA), a compound of cinnamon is another ligand. We hypothesize whether AMPK activation and adiponectin secretion by tCA is transmitted by GPR signaling. Differentiated 3T3-L1 cells were incubated with pertussis toxin (PTX), an inhibitor of Gi/Go-protein-coupling, and treated with different tCA concentrations. Treatment with tCA increased adiponectin and the pAMPK/AMPK ratio (p ≤ 0.001). PTX incubation abolished the increased pAMPK/AMPK ratio and adiponectin secretion. The latter remained increased compared to controls (p ≤ 0.002). tCA treatment stimulated adiponectin secretion and AMPK activation; the inhibitory effect of PTX suggests GPR is involved in tCA stimulated signaling. PMID:24557583

  20. Predictive role of adiponectin and high-sensitivity C-reactive protein for prediction of cardiovascular event in an Iranian cohort Study: The Isfahan Cohort Study

    PubMed Central

    Kazemi-Saleh, Davoud; Koosha, Pooya; Sadeghi, Masoumeh; Sarrafzadegan, Nizal; Karbasi-Afshar, Reza; Boshtam, Mansoureh; Oveis-Gharan, Shahram

    2016-01-01

    BACKGROUND Numerous studies have been conducted on the predictive effects of high-sensitivity C-reactive protein (hs-CRP) on cardiovascular events. Few studies have been conducted to investigate the effects of adiponectin for the prediction of the incident of cardiovascular events in the Middle East area. This study compared the predictive effect of hs-CRP and adiponectin on healthy volunteers for the prediction of cerebrovascular disease (CVD). METHODS This nested case-control in original Isfahan Cohort Study (ICS) was conducted from 2001 to 2011. Participants were selected from ICS. The case group included participants with CVD while the control group included participants without CVD. The level of hs-CRP and adiponectin was measured in the blood samples collected in the year 2007. Thereafter, the statistical analyses were performed to determine the predictive value of hs-CRP and adiponectin in CVD prediction. RESULTS The results showed that before the elimination of diabetes effect; there was a significant difference between the two groups, in terms of the mean of adiponectin (P = 0.019) and no significant difference was observed in hs-CRP levels (P = 0.673). However, after eliminating the factor of diabetes, there was no significant difference between the case and control groups in adiponectin and hs-CRP levels (P = 0.184, P = 0.946). The results showed that the odds ratio (OR) of the adiponectin level was 0.879 [95% confidence interval (CI): 0.719-1.075, P = 0.210] while the OR of hs-CRP was 1.045 (95% CI: 0.922-1.185, P = 0.491). Furthermore, it was shown that after adjustment for age, sex, and diabetes; the OR of adiponectine was 0.875 (95% CI: 0.701-1.091, P = 0.235) and that of hs-CRP was 1.068 (95% CI: 0.935-1.219, P = 0.333). CONCLUSION The results show that adiponectin and hs-CRP cannot be predictors for cardiovascular events in a healthy population. Risk factors such as diabetes limit the use of adiponectin as a CVD predictor. PMID:27752270

  1. Adiponectin Lowers Glucose Production by Increasing SOGA

    PubMed Central

    Cowerd, Rachael B.; Asmar, Melissa M.; Alderman, J. McKee; Alderman, Elizabeth A.; Garland, Alaina L.; Busby, Walker H.; Bodnar, Wanda M.; Rusyn, Ivan; Medoff, Benjamin D.; Tisch, Roland; Mayer-Davis, Elizabeth; Swenberg, James A.; Zeisel, Steven H.; Combs, Terry P.

    2010-01-01

    Adiponectin is a hormone that lowers glucose production by increasing liver insulin sensitivity. Insulin blocks the generation of biochemical intermediates for glucose production by inhibiting autophagy. However, autophagy is stimulated by an essential mediator of adiponectin action, AMPK. This deadlock led to our hypothesis that adiponectin inhibits autophagy through a novel mediator. Mass spectrometry revealed a novel protein that we call suppressor of glucose by autophagy (SOGA) in adiponectin-treated hepatoma cells. Adiponectin increased SOGA in hepatocytes, and siRNA knockdown of SOGA blocked adiponectin inhibition of glucose production. Furthermore, knockdown of SOGA increased late autophagosome and lysosome staining and the secretion of valine, an amino acid that cannot be synthesized or metabolized by liver cells, suggesting that SOGA inhibits autophagy. SOGA decreased in response to AICAR, an activator of AMPK, and LY294002, an inhibitor of the insulin signaling intermediate, PI3K. AICAR reduction of SOGA was blocked by adiponectin; however, adiponectin did not increase SOGA during PI3K inhibition, suggesting that adiponectin increases SOGA through the insulin signaling pathway. SOGA contains an internal signal peptide that enables the secretion of a circulating fragment of SOGA, providing a surrogate marker for intracellular SOGA levels. Circulating SOGA increased in parallel with adiponectin and insulin activity in both humans and mice. These results suggest that adiponectin-mediated increases in SOGA contribute to the inhibition of glucose production. PMID:20813965

  2. 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.

  3. Adiponectin protects the rats liver against chronic intermittent hypoxia induced injury through AMP-activated protein kinase pathway

    PubMed Central

    Ding, Wenxiao; Zhang, Qiang; Dong, Yanbin; Ding, Ning; Huang, Hanpeng; Zhu, Xianji; Hutchinson, Sean; Gao, Xingya; Zhang, Xilong

    2016-01-01

    This study was performed to assess the effect of chronic intermittent hypoxia (CIH) on the liver, the associated mechanisms and the potential therapeutic roles of adiponectin (Ad). Sixty rats were randomly assigned to four groups: the normal control (NC), NC and Ad supplement (NC + Ad), CIH, and CIH and Ad supplement (CIH + Ad) groups. The rats in the CIH and CIH + Ad groups were exposed to a hypoxic environment for 4 months. Rats in the NC + Ad and CIH + Ad groups were also treated with an intravenous injection of Ad (10 ug), twice a week. The plasma levels of hepatic enzymes, serum triglyceride, liver triglyceride, fasting blood glucose and hepatic cell apoptosis in hepatic tissue, were higher in the CIH group than in the NC and NC + Ad groups. However, the Ad supplementation in the CIH + Ad group rescued the hepatic tissue insult by activating the AMP-activated protein kinase (AMPK) pathway. In conclusion, Ad could protect against CIH-induced hepatic injury partly through the AMPK pathway. PMID:27678302

  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. Associations of erythrocyte membrane fatty acids with the concentrations of C-reactive protein, interleukin 1 receptor antagonist and adiponectin in 1373 men.

    PubMed

    Takkunen, M J; de Mello, V D F; Schwab, U S; Ågren, J J; Kuusisto, J; Uusitupa, M I J

    2014-10-01

    Dietary and endogenous fatty acids could play a role in low-grade inflammation. In this cross-sectional study the proportions of erythrocyte membrane fatty acids (EMFA) and the concentrations of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra) and adiponectin were measured and their confounder-adjusted associations examined in 1373 randomly selected Finnish men aged 45-70 years participating in the population based Metsim study in Eastern Finland. The sum of n-6 EMFAs, without linoleic acid (LA), was positively associated with concentrations of CRP and IL-1Ra (r partial=0.139 and r partial=0.115, P<0.001). These associations were especially strong among lean men (waist circumference <94 cm; r partial=0.156 and r partial=0.189, P<0.001). Total n-3 EMFAs correlated inversely with concentrations of CRP (r partial=-0.098, P<0.001). Palmitoleic acid (16:1n-7) correlated positively with CRP (r partial=0.096, P<0.001). Cis-vaccenic acid (18:1n-7) was associated with high concentrations of adiponectin (r partial=0.139, P<0.001). In conclusion, n-6 EMFAs, except for LA, correlated positively with the inflammatory markers. Palmitoleic acid was associated with CRP, whereas, interestingly, its elongation product, cis-vaccenic acid, associated with anti-inflammatory adiponectin.

  8. Degradation of cellular and viral Fos proteins.

    PubMed

    Acquaviva, C; Ferrara, P; Bossis, G; Brockly, F; Salvat, C; Jariel-Encontre, I; Piechaczyk, M

    2001-01-01

    c-Fos proto-oncoprotein is a short-lived transcription factor with oncogenic potential. We have shown that it is massively degraded by the proteasome in vivo under various experimental conditions. Other proteolytic systems including lysosomes and calpains, might, however, also marginally operate on it. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for its addressing to the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome- and ubiquitin-dependent in vivo. Interestingly, several lines of evidence indicate that the addressing of c-Fos and c-Jun to the proteasome is, at least in part, governed by different mechanisms. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of c-Fos main destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to an unidentified proteolytic system. This observation is consistent with the idea that Fos-expressing viruses have evolved to ensure control protein levels to avoid high protein accumulation-linked apoptosis. In conclusion, the unveiling of the complex mechanism network responsible for the degradation of AP-1 family members is still at its beginning and a number of issues regarding the regulation of this process and the addressing to the proteasome are still unresolved.

  9. Multiple degradation pathways for Fos family proteins.

    PubMed

    Acquaviva, Claire; Bossis, Guillaume; Ferrara, Patrizia; Brockly, Frederique; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2002-11-01

    c-Fos protooncoprotein is a short-lived transcription factor with oncogenic potential. It is massively degraded by the proteasome in vivo under various experimental conditions. Those include consititutive expression in exponentially growing cells and transient induction in cells undergoing the G0/G1 phase transition upon stimulation by serum. Though there is evidence that c-Fos can be ubiquitinylated in vitro, the unambigous demonstration that prior ubiquitinylation is necessary for degradation by the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome dependent. However, several lines of evidence indicate that the mechanisms by which it addresses the proteasome are different from those operating on c-Fos. Moreover, genetic analysis has indicated that c-Fos is addressed to the proteasome via pathways that differ depending on the conditions of expression. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses in mutated forms, which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of one of the main c-Fos destabilizers but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. However, although viral Fos proteins have acquired full resistance to proteasomal degradation, stabilization is limited because the mutations they have accumulated, during or after c-fos gene transduction, confer sensitivity to an unidentified proteolytic system(s). This observation is consistent with the idea that fos-expressing viruses have evolved expression machineries to ensure controlled protein levels in order to maintain an optimal balance between prooncogenic and proapoptotic activities of v-Fos proteins.

  10. Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells.

    PubMed

    Ogunwobi, Olorunseun Olatunji; Beales, Ian L P

    2006-05-15

    Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

  11. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    PubMed

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  12. 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

  13. Non-degradative Ubiquitination of Protein Kinases.

    PubMed

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

    2016-06-01

    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.

  14. Relationship between adiponectin and fertility in the female pig.

    PubMed

    Campos, Danila B; Albornoz, Marcelo; Papa, Paula C; Palin, Marie-France; Bordignon, Vilceu; Murphy, Bruce D

    2015-03-01

    Adiponectin isoforms may mediate different aspects of the pleiotropic function of the protein, including the reproductive process. We examined the pattern of circulating adiponectin and adiponectin system expression in fat and ovarian tissues of hyperfertile and subfertile sows. We demonstrated the presence of five different isoforms of adiponectin (90, 158, 180, 250 and >250kDa) in the circulation and identified a subgroup of subfertile females that displayed reduced abundance of all adiponectin isoforms as well as a lack of the 250-kDa adiponectin isoform in both serum and follicular fluid. Subfertility in these animals was associated with fewer large follicles and corpora lutea in the ovaries, as well as lower concentrations of 17β-oestradiol in the follicular fluid of large follicles. In addition, subfertile females showed higher adiponectin mRNA in fat tissue and altered mRNA and protein expression of adiponectin and its receptors in the ovary. Changes in the abundance and pattern of circulating adiponectin isoforms have been associated with reproductive disorders in animals and humans, including polycystic ovarian syndrome (PCOS). Our findings suggest that the adiponectin system may play an important role in controlling ovarian function and influencing porcine fertility.

  15. 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.

  16. 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

  17. Glycidol degrades scrapie mouse prion protein.

    PubMed

    Yamamoto, M; Horiuchi, M; Ishiguro, N; Shinagawa, M; Matsuo, T; Kaneko, K

    2001-09-01

    Agents of transmissible spongiform encephalopathy (prion) are known to be extremely resistant to physicochemical inactivation procedures such as heat, radiation, chemical disinfectants such as detergents, alcohols, glutaraldehyde, formalin, and so on. Because of its remarkable resistance, it is difficult to inactivate prion. Chemical inactivation seems to be a practical method because it is applicable to large or fixed surfaces and complicated equipment. Here, three epoxides: beta-propiolactone, propylene oxide, and glycidol (GLD) were examined of their inactivation ability against scrapie-mouse prion protein (PrP(Sc)) under various conditions of chemical concentration, incubation time, and temperature. Among these chemicals, GLD worked most effectively and degraded PrP into small fragments. As a result of the bioassay, treatment with 3% GLD for 5 hr and 5% GLD for 2, 5 hr or 12 hr at room temperature prolonged the mean incubation time by 44, 30, 110 and 73 days, respectively. From dose-incubation time standard curve, the decrease in infectivity titers were estimated as 10(3) or more. Therefore, degradation of PrP(Sc) by GLD decreased the scrapie infectivity. It is also suggested that pH and salt concentrations influence the effect of GLD. Although further study is necessary to determine the optimal condition, GLD may be a potential prion disinfectant.

  18. 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

  19. 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

  20. Specific dietary patterns and concentrations of adiponectin

    PubMed Central

    Izadi, Vajihe; Azadbakht, Leila

    2015-01-01

    Background: One of the adipokines mostly secreted from adipose tissue is adiponectin. Adiponectin is well known as the anti-diabetic, anti-obesity and cardio-protective factor. Present study focused on the review the previous studies about relationship between adherence to healthy dietary pattern, independent of one or two special dietary components, and concentration of adiponectin. Materials and Methods: We searched in PubMed search engine from 2003 to July 2014 using the following key words: Healthy dietary pattern, mediterranean dietary pattern, dietary pattern, diet intervention and adiponectin and adipokines. Then, we recruited 10 articles to review in the present study. Results: Cohort studies that are examined this relationship among women showed the strong positive association in this regard. According to cross-sectional studies adherence to healthy dietary pattern like Mediterranian intervention with moderate weight loss had a positive association with concentration of adiponectin. Conclusion: It seems that adherents to the healthy dietary patterns have great levels of circulating adiponectin. However, it is not clear that whether the separate components of healthy dietary patterns like good sources of fats or protein or fibers mostly have important roles in these beneficial effects of such dietary patterns or not. PMID:25983773

  1. Expression of adiponectin receptors in mouse adrenal glands and the adrenocortical Y-1 cell line: adiponectin regulates steroidogenesis.

    PubMed

    Li, Ping; Sun, Fei; Cao, Huang-Ming; Ma, Qin-Yun; Pan, Chun-Ming; Ma, Jun-Hua; Zhang, Xiao-Na; Jiang, He; Song, Huai-Dong; Chen, Ming-Dao

    2009-12-25

    Obesity is frequently associated with malfunctions of the hypothalamus-pituitary-adrenal (HPA) axis and hyperaldosteronism, but the mechanism underlying this association remains unclear. Since the adrenal glands are embedded in adipose tissue, direct cross-talk between adipose tissue and the adrenal gland has been proposed. A previous study found that adiponectin receptor mRNA was expressed in human adrenal glands and aldosterone-producing adenoma (APA). However, the expression of adiponectin receptors in adrenal glands has not been confirmed at the protein level or in other species. Furthermore, it is unclear whether adiponectin receptors expressed in adrenal cells are functional. We found, for the first time, that adiponectin receptor (AdipoR1 and AdipoR2) mRNA and protein were expressed in mouse adrenal and adrenocortical Y-1 cells. However, adiponectin itself was not expressed in mouse adrenal or Y-1 cells. Furthermore, adiponectin acutely reduced basal levels of corticosterone and aldosterone secretion. ACTH-induced steroid secretion was also inhibited by adiponectin, and this was accompanied by a parallel change in the expression of the key genes involved in steroidogenesis. These findings indicate that adiponectin may take part in the modulation of steroidogenesis. Thus, adiponectin is likely to have physiological and/or pathophysiological significance as an endocrine regulator of adrenocortical function.

  2. T-cadherin Is Essential for Adiponectin-mediated Revascularization*

    PubMed Central

    Parker-Duffen, Jennifer L.; Nakamura, Kazuto; Silver, Marcy; Kikuchi, Ryosuke; Tigges, Ulrich; Yoshida, Sumiko; Denzel, Martin S.; Ranscht, Barbara; Walsh, Kenneth

    2013-01-01

    Adipose tissue secretes protein factors that have systemic actions on cardiovascular tissues. Previous studies have shown that ablation of the adipocyte-secreted protein adiponectin leads to endothelial dysfunction, whereas its overexpression promotes wound healing. However, the receptor(s) mediating the protective effects of adiponectin on the vasculature is not known. Here we examined the role of membrane protein T-cadherin, which localizes adiponectin to the vascular endothelium, in the revascularization response to chronic ischemia. T-cadherin-deficient mice were analyzed in a model of hind limb ischemia where blood flow is surgically disrupted in one limb and recovery is monitored over 28 days by laser Doppler perfusion imaging. In this model, T-cadherin-deficient mice phenocopy adiponectin-deficient mice such that both strains display an impaired blood flow recovery compared with wild-type controls. Delivery of exogenous adiponectin rescued the impaired revascularization phenotype in adiponectin-deficient mice but not in T-cadherin-deficient mice. In cultured endothelial cells, T-cadherin deficiency by siRNA knockdown prevented the ability of adiponectin to promote cellular migration and proliferation. These data highlight a previously unrecognized role for T-cadherin in limb revascularization and show that it is essential for mediating the vascular actions of adiponectin. PMID:23824191

  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. 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

  5. SNIPER peptide-mediated degradation of endogenous proteins.

    PubMed

    Fan, Xuelai; Wang, Yu Tian

    2015-03-02

    Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method (SNIPER: Selective Native Protein Eradication) to degrade any given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway. This unit presents a typical strategy in the design and validation of a protein-knockdown peptide.

  6. 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

  7. 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

  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. Hepatitis B Virus Protein X Induces Degradation of Talin-1

    PubMed Central

    van de Klundert, Maarten A. A.; van den Biggelaar, Maartje; Kootstra, Neeltje A.; Zaaijer, Hans L.

    2016-01-01

    In the infected human hepatocyte, expression of the hepatitis B virus (HBV) accessory protein X (HBx) is essential to maintain viral replication in vivo. HBx critically interacts with the host damaged DNA binding protein 1 (DDB1) and the associated ubiquitin ligase machinery, suggesting that HBx functions by inducing the degradation of host proteins. To identify such host proteins, we systematically analyzed the HBx interactome. One HBx interacting protein, talin-1 (TLN1), was proteasomally degraded upon HBx expression. Further analysis showed that TLN1 levels indeed modulate HBV transcriptional activity in an HBx-dependent manner. This indicates that HBx-mediated TLN1 degradation is essential and sufficient to stimulate HBV replication. Our data show that TLN1 can act as a viral restriction factor that suppresses HBV replication, and suggest that the HBx relieves this restriction by inducing TLN1 degradation. PMID:27775586

  10. Adiponectin gene polymorphisms: Association with childhood obesity.

    PubMed

    Fraga, Vanêssa Gomes; Gomes, Karina Braga

    2014-03-01

    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. PMID:27625863

  11. 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. PMID:27625863

  12. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    PubMed Central

    Jackson, Matthew P.

    2016-01-01

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

  13. 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

  14. Protein oxidation and degradation caused by particulate matter

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  15. Protein oxidation and degradation caused by particulate matter

    PubMed Central

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

    2016-01-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM. PMID:27644844

  16. Protein oxidation and degradation caused by particulate matter.

    PubMed

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

    2016-01-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM. PMID:27644844

  17. 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

  18. Expression of adiponectin and adiponectin receptors 1 and 2 in the porcine uterus, conceptus, and trophoblast during early pregnancy.

    PubMed

    Smolinska, Nina; Maleszka, Anna; Dobrzyn, Kamil; Kiezun, Marta; Szeszko, Karol; Kaminski, Tadeusz

    2014-10-15

    Adiponectin, one of the several adipocytokines secreted mainly by the adipose tissue, plays an important role in regulating energy homeostasis and controls female fertility. Female reproductive functions are closely associated with nutritional status, and adiponectin seems to be an important factor linking the regulation of metabolic homeostasis with reproductive processes. The biological activity of adiponectin is mediated by two distinct receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). The objective of this study was to determine the presence of and changes in the gene and protein expression pattern of adiponectin and its receptors in the porcine uterus during early pregnancy and on Days 10 to 11 of the estrous cycle and in the conceptus and trophoblast. The highest level of adiponectin transcript was observed on Days 15 to 16 of gestation, Days 10 to 11 of the cycle in the endometrium, and Days 15 to 16 of gestation in the myometrium. The highest expression of AdipoR1 and AdipoR2 genes was detected on Days 10 to 11 of gestation in the endometrium, and Days 12 to 13 in the myometrium. The highest content of adiponectin protein was noted on Days 12 to 13 and 30 to 32 of gestation in the endometrium and Days 10 to 11 of the cycle in the myometrium. The expression of adiponectin protein was higher on Days 27 to 28 and 30 to 32 in the conceptuses. AdipoR1 protein content in the myometrium was highest on Days 12 to 13 and 30 to 32. In contrast, in the endometrium, it was more constant. The highest content of AdipoR2 protein was detected on Days 15 to 16 and 30 to 32 of gestation, Days 10 to 11 of the cycle in the endometrium, and Days 10 to 11 of gestation in the myometrium. In the conceptuses, the highest AdipoR1 protein content was observed on Days 15 to 16, and the highest AdipoR2 protein expression was determined on Days 15 to 16 and 27 to 28. In the trophoblasts, AdipoR1 protein content was higher on Days 27 to 28 than on Days 30

  19. 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.

  20. 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.

  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. 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

  3. Development of target protein-selective degradation inducer for protein knockdown.

    PubMed

    Itoh, Yukihiro; Ishikawa, Minoru; Kitaguchi, Risa; Sato, Shinichi; Naito, Mikihiko; Hashimoto, Yuichi

    2011-05-15

    Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER (Specific and Nongenetic Inhibitor-of-apoptosis-proteins (IAPs)-dependent Protein ERaser) degrades both the target proteins and IAPs. Here, we designed a small-molecular amide-type SNIPER to overcome this issue. As proof of concept, we synthesized and biologically evaluated an amide-type SNIPER which induces selective degradation of cellular retinoic acid binding protein II (CRABP-II), but not IAPs. Such small-molecular, amide-type SNIPERs that induce target protein-selective degradation without affecting IAPs should be effective tools to study the biological roles of target proteins in living cells.

  4. 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

  5. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway

    SciTech Connect

    Luo Xianghang; Guo Lijuan; Yuan Lingqing; Xie Hui; Zhou Houde; Wu Xianping; Liao Eryuan . E-mail: eyliao1207@21cn.com

    2005-09-10

    Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin.

  6. Acid-degradable polyurethane particles for protein-based vaccines

    PubMed Central

    Bachelder, Eric M.; Beaudette, Tristan T.; Broaders, Kyle E.; Paramonov, Sergey E.; Dashe, Jesse; Fréchet, Jean M. J.

    2009-01-01

    Acid-degradable particles containing a model protein antigen, ovalbumin, were prepared from a polyurethane with acetal moieties embedded throughout the polymer, and characterized by dynamic light scattering and transmission electron microscopy. The small molecule degradation by-product of the particles was synthesized and tested in vitro for toxicity indicating an LC50 of 12,500 μg/ml. A new liquid chromatography-mass spectrometry technique was developed to monitor the in vitro degradation of these particles. The degradation by-product inside RAW macrophages was at its highest level after 24 hours of culture and was efficiently exocytosed until it was no longer detectable after four days. When tested in vitro, these particles induced a substantial increase in the presentation of the immunodominant ovalbumin-derived peptide SIINFEKL in both macrophages and dendritic cells. In addition, vaccination with these particles generated a cytotoxic T-lymphocyte response that was superior to both free ovalbumin and particles made from an analogous but slower-degrading acid-labile polyurethane polymer. Overall, we present a fully degradable polymer system with non-toxic by-products, which may find use in various biomedical applications including protein-based vaccines. PMID:18710254

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Controlling Protein Activity and Degradation Using Blue Light.

    PubMed

    Lutz, Anne P; Renicke, Christian; Taxis, Christof

    2016-01-01

    Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light). PMID:26965116

  12. 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

  13. Abundance of adiponectin system and G-protein coupled receptor GPR109A mRNA in adipose tissue and liver of F2 offspring cows of Charolais × German Holstein crosses that differ in body fat accumulation.

    PubMed

    Mielenz, M; Kuhla, B; Hammon, H M

    2013-01-01

    In addition to its role in energy storage, adipose tissue (AT) is an important endocrine organ and it secretes adipokines. The adipokine adiponectin improves insulin sensitivity by activation of its receptors AdipoR1 and AdipoR2. Lipolysis in AT is downregulated by the G-protein coupled receptor (GPR109A), which binds the endogenous ligand β-hydroxybutyrate (BHBA). Insulin sensitivity is reduced during the transition from late pregnancy to early lactation in dairy cattle and BHBA is increased postpartum, implying the involvement of the adiponectin system and GPR109A in this process. The aim of the current investigation was to study the effect of the genetic background of cows on the mRNA abundance of the adiponectin system, as well as GPR109A, in an F(2) population of 2 Charolais × German Holstein families. These families were deduced from full- and half-sibs sharing identical but reciprocal paternal and maternal Charolais grandfathers. The animals of the 2 families showed significant differences in fat accretion and milk secretion and were designated fat-type (high fat accretion but low milk production) and lean-type (low fat accretion but high milk production). The mRNA of the adiponectin system and GPR109A were quantified by real-time PCR in different fat depots (subcutaneous from back, mesenteric, kidney) and liver. The mRNA data were correlated with AT masses (intermuscular topside border fat, kidney, mesenteric, omental, total inner fat mass, total subcutaneous fat mass, and total fat mass) and blood parameters (glucose, nonesterified fatty acids, BHBA, urea, insulin, and glucagon). The abundance of adiponectin system mRNA was higher in discrete AT depots of fat-type cows [adiponectin mRNA in mesenteric fat (trend), AdipoR1 in kidney and mesenteric AT, and AdipoR2 in subcutaneous fat (trend)] than in lean-type cows. More GPR109A mRNA was found in kidney fat of the lean-type family than in that of the fat-type family. In liver, the abundance of AdipoR2 and

  14. 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

  15. 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

  16. ADAMTS-12 Associates with and Degrades Cartilage Oligomeric Matrix Protein*

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Xu, Ke; Luan, Yi; Ilalov, Kiril; Sehgal, Bantoo; Yu, Shuang; Howell, Ronald D.; Cesare, Paul E. Di

    2006-01-01

    Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis. PMID:16611630

  17. The complex role of adiponectin in chronic kidney disease.

    PubMed

    Jia, Ting; Carrero, Juan Jesús; Lindholm, Bengt; Stenvinkel, Peter

    2012-10-01

    Although adiponectin, an adipocytokine released from adipose tissue, is thought to have anti-atherogenic, anti-inflammatory, and insulin-sensitizing effects, it appears that high, rather than low, circulating levels of adiponectin predict increased mortality in chronic kidney disease (CKD) patients in whom the circulating levels may rise to about three times higher than the levels in healthy subjects. As it could be hypothesized that in the uremic milieu high adiponectin levels reflect protein-energy wasting, lower residual renal function and/or volume overload, this may explain, at least in part, the observed paradoxical link between hyperadiponectinemia and poor outcome in CKD. To determine the biological consequences of high circulating adiponectin levels on carbohydrate and insulin metabolism as well as relations with cardiovascular function and mortality in the uremic milieu, further studies need to take into account both high-, and low-molecular weight adiponectin moieties as well as the role of adiponectin receptors. This brief review summarizes some of the recent advances in our understanding of the complex and context-sensitive role of this elusive and intriguing adipokine in the uremic milieu.

  18. 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.

  19. Expression of adiponectin and adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine uterus during the oestrous cycle.

    PubMed

    Smolinska, Nina; Dobrzyn, Kamil; Maleszka, Anna; Kiezun, Marta; Szeszko, Karol; Kaminski, Tadeusz

    2014-04-01

    Adiponectin is a hormone secreted primarily by white adipose tissue. Recent studies have shown that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in different reproductive tissues, including the ovary and uterus. This newly discovered endocrine system plays an important role in the regulation of reproductive processes. The expression of the adiponectin system in the porcine uterus during the oestrous cycle has not been researched to date. The aim of the present study was to investigate the presence and changes in adiponectin system expression in the porcine uterus on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. The expression of the adiponectin gene was highest on days 14-16 and 2-3 in the endometrium and myometrium, respectively. In the endometrium, the content of AdipoR1 and AdipoR2 mRNAs was highest on days 10-12, whereas significantly higher expression levels of both genes were noted in the myometrium on days 17-19. The highest content of adiponectin and AdipoR1 protein in the endometrium was reported on days 2-3. In the myometrium, the expression levels of both receptor proteins were significantly higher on days 17-19. Adiponectin system proteins were localized in endometrial epithelial glandular cells, luminal epithelial cells and stromal cells as well as in longitudinal and circular muscles of the myometrium. This study demonstrated the presence of adiponectin, AdipoR1 and AdipoR2 genes and proteins in the porcine uterus and the effect of the stage of the oestrous cycle on the expression of the adiponectin system. Our results suggest that locally synthesized adiponectin directly affects uterine functions.

  20. 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

  1. Neuronal nitric oxide synthase-dependent elevation in adiponectin in the rostral ventrolateral medulla underlies g protein-coupled receptor 18-mediated hypotension in conscious rats.

    PubMed

    Penumarti, Anusha; Abdel-Rahman, Abdel A

    2014-10-01

    Direct activation of the endocannabinoid receptor G protein-coupled receptor 18 (GPR18) in the rostral ventrolateral medulla (RVLM) of conscious rats by abnormal cannabidiol (Abn CBD; trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) elevates local nitric oxide (NO) and adiponectin (ADN) levels and reduces oxidative stress and blood pressure (BP). However, the molecular mechanisms for GPR18-mediated neurochemical responses, including the nitric oxide synthase isoform that generates NO, and their potential causal link to the BP reduction are not known. We hypothesized that GPR18-mediated enhancement of Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and neuronal nitric oxide synthase (nNOS) phosphorylation, triggered by a reduction in cAMP, accounts for the NO/ADN-dependent reductions in RVLM oxidative stress and BP. Intra-RVLM GPR18 activation (Abn CBD; 0.4 μg) enhanced RVLM Akt, ERK1/2, and nNOS phosphorylation as well as ADN levels during the hypotensive response. Prior GPR18 blockade with O-1918 (1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]benzene) produced the opposite effects and abrogated Abn CBD-evoked neurochemical and BP responses. Pharmacological inhibition of RVLM phosphoinositide 3-kinase (PI3K)/Akt (wortmannin), ERK1/2 (PD98059 [2-​(2-​amino-​3-​methoxyphenyl)-​4H-​1-​benzopyran-​4-​one]), or nNOS (N(ω)-propyl-l-arginine), or activation of adenylyl cyclase (forskolin) virtually abolished intra-RVLM Abn CBD-evoked hypotension and the increases in Akt, ERK1/2, and nNOS phosphorylation and in ADN levels in the RVLM. Our pharmacological and neurochemical findings support a pivotal role for PI3K, Akt, ERK1/2, nNOS, and adenylyl cyclase, via modulation of NO, ADN, and cAMP levels, in GPR18 regulation of the RVLM redox state and BP in conscious rats.

  2. 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

  3. 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}.

  4. 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

  5. Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

    PubMed Central

    Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

    2012-01-01

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

  6. Adiponectin deficiency promotes tumor growth in mice by reducing macrophage infiltration.

    PubMed

    Sun, Yutong; Lodish, Harvey F

    2010-08-05

    Adiponectin is an adipocyte-derived plasma protein that has been implicated in regulating angiogenesis, but the role of adiponectin in regulating this process is still controversial. In this study, in order to determine whether adiponectin affects tumor growth and tumor induced vascularization, we implanted B16F10 melanoma and Lewis Lung Carcinoma cells subcutaneously into adiponectin knockout and wild-type control mice, and found that adiponectin deficiency markedly promoted the growth of both tumors. Immunohistochemical analyses indicated that adiponectin deficiency reduced macrophage recruitment to the tumor, but did not affect cancer cell mitosis, apoptosis, or tumor-associated angiogenesis. In addition, treatment with recombinant adiponectin did not affect the proliferation of cultured B16F10 tumor cells. Importantly, the restoration of microphage infiltration at an early stage of tumorigenesis by means of co-injection of B16F10 cells and macrophages reversed the increased tumor growth in adiponectin knockout mice. Thus, we conclude that the enhanced tumor growth observed in adiponectin deficient mice is likely due to the reduction of macrophage infiltration rather than enhanced angiogenesis.

  7. 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

  8. The ends and means of artificially induced targeted protein degradation.

    PubMed

    Prabha, C Ratna; Mukherjee, Soumya; Raman, Renuka; Kulkarni, Swapnali

    2012-12-01

    Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology.

  9. Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation*

    PubMed Central

    Huang, Min; Whang, Patrick; Chodaparambil, Jayanth V.; Pollyea, Daniel A.; Kusler, Brenda; Xu, Liwen; Felsher, Dean W.; Mitchell, Beverly S.

    2011-01-01

    Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H2O2) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H2O2 impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-l-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation. PMID:21242306

  10. 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.

  11. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-01

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  12. 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

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

    PubMed

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

    2015-11-01

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

  14. 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

  15. 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.

  16. 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).

  17. Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury

    PubMed Central

    Liu, Xu-Hui; Yang, Yue-Wu; Dai, Hai-Tao; Cai, Song-Wang; Chen, Rui-Han; Ye, Zhi-Qiang

    2015-01-01

    AIM: To determine the potential protective role of adiponectin in intestinal ischemia reperfusion (I/R) injury. METHODS: A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzyme-linked immunosorbent assay (ELISA). The serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The production of malondialdehyde (MDA) and superoxide dismutase (SOD) and villous injury scores were also measured. RESULTS: Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase (AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1 (HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. CONCLUSION: Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway. PMID:26715807

  18. 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

  19. Higher Levels of Adiponectin in Vascular Endothelial Cells are Associated with Greater Brachial Artery Flow-mediated Dilation in Older Adults

    PubMed Central

    Yoo, Jeung-Ki; Hwang, Moon-Hyon; Luttrell, Meredith J.; Kim, Han-Kyul; Meade, Thomas H.; English, Mark; Segal, Mark S.; Christou, Demetra D.

    2015-01-01

    Adiponectin, an adipocyte-derived protein, exerts anti-atherosclerotic effects on the vascular endothelium. Recently adiponectin protein has been reported in murine vascular endothelial cells, however, whether adiponectin is present in human vascular endothelial cells remains unexplored. We sought to examine 1) adiponectin protein in vascular endothelial cells collected from older adults free of overt cardiovascular disease; 2) the relation between endothelial cell adiponectin and in vivo vascular endothelial function; and 3) the relation between endothelial cell adiponectin, circulating (plasma) adiponectin and related factors. We measured vascular endothelial function (brachial artery flow-mediated dilation using ultrasonography), vascular endothelial cell adiponectin (biopsy coupled with quantitative immunofluorescence) and circulating adiponectin (Mercodia, ELISA) in older, sedentary, non-smoking, men and women (55 – 79 years). We found that higher endothelial cell adiponectin was related with greater flow-mediated dilation (r=0.43, P<0.05) and greater flow-mediated dilation normalized for shear stress (r=0.56, P<0.01), but was not related with vascular smooth muscle responsiveness to nitric oxide (r=0.04, P=0.9). Vascular endothelial cell adiponectin was not related with circulating adiponectin (r=−0.14, P=0.6). Endothelial cell and circulating adiponectin were differentially associated with adiposity, metabolic and other factors, but both were inversely associated with renal function (r=0.44 to 0.62, P ≤ 0.04). In conclusion, higher endothelial cell adiponectin levels are associated with higher vascular endothelial function, independent of circulating adiponectin levels in older adults. PMID:25572013

  20. 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

  1. Long term intake of 0.1% ethanol decreases serum adiponectin by suppressing PPARγ expression via p38 MAPK pathway.

    PubMed

    Tian, Chong; Jin, Xin; Ye, Xiaolei; Wu, Hongmei; Ren, Weiye; Zhang, Rui; Long, Jia; Ying, Chenjiang

    2014-03-01

    Light alcohol consumption was reported to be negatively associated with insulin resistance and risk of cardiovascular diseases; however, the results were inconsistent. We here investigate whether long term intake of low-concentration ethanol can affect adiponectin levels. Male Wistar rats were exposed to 0.1% ethanol in drinking water for 26weeks. Visceral adipose tissue (VAT) was cultured and treated with ethanol, SB203580, GW9662, or rosiglitazone. Adiponectin in serum and culture supernatant were measured by ELISA, mRNA levels of adiponectin and PPARγ were determined by RT-PCR, and protein expressions of PPARγ, p38 MAPK and phospho-p38 MAPK were determined by Western blot. In vivo, ethanol decreased the mRNA of adiponectin in VAT and serum adiponectin significantly. Decreased PPARγ and increased activation of p38 MAPK were observed in ethanol treated group. In vitro, SB203580 increased the adiponectin and PPARγ levels in normal DMEM cultured VAT and ameliorated ethanol-induced decrease of adiponectin and PPARγ expressions. GW9662 also decreased the adiponectin levels; Both ethanol and GW9662 weakened the rosiglitazone-induced elevation of adiponectin levels in cultured VAT. These data suggest that long term intake of 0.1% ethanol down-regulated adiponectin levels, and the regulation of PPARγ via p38 MAPK pathway plays an important role in the mechanism underneath.

  2. 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.

  3. Adiponectin, leptin, and yoga practice.

    PubMed

    Kiecolt-Glaser, Janice K; Christian, Lisa M; Andridge, Rebecca; Hwang, Beom Seuk; Malarkey, William B; Belury, Martha A; Emery, Charles F; Glaser, Ronald

    2012-12-01

    To address the mechanisms underlying hatha yoga's potential stress-reduction benefits, we compared adiponectin and leptin data from well-matched novice and expert yoga practitioners. These adipocytokines have counter-regulatory functions in inflammation; leptin plays a proinflammatory role, while adiponectin has anti-inflammatory properties. Fifty healthy women (mean age=41.32, range=30-65), 25 novices and 25 experts, provided fasting blood samples during three separate visits. Leptin was 36% higher among novices compared to experts, P=.008. Analysis of adiponectin revealed a borderline effect of yoga expertise, P=.08; experts' average adiponectin levels were 28% higher than novices across the three visits. In contrast, experts' average adiponectin to leptin ratio was nearly twice that of novices, P=.009. Frequency of self-reported yoga practice showed significant negative relationships with leptin; more weeks of yoga practice over the last year, more lifetime yoga sessions, and more years of yoga practice were all significantly associated with lower leptin, with similar findings for the adiponectin to leptin ratio. Novices and experts did not show even marginal differences on behavioral and physiological dimensions that might represent potential confounds, including BMI, central adiposity, cardiorespiratory fitness, and diet. Prospective studies addressing increased risk for type II diabetes, hypertension, and cardiovascular disease have highlighted the importance of these adipocytokines in modulating inflammation. Although these health risks are clearly related to more extreme values then we found in our healthy sample, our data raise the possibility that longer-term and/or more intensive yoga practice could have beneficial health consequences by altering leptin and adiponectin production. PMID:22306535

  4. Neurogenesis-independent antidepressant-like effects of enriched environment is dependent on adiponectin.

    PubMed

    Nicolas, Sarah; Veyssière, Julie; Gandin, Carine; Zsürger, Nicole; Pietri, Mariel; Heurteaux, Catherine; Glaichenhaus, Nicolas; Petit-Paitel, Agnès; Chabry, Joëlle

    2015-07-01

    Environmental enrichment (EE) that combines voluntary physical exercise, sensory and social stimuli, causes profound changes in rodent brain at molecular, anatomical and behavioral levels. Here, we show that EE efficiently reduces anxiety and depression-like behaviors in a mouse model of depression induced by long-term administration of corticosterone. Mechanisms underlying EE-related beneficial effects remain largely unexplored; however, our results point toward adiponectin, an adipocyte-secreted protein, as a main contributor. Indeed, adiponectin-deficient (adipo(-/-)) mice did not benefit from all the EE-induced anxiolytic and antidepressant-like effects as evidenced by their differential responses in a series of behavioral tests. Conversely, a single intravenous injection of exogenous adiponectin restored the sensitivity of adipo(-/-) mice to EE-induced behavioral benefits. Interestingly, adiponectin depletion did not prevent the hippocampal neurogenesis induced by EE. Therefore, antidepressant properties of adiponectin are likely to be related to changes in signaling in the hypothalamus rather than through hippocampal-neurogenesis mechanisms. Additionally, EE did not modify the plasma levels of adiponectin but may favor the passage of adiponectin from the blood to the cerebrospinal fluid. Our findings provide advances in the understanding of the anxiolytic and antidepressant-like effects of EE and highlight adiponectin as a pivotal mediator.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Degradation of Proteins Artificially Introduced into Vacuoles of Chara australis1

    PubMed Central

    Moriyasu, Yuji; Tazawa, Masashi

    1988-01-01

    When an exogenous protein, bovine serum albumin, was introduced into the vacuole of a Chara australis internodal cell, it was degraded with time. This degradation proceeded only in the vacuole as far as could be observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Degradation was inhibited by protease inhibitors such as antipain and leupeptin. Endogenous proteins introduced into the vacuole were also degraded there. Furthermore, intravacuolar cytoplasmic drops, which were often formed by cell ligation, seemed to be degraded in the vacuole. However, bovine serum albumin degradation did not proceed when mixed with isolated vacuolar sap. These results show that the vacuole in the Chara internodal cell has the capacity to degrade cellular proteins, but that cytoplasmic support is needed for this degrading activity to be maintained. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:16666427

  10. 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.

  11. 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

  12. Arabidopsis DELLA protein degradation is controlled by a type-one protein phosphatase, TOPP4.

    PubMed

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

    2014-07-01

    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.

  13. 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

  14. 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

  15. 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...

  16. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome

    PubMed Central

    Kadowaki, Takashi; Yamauchi, Toshimasa; Kubota, Naoto; Hara, Kazuo; Ueki, Kohjiro; Tobe, Kazuyuki

    2006-01-01

    Adiponectin is an adipokine that is specifically and abundantly expressed in adipose tissue and directly sensitizes the body to insulin. Hypoadiponectinemia, caused by interactions of genetic factors such as SNPs in the Adiponectin gene and environmental factors causing obesity, appears to play an important causal role in insulin resistance, type 2 diabetes, and the metabolic syndrome, which are linked to obesity. The adiponectin receptors, AdipoR1 and AdipoR2, which mediate the antidiabetic metabolic actions of adiponectin, have been cloned and are downregulated in obesity-linked insulin resistance. Upregulation of adiponectin is a partial cause of the insulin-sensitizing and antidiabetic actions of thiazolidinediones. Therefore, adiponectin and adiponectin receptors represent potential versatile therapeutic targets to combat obesity-linked diseases characterized by insulin resistance. This Review describes the pathophysiology of adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. PMID:16823476

  17. 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

  18. [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.

  19. 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

  20. Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

    PubMed

    Yu, Geng; Rosenberg, Julian N; Betenbaugh, Michael J; Oyler, George A

    2015-12-01

    Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring.

  1. 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

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

    PubMed

    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

  3. 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

  4. 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.

  5. Adiponectin and thiazolidinedione targets CRTC2 to regulate hepatic gluconeogenesis.

    PubMed

    Yoon, Young Sil; Ryu, Dongryeol; Lee, Min Woo; Hong, Sungpyo; Koo, Seung Hoi

    2009-08-31

    During fasting periods, hepatic glucose production is enhanced by glucagon to provide fuels for other organs. This process is mediated via cAMP-dependent induction of the CREB regulated transcriptional coactivator (CRTC) 2, a critical transcriptional activator for hepatic gluconeogenesis. We have previously shown that CRTC2 activity is regulated by AMP activated protein kinase (AMPK) family members. Here we show that adiponectin and thiazolidinedione directly regulate AMPK to modulate CRTC2 activity in hepatocytes. Adiponectin or thiazolidinedione lowered glucose production from primary hepatocytes. Treatment of both reagents reduced gluconeogenic gene expression as well as cAMP-mediated induction of CRE reporter, suggesting that these reagents directly affect CREB/CRTC2- dependent transcription. Furthermore, adiponectin or thiazolidinedione mediated repression of CRE activity is largely blunted by co-expression of phosphorylation defective mutant CRTC2, underscoring the importance of serine 171 residue of this factor. Taken together, we propose that adiponectin and thiazolidinedione promote the modulation of AMPK-dependent CRTC2 activity to influence hepatic gluconeogenesis.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation.

    PubMed

    Winter, Georg E; Buckley, Dennis L; Paulk, Joshiawa; Roberts, Justin M; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E

    2015-06-19

    The development of effective pharmacological inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-molecule antagonists disrupt the activity of only one domain in the target protein. We devised a chemical strategy that promotes ligand-dependent target protein degradation using as an example the transcriptional coactivator BRD4, a protein critical for cancer cell growth and survival. We appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compound, dBET1, induced highly selective cereblon-dependent BET protein degradation in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degradation of the cytosolic protein FKBP12. This chemical strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.

  13. 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.

  14. 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

  15. 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.

  16. Adiponectin and noncardiovascular death: a nested case-control study.

    PubMed

    Matsumoto, Masatoshi; Ishikawa, Shizukiyo; Kajii, Eiji

    2008-06-01

    This study is to evaluate the associations between adiponectin level and noncardiovascular death and to test a hypothesis that adiponectin level reflects the degree of systemic wasting that precedes death. A nested case-control study was conducted involving 5243 subjects, drawn from 12490 subjects of the Jichi Medical School Cohort Study, whose blood samples had been drawn between 1992 and 1995. Over an average of 10.8 years of follow-up, 103 cases with noncardiovascular death and 565 controls without history/event/death of any cardiovascular disease were identified. Odds ratios (ORs) were estimated relative to the lowest quintile of adiponectin level. The risks for noncardiovascular death of the second lowest quintile and the highest quintile of adiponectin level were significantly higher than that of the lowest quintile when adjusted for age and sex (model 1) (OR, 2.38 [95% confidence interval (CI), 1.12-5.06] and 2.16 [1.01-4.80]). All the statistical significances disappeared when adjusted further for body mass index and C-reactive protein level (model 2). When excluding cases with cancer death, the odds for death in the highest 2 quintiles were significantly higher than those in the lowest quintile in model 1 (OR, 2.80 [95% CI, 1.04-7.59] and 3.74 [1.38-10.18]). The significant difference between the highest vs the lowest quintile remained significant in model 2 and even after adjusting further for smoking, diabetes, and total cholesterol level (model 3) (OR, 3.28 [95% CI, 1.02-10.51] and 3.98 [1.21-13.13]). Adiponectin levels had linear associations with the risks of noncardiovascular noncancer death in models 1, 2, and 3 (OR per 1 SD increase in log-adiponectin, 1.72 [95% CI, 1.23-2.40], 1.89 [1.23-2.91], and 2.01 [1.29-3.15]). Adiponectin is an independent indicator of noncardiovascular mortality that may relate with systemic wasting.

  17. 5-Hydroxytryptamine 2A receptor signaling cascade modulates adiponectin and plasminogen activator inhibitor 1 expression in adipose tissue.

    PubMed

    Uchida-Kitajima, Shoko; Yamauchi, Toshimasa; Takashina, Youko; Okada-Iwabu, Miki; Iwabu, Masato; Ueki, Kohjiro; Kadowaki, Takashi

    2008-09-01

    Knowledge of the regulatory factors associated with down-regulation of adiponectin gene expression and up-regulation of PAI-1 gene expression is crucial to understand the pathophysiological basis of obesity and metabolic diseases, and could establish new treatment strategies for these conditions. We showed that expression of 5-HT(2A) receptors was up-regulated in hypertrophic 3T3-L1 adipocytes, which exhibited decreased expression of adiponectin and increased expression of PAI-1. 5-HT(2A) receptor antagonists and suppression of 5-HT(2A) receptor gene expression enhanced adiponectin expression. Activation of Gq negatively regulated adiponectin expression, and inhibition of mitogen-activated protein kinase reversed the Gq-induced effect. Moreover, the 5-HT(2A) receptor blockade reduced PAI-1 expression. These findings indicate that antagonism of 5-HT(2A) receptors in adipocytes could improve the obesity-linked decreases in adiponectin expression and increases in PAI-1 expression.

  18. Maternal diet and cord blood leptin and adiponectin concentrations at birth

    PubMed Central

    Mantzoros, Christos S.; Sweeney, Laura; Williams, Catherine J.; Oken, Emily; Kelesidis, Theodoros; Rifas-Shiman, Sheryl L.; Gillman, Matthew W.

    2010-01-01

    Background & Aims The purpose of this study was to determine the effects of total energy intake, macronutrient intake, and maternal adherence to Mediterranean diet or Alternative Healthy Eating Index (AHEI) on cord blood leptin and adiponectin levels, which have been associated with childhood adiposity. Methods We used multivariable linear regression to assess associations of maternal diet, averaged over 1st and 2nd trimesters, with cord blood adipokines of 780 women from the prospective cohort study Project Viva. Results Mean (SD) energy intake during pregnancy was 2135 (596) kcal. Mean (SD) cord blood levels of leptin and adiponectin were 9.0 (6.6) ng/ml and 28.6 (6.7) μg/ml, respectively. Neither closer adherence to a Mediterranean/AHEI pattern diet nor energy intake was associated with either cord blood leptin or adiponectin. Protein intake was associated with both marginally lower leptin (−0.22 ng/ml [95% CI −0.41, −0.02] for each 1% of energy) and adiponectin (−0.25 μg/ml [95% CI −0.48, −0.02]). Conclusions Closer adherence to a Mediterranean/AHEI pattern diet during pregnancy was not associated with cord blood leptin or adiponectin. Maternal protein intake was weakly but significantly associated with lower cord blood leptin and adiponectin. PMID:20363059

  19. Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response

    PubMed Central

    van Stijn, Caroline M. W.; Kim, Jason; Lusis, Aldons J.; Barish, Grant D.; Tangirala, Rajendra K.

    2015-01-01

    Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40–60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 µg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-α, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by up-regulating liver X receptor-α (LXRα) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.—van Stijn, C. M. W., Kim, J., Lusis, A. J., Barish, G.D., Tangirala, R. K. Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response. PMID:25392268

  20. Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

    PubMed

    Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

    2015-03-15

    Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Balance Between Folding and Degradation for Hsp90-Dependent Client Proteins: A Key Role for CHIP

    PubMed Central

    Kundrat, Lenka; Regan, Lynne

    2011-01-01

    Cells must regulate the synthesis and degradation of their proteins to maintain a balance that is appropriate for their specific growth conditions. Here we present the results of an investigation of the balance between protein folding and degradation for mammalian chaperone Hsp90-dependent client proteins. The central players are the molecular chaperones Hsp70 and Hsp90, the co-chaperone HOP, and ubiquitin ligase, CHIP. Hsp70 and Hsp90 bind to HOP thus forming a ternary folding complex whereas the binding of CHIP to the chaperones has previously been shown to lead to ubiquitination and ultimately to degradation of the client proteins as well as the chaperones. To understand the folding/degradation balance in more detail, we characterized the stoichiometries of the CHIP-Hsp70 and CHIP-Hsp90 complexes and measured the corresponding dissociation constants to be ~ 1 µM and ~ 4.5 µM respectively. We quantified the rate of ubiquitination of various substrates by CHIP in vitro. We further determined that the folding and degradation machineries cannot coexist in one complex. Lastly, we measured the in vivo concentrations of Hsp70, Hsp90, HOP, and CHIP under normal conditions and when client proteins are being degraded due to inhibition of the folding pathway. These in vivo measurements along with the in vitro data allowed us to calculate the approximate cellular concentrations of the folding and degradation complexes under both conditions and formulate a quantitative model for the balance between protein folding and degradation as well as an explanation for the shift to client protein degradation when the folding pathway is inhibited. PMID:20704274

  6. 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

  7. 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.

  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. Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles.

    PubMed

    Ma, Zhifang; Bai, Jing; Jiang, Xiue

    2015-08-19

    Established nanobio interactions face the challenge that the formation of nanoparticle-protein corona complexes shields the inherent properties of the nanoparticles and alters the manner of the interactions between nanoparticles and biological systems. Therefore, many studies have focused on protein corona-mediated nanoparticle binding, internalization, and intracellular transportation. However, there are a few studies to pay attention to if the corona encounters degradation after internalization and how the degradation of the protein corona affects cytotoxicity. To fill this gap, we prepared three types of off/on complexes based on gold nanoparticles (Au NPs) and dye-labeled serum proteins and studied the extracellular and intracellular proteolytic processes of protein coronas as well as their accompanying effects on cytotoxicity through multiple evaluation mechanisms, including cell viability, adenosine triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). The proteolytic process was confirmed by recovery of the fluorescence of the dye-labeled protein molecules that was initially quenched by Au NPs. Our results indicate that the degradation rate of protein corona is dependent on the type of the protein based on systematical evaluation of the extracellular and intracellular degradation processes of the protein coronas formed by human serum albumin (HSA), γ-globulin (HGG), and serum fibrinogen (HSF). Degradation is the fastest for HSA corona and the slowest for HSF corona. Notably, we also find that the Au NP-HSA corona complex induces lower cell viability, slower ATP production, lower MMP, and higher ROS levels. The cytotoxicity of the nanoparticle-protein corona complex may be associated with the protein corona degradation process. All of these results will enrich the database of cytotoxicity induced by nanomaterial-protein corona complexes.

  10. Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles.

    PubMed

    Ma, Zhifang; Bai, Jing; Jiang, Xiue

    2015-08-19

    Established nanobio interactions face the challenge that the formation of nanoparticle-protein corona complexes shields the inherent properties of the nanoparticles and alters the manner of the interactions between nanoparticles and biological systems. Therefore, many studies have focused on protein corona-mediated nanoparticle binding, internalization, and intracellular transportation. However, there are a few studies to pay attention to if the corona encounters degradation after internalization and how the degradation of the protein corona affects cytotoxicity. To fill this gap, we prepared three types of off/on complexes based on gold nanoparticles (Au NPs) and dye-labeled serum proteins and studied the extracellular and intracellular proteolytic processes of protein coronas as well as their accompanying effects on cytotoxicity through multiple evaluation mechanisms, including cell viability, adenosine triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). The proteolytic process was confirmed by recovery of the fluorescence of the dye-labeled protein molecules that was initially quenched by Au NPs. Our results indicate that the degradation rate of protein corona is dependent on the type of the protein based on systematical evaluation of the extracellular and intracellular degradation processes of the protein coronas formed by human serum albumin (HSA), γ-globulin (HGG), and serum fibrinogen (HSF). Degradation is the fastest for HSA corona and the slowest for HSF corona. Notably, we also find that the Au NP-HSA corona complex induces lower cell viability, slower ATP production, lower MMP, and higher ROS levels. The cytotoxicity of the nanoparticle-protein corona complex may be associated with the protein corona degradation process. All of these results will enrich the database of cytotoxicity induced by nanomaterial-protein corona complexes. PMID:26200209

  11. A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

    PubMed Central

    Wu, Peng; Lu, Ming-xing; Cui, Xiao-tian; Yang, He-qing; Yu, Shen-liang; Zhu, Jian-bin; Sun, Xiao-li; Lu, Boxun

    2016-01-01

    Aim: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies. Methods: The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads. Results: We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers. Conclusion: We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies. PMID:27264314

  12. 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).

  13. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame. PMID:24418875

  14. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame.

  15. 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.

  16. Transcriptomics profiling study of breast cancer from Kingdom of Saudi Arabia revealed altered expression of Adiponectin and Fatty Acid Binding Protein4: Is lipid metabolism associated with breast cancer?

    PubMed Central

    2015-01-01

    Background Breast cancer incidence rates are increasing at an alarming rate among Saudi Arabian females. Most molecular genetic discoveries on breast cancer and other cancers have arisen from studies examining European and American patients. However, possibility of specific changes in molecular signature among cancer patients of diverse ethnic groups remains largely unexplored. We performed transcriptomic profiling of surgically-resected breast tumors from 45 patients based in the Western region of Saudi Arabia using Affymetrix Gene 1.0 ST chip. Pathway and biological function-based clustering was apparent across the tissue samples. Results Pathway analysis revealed canonical pathways that had not been previously implicated in breast cancer. Biological network analysis of differentially regulated genes revealed that Fatty acid binding protein 4, adipocyte (FABP4), adiponectin (ADIPOQ), and retinol binding protein 4 (RBP4) were most down regulated genes, sharing strong connection with the other molecules of lipid metabolism pathway. The marked biological difference in the signatures uncovered between the USA and Saudi samples underpins the importance of this study. Connectivity Map identified compounds that could reverse an observed gene expression signature Conclusions This study describes, to our knowledge, the first genome-wide profiling of breast cancer from Saudi ethnic females. We demonstrate the involvement of the lipid metabolism pathway in the pathogenesis of breast cancer from this region. This finding also highlights the need for strategies to curb the increasing rates of incidence of this disease by educating the public about life-style risk factors such as unhealthy diet and obesity. PMID:25923423

  17. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation

    PubMed Central

    Seo, Ji Hae; Park, Ji-Hyeon; Lee, Eun Ji; Vo, Tam Thuy Lu; Choi, Hoon; Kim, Jun Yong; Jang, Jae Kyung; Wee, Hee-Jun; Lee, Hye Shin; Jang, Se Hwan; Park, Zee Yong; Jeong, Jaeho; Lee, Kong-Joo; Seok, Seung-Hyeon; Park, Jin Young; Lee, Bong Jin; Lee, Mi-Ni; Oh, Goo Taeg; Kim, Kyu-Won

    2016-01-01

    Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress. PMID:27708256

  18. Population-specific coding variant underlies genome-wide association with adiponectin level

    PubMed Central

    Croteau-Chonka, Damien C.; Wu, Ying; Li, Yun; Fogarty, Marie P.; Lange, Leslie A.; Kuzawa, Christopher W.; McDade, Thomas W.; Borja, Judith B.; Luo, Jingchun; AbdelBaky, Omar; Combs, Terry P.; Adair, Linda S.; Lange, Ethan M.; Mohlke, Karen L.

    2012-01-01

    Adiponectin is a protein hormone that can affect major metabolic processes including glucose regulation and fat metabolism. Our previous genome-wide association (GWA) study of circulating plasma adiponectin levels in Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS) detected a 100 kb two-SNP haplotype at KNG1–ADIPOQ associated with reduced adiponectin (frequency = 0.050, P = 1.8 × 10−25). Subsequent genotyping of CLHNS young adult offspring detected an uncommon variant [minor allele frequency (MAF) = 0.025] located ∼800 kb from ADIPOQ that showed strong association with lower adiponectin levels (P = 2.7 × 10−15, n = 1695) and tagged a subset of KNG1–ADIPOQ haplotype carriers with even lower adiponectin levels. Sequencing of the ADIPOQ-coding region detected variant R221S (MAF = 0.015, P = 2.9 × 10−69), which explained 17.1% of the variance in adiponectin levels and largely accounted for the initial GWA signal in Filipinos. R221S was not present in 12 514 Europeans with previously sequenced exons. To explore the mechanism of this substitution, we re-measured adiponectin level in 20 R221S offspring carriers and 20 non-carriers using two alternative antibodies and determined that the presence of R221S resulted in artificially low quantification of adiponectin level using the original immunoassay. These data provide an example of an uncommon variant responsible for a GWA signal and demonstrate that genetic associations with phenotypes measured by antibody-based quantification methods can be affected by uncommon coding SNPs residing in the antibody target region. PMID:22010046

  19. 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

  20. Effects of pasteurization on adiponectin and insulin concentrations in donor human milk.

    PubMed

    Ley, Sylvia H; Hanley, Anthony J; Stone, Debbie; O'Connor, Deborah L

    2011-09-01

    Although pasteurization is recommended before distributing donor human milk in North America, limited data are available on its impact on metabolic hormones in milk. We aimed to investigate the effects of pasteurization on adiponectin and insulin concentrations in donor human milk. The study investigates concentrations of components in donor human milk before and after Holder pasteurization. After the guidelines of the Human Milk Bank Association of North America, human milk samples were pooled to produce 17 distinct batches (4 individuals per batch) and pasteurized at 62.5°C for 30 min. Adiponectin, insulin, energy, fat, total protein, and glucose concentrations were measured pre- and postpasteurization. Pasteurization reduced milk adiponectin and insulin by 32.8 and 46.1%, respectively (both p < 0.0001). Adiponectin and insulin were significantly correlated with energy and fat milk composition (r = 0.36-0.47; all p < 0.05). Pasteurization effects on milk hormone concentrations remained significant after adjusting for fat and energy (beta ± SEE: -4.11 ± 1.27, p = 0.003 for adiponectin; -70.0 ± 15.0, p < 0.0001 for insulin). Holder pasteurization reduced adiponectin and insulin concentrations in donor human milk. In view of emerging knowledge on the importance of milk components, continued work to find the optimal pasteurization process that mitigates risks but promotes retention of bioactive components is needed.

  1. A novel ER J-protein DNAJB12 accelerates ER-associated degradation of membrane proteins including CFTR.

    PubMed

    Yamamoto, Yo-hei; Kimura, Taiji; Momohara, Shuku; Takeuchi, Masato; Tani, Tokio; Kimata, Yukio; Kadokura, Hiroshi; Kohno, Kenji

    2010-01-01

    Cytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membrane-spanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.

  2. 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

  3. Effects of synchronicity of carbohydrate and protein degradation on rumen fermentation characteristics and microbial protein synthesis.

    PubMed

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

    2013-03-01

    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.

  4. Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18.

    PubMed

    Stewart, Deborah; Kazemi, Shirin; Li, Suiyang; Massimi, Paola; Banks, Lawrence; Koromilas, Antonis E; Matlashewski, Greg

    2004-06-01

    Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types. PMID:15166424

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Negative Skeletal Effects of Locally Produced Adiponectin

    PubMed Central

    Abbott, Marcia J.; Roth, Theresa M.; Ho, Linh; Wang, Liping; O’Carroll, Dylan; Nissenson, Robert A.

    2015-01-01

    Epidemiological studies show that high circulating levels of adiponectin are associated with low bone mineral density. The effect of adiponectin on skeletal homeostasis, on osteoblasts in particular, remains controversial. We investigated this issue using mice with adipocyte-specific over-expression of adiponectin (AdTg). MicroCT and histomorphometric analysis revealed decreases (15%) in fractional bone volume in AdTg mice at the proximal tibia with no changes at the distal femur. Cortical bone thickness at mid-shafts of the tibia and at the tibiofibular junction was reduced (3–4%) in AdTg mice. Dynamic histomorphometry at the proximal tibia in AdTg mice revealed inhibition of bone formation. AdTg mice had increased numbers of adipocytes in close proximity to trabecular bone in the tibia, associated with increased adiponectin levels in tibial marrow. Treatment of BMSCs with adiponectin after initiation of osteoblastic differentiation resulted in reduced mineralized colony formation and reduced expression of mRNA of osteoblastic genes, osterix (70%), Runx2 (52%), alkaline phosphatase (72%), Col1 (74%), and osteocalcin (81%). Adiponectin treatment of differentiating osteoblasts increased expression of the osteoblast genes PPARγ (32%) and C/ebpα (55%) and increased adipocyte colony formation. These data suggest a model in which locally produced adiponectin plays a negative role in regulating skeletal homeostasis through inhibition of bone formation and by promoting an adipogenic phenotype. PMID:26230337

  11. 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

  12. ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C.

    PubMed

    Dong, Mei; Bridges, James P; Apsley, Karen; Xu, Yan; Weaver, Timothy E

    2008-06-01

    Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. In this study, genes specifically induced in response to transient expression of two disease-associated mutations were identified by microarray analyses. Immunoglobulin heavy chain binding protein (BiP) and two heat shock protein 40 family members, endoplasmic reticulum-localized DnaJ homologues ERdj4 and ERdj5, were significantly elevated and exhibited prolonged and specific association with the misfolded proprotein; in contrast, ERdj3 interacted with BiP, but it did not associate with either wild-type or mutant SP-C. Misfolded SP-C, ERdj4, and ERdj5 coprecipitated with p97/VCP indicating that the cochaperones remain associated with the misfolded proprotein until it is dislocated to the cytosol. Knockdown of ERdj4 and ERdj5 expression increased ER retention and inhibited degradation of misfolded SP-C, but it had little effect on the wild-type protein. Transient expression of ERdj4 and ERdj5 in X-box binding protein 1(-/-) mouse embryonic fibroblasts substantially restored rapid degradation of mutant SP-C proprotein, whereas transfection of HPD mutants failed to rescue SP-C endoplasmic reticulum-associated protein degradation. ERdj4 and ERdj5 promote turnover of misfolded SP-C and this activity is dependent on their ability to stimulate BiP ATPase activity. PMID:18400946

  13. 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

  14. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  15. Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A.

    PubMed

    Weidler, Guido; Zur Oven-Krockhaus, Sven; Heunemann, Michael; Orth, Christian; Schleifenbaum, Frank; Harter, Klaus; Hoecker, Ute; Batschauer, Alfred

    2012-06-01

    The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.

  16. Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation.

    PubMed

    Mahammad, Saleemulla; Murthy, S N Prasanna; Didonna, Alessandro; Grin, Boris; Israeli, Eitan; Perrot, Rodolphe; Bomont, Pascale; Julien, Jean-Pierre; Kuczmarski, Edward; Opal, Puneet; Goldman, Robert D

    2013-05-01

    Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN gene (encoding for gigaxonin), which is predicted to be an E3 ligase adaptor. In GAN, aggregates of intermediate filaments (IFs) represent the main pathological feature detected in neurons and other cell types, including patients' dermal fibroblasts. The molecular mechanism by which these mutations cause IFs to aggregate is unknown. Using fibroblasts from patients and normal individuals, as well as Gan-/- mice, we demonstrated that gigaxonin was responsible for the degradation of vimentin IFs. Gigaxonin was similarly involved in the degradation of peripherin and neurofilament IF proteins in neurons. Furthermore, proteasome inhibition by MG-132 reversed the clearance of IF proteins in cells overexpressing gigaxonin, demonstrating the involvement of the proteasomal degradation pathway. Together, these findings identify gigaxonin as a major factor in the degradation of cytoskeletal IFs and provide an explanation for IF aggregate accumulation, the subcellular hallmark of this devastating human disease.

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

    SciTech Connect

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

    2011-05-27

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

  18. 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

  19. 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

  20. Mechanisms of HIV protein degradation into epitopes: implications for vaccine design.

    PubMed

    Rucevic, Marijana; Boucau, Julie; Dinter, Jens; Kourjian, Georgio; Le Gall, Sylvie

    2014-08-21

    The degradation of HIV-derived proteins into epitopes displayed by MHC-I or MHC-II are the first events leading to the priming of HIV-specific immune responses and to the recognition of infected cells. Despite a wealth of information about peptidases involved in protein degradation, our knowledge of epitope presentation during HIV infection remains limited. Here we review current data on HIV protein degradation linking epitope production and immunodominance, viral evolution and impaired epitope presentation. We propose that an in-depth understanding of HIV antigen processing and presentation in relevant primary cells could be exploited to identify signatures leading to efficient or inefficient epitope presentation in HIV proteomes, and to improve the design of immunogens eliciting immune responses efficiently recognizing all infected cells.

  1. 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.

  2. 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

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

    PubMed

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

    2015-12-02

    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 (UPR(mt)). 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 UPR(mt). 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 UPR(mt), this pathway may preferentially act to promote chaperone mediated refolding of proteins.

  4. 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

  5. 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

  6. Serum Adiponectin and Indices of Cardiovascular Risk in Young Women with Excessive Body Mass

    PubMed Central

    Sypniewska, G.; Rajewski, P.; Gruszka, M.

    2010-01-01

    Adiponectin reduces oxidative stress, the release of C-reactive protein and influences on the process of atherogenesis reducing lipid accumulation in the blood vessels. The findings on the association of adiponectin with cardiovascular risk are contradictory. This study aimed to assess the relationship between adiponectin and indices of cardiovascular risk in women with excessive body mass. Adiponectin, hsCRP and lipids were measured in blood samples obtained from normoglycemic women with excessive body mass (n=52;BMI≥25 kg/m2) aged 25-40 yrs and age-matched healthy controls (n=36; BMI<25kg/m2). All subjects underwent blood pressure examination and anthropometric measurements. Median concentration of adiponectin in the serum in women with excessive body mass was significantly lower than in women with normal weight (10,8 vs 15,5 µg/ml; p<0,01). Similarly, median serum concentration of triglycerides, hsCRP and blood pressure values were significantly higher and HDL-cholesterol significantly lower in women with BMI≥25 kg/m2 in comparison to these with normal BMI, however only HDL-C and hsCRP were found to be beyond widely accepted cut-offs. Hypoadiponectinemia in women with excessive body mass (adiponectin concentration below the 5th percentile in the control group) was associated predominantly with abnormally increased median values of hsCRP and blood pressure. Concentrations of total cholesterol, non-HDL-C and LDL-C were also significantly higher in women with excessive body mass and hypoadiponectinemia, however still within the reference range. Our results suggest that adiponectin may be used as a prognostic marker of cardiovascular risk in women with excessive body mass.

  7. 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.

  8. 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

  9. Low Adiponectin Concentration in Pregnancy Predicts Postpartum Insulin Resistance, Beta-cell Dysfunction, and Fasting Glycaemia

    PubMed Central

    Retnakaran, R; Qi, Y; Connelly, PW; Sermer, M; Hanley, AJ; Zinman, B

    2010-01-01

    Aims/Hypothesis The postpartum following gestational diabetes (GDM) is characterized by subtle metabolic defects, including beta-cell dysfunction that is believed to mediate the increased future risk of type 2 diabetes in this patient population. Recently, low circulating levels of adiponectin and increased leptin and C-reactive protein (CRP) have emerged as novel diabetic risk factors, although their relevance to GDM and subsequent diabetes has not been characterized. Thus, we sought to determine whether adiponectin, leptin and CRP in pregnancy relate to the postpartum metabolic defects linking GDM with type 2 diabetes. Methods 487 women underwent metabolic characterization, including oral glucose tolerance test (OGTT), in pregnancy and at 3-months postpartum. Based on the antepartum OGTT, there were 137 women with GDM, 91 with gestational impaired glucose tolerance, and 259 with normal glucose tolerance. Results Adiponectin levels were lowest (p<0.0001) and CRP levels highest (p=0.0008) in women with GDM. Leptin did not differ between the glucose tolerance groups (p=0.4483). Adiponectin (r=0.41,p<0.0001), leptin (r=−0.36,p<0.0001) and CRP (r=−0.30,p<0.0001) in pregnancy were all associated with postpartum insulin sensitivity (ISOGTT). Intriguingly, adiponectin was also related to postpartum beta-cell function (insulinogenic index/HOMA-IR) (r=0.16,p=0.0009). Indeed, on multiple linear regression analyses, adiponectin in pregnancy independently predicted both postpartum insulin sensitivity (t=3.97,p<0.0001) and beta-cell function (t=2.37,p=0.0181), even after adjustment for GDM. Furthermore, adiponectin emerged as a significant negative independent determinant of postpartum fasting glucose (t=−3.01,p=0.0027). Conclusions Hypoadiponectinemia in pregnancy predicts postpartum insulin resistance, beta-cell dysfunction, and fasting glycaemia, and hence may be relevant to the pathophysiology relating GDM with type 2 diabetes. PMID:19937225

  10. Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

    PubMed Central

    Kwak, Su-Jin; Hong, Seung-Hyun; Bajracharya, Rijan; Yang, Se-Yeol; Lee, Kyu-Sun; Yu, Kweon

    2013-01-01

    Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application. PMID:23874700

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

    PubMed

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

    2015-08-01

    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.

  12. 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

  13. Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2.

    PubMed

    Foo, Roger S-Y; Chan, Lennard K W; Kitsis, Richard N; Bennett, Martin R

    2007-02-23

    Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.

  14. 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

  15. Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation.

    PubMed

    Kennedy, Susan A; Scaife, Caitríona; Dunn, Michael J; Wood, Alfred E; Watson, R William G

    2011-06-01

    Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.

  16. Involvement of protein degradation by the ubiquitin proteasome system in opiate addictive behaviors.

    PubMed

    Massaly, Nicolas; Dahan, Lionel; Baudonnat, Mathieu; Hovnanian, Caroline; Rekik, Khaoula; Solinas, Marcello; David, Vincent; Pech, Stéphane; Zajac, Jean-Marie; Roullet, Pascal; Mouledous, Lionel; Frances, Bernard

    2013-03-01

    Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction.

  17. 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

  18. 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.

  19. ADIPOQ, ADIPOR1, and ADIPOR2 Polymorphisms in Relation to Serum Adiponectin Levels and Body Mass Index in Black and White Women

    PubMed Central

    Cohen, Sarah S.; Gammon, Marilie D.; North, Kari E.; Millikan, Robert C.; Lange, Ethan M.; Williams, Scott M.; Zheng, Wei; Cai, Qiuyin; Long, Jirong; Smith, Jeffrey R.; Signorello, Lisa B.; Blot, William J.; Matthews, Charles E.

    2012-01-01

    Adiponectin is an adipose-secreted protein with influence on several physiologic pathways including those related to insulin sensitivity, inflammation, and atherogenesis. Adiponectin levels are highly heritable and several single nucleotide polymorphisms (SNPs) in adiponectin-related genes (ADIPOQ, ADIPOR1, ADIPOR2) have been examined in relation to circulating adiponectin levels and obesity phenotypes, but despite differences in adiponectin levels and obesity prevalence by race, few studies have included black participants. Using cross-sectional interview data and blood samples collected from 990 black and 977 white women enrolled in the Southern Community Cohort Study from 2002 to 2006, we examined 25 SNPs in ADIPOQ, 19 in ADIPOR1, and 27 in ADIPOR2 in relation to serum adiponectin levels and body mass index (BMI) using race-stratified linear regression models adjusted for age and percentage African ancestry. SNP rs17366568 in ADIPOQ was significantly associated with serum adiponectin levels in white women only (adjusted mean adiponectin levels = 15.9 for G/G genotype, 13.7 for A/G, and 9.3 for A/A, p=0.00036). No other SNPs were associated with adiponectin or BMI among blacks or whites. Because adiponectin levels as well as obesity are highly heritable and vary by race but associations with polymorphisms in the ADIPOQ, ADIPOR1, and ADIPOR2 genes have been few in this and other studies, future work including large populations from diverse racial groups is needed to detect additional genetic variants that influence adiponectin and BMI. PMID:21273992

  20. 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

  1. 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

  2. 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.

  3. 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

  4. Dimerization-driven degradation of C. elegans and human E proteins

    PubMed Central

    Sallee, Maria D.; Greenwald, Iva

    2015-01-01

    E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix–loop–helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure–function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts. PMID:26159995

  5. 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

  6. Efficiency of enteral nitrogen support in surgical patients: small peptides v non-degraded proteins.

    PubMed Central

    Ziegler, F; Ollivier, J M; Cynober, L; Masini, J P; Coudray-Lucas, C; Levy, E; Giboudeau, J

    1990-01-01

    In a prospective study, 12 intensive care patients, after abdominal surgery, received three alternate six-day courses of two enteral diets with identical nitrogen (0.3 g N/kg per day) and energy (60 kcal/kg per day) supply. The protein hydrolysate (PH) diet contained enzyme-hydrolysed casein and lactoserum (60% small peptides), while the non-degraded protein (NDP) diet contained a nitrogen source of similar amino acid composition, but in the form of non-degraded proteins. The patients were randomised to receive either PH-NDP-PH or NDP-PH-NDP. Parameters reflecting protein metabolism were assessed in the plasma, urine, and stomal effluent on days 1, 6, 12, and 18, three hours after stopping the nutrition (t0), and one hour after restarting it (t1). Comparisons of t1 and t0 values showed that 13 amino acids (including the eight essential amino acids) increased significantly with the protein hydrolysate diet, but only two increased with the non-degraded protein diet. Similarly, with protein hydrolysate, insulin-aemia at t1 was significantly higher than at t0 and correlated with plasma leucine, phenylalanine, alanine, and lysine concentrations. In addition, significant improvements in plasma albumin, transferrin, and retinol binding protein concentrations were seen with protein hydrolysate, together with a significant decrease in the plasma phenylalanine/tyrosine ratio and urinary 3-methylhistidine excretion. We conclude that in patients in intensive care after abdominal surgery enteral support containing small peptides is more effective than an equivalent diet containing whole proteins in restoring plasma amino acid and protein levels. PMID:2123819

  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. 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.

  9. 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.

  10. Beclin 2 Functions in Autophagy, Degradation of G Protein-Coupled Receptors, and Metabolism

    PubMed Central

    He, Congcong; Wei, Yongjie; Sun, Kai; Li, Binghua; Dong, Xiaonan; Zou, Zhongju; Liu, Yang; Kinch, Lisa N.; Khan, Shaheen; Sinha, Sangita; Xavier, Ramnik J.; Grishin, Nick V.; Xiao, Guanghua; Eskelinen, Eeva-Liisa; Scherer, Philipp E.; Whistler, Jennifer L.; Levine, Beth

    2013-01-01

    Summary The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a novel converging regulator of autophagy and GPCR turnover, and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking and metabolism. PMID:23954414

  11. 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

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

    PubMed Central

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

    2014-01-01

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. PMID:25422373

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

    PubMed Central

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

    2014-01-01

    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. 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

  16. Ruminal degradation, amino acid composition, and intestinal digestibility of the residual components of five protein supplements.

    PubMed

    Maiga, H A; Schingoethe, D J; Henson, J E

    1996-09-01

    Two ruminally cannulated Holstein cows (approximately 202 DIM) were used to determine the in situ degradability of five protein supplements: blood meal, meat and bone meal, corn gluten meal, expeller soybean meal, and solvent extracted soybean meal. Dacron bags containing 4 g of each supplement in duplicate were soaked in water and then incubated in the rumen for 0, 3, 6, 12, 18, and 24 h for 3 d. Four extra sample bags of each supplement were incubated in the rumen for 12 h to determine the in vitro intestinal digestibility and AA analysis of the residues. Protein supplements were also analyzed for their AA content. Ruminal degradability of individual supplements varied. Solvent soybean meal was the most degradable, and blood meal was the least degradable. Specific first-limiting essential AA were isoleucine for blood meal and meat and bone meal, lysine for corn gluten meal, and methionine for the soybean meals. The RUP fraction in solvent-extracted and expeller soybean meals tended to be more intestinally digestible than did the protein in blood meal and meat and bone meal. In general, all protein supplements, except solvent-extracted soybean meal, were high in RUP and had the potential to provide good quality AA to complement microbial AA for production.

  17. 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…

  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. Targeted proteomics analysis of protein degradation in plant signaling on an LTQ-Orbitrap mass spectrometer.

    PubMed

    Majovsky, Petra; Naumann, Christin; Lee, Chil-Woo; Lassowskat, Ines; Trujillo, Marco; Dissmeyer, Nico; Hoehenwarter, Wolfgang

    2014-10-01

    Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.

  20. 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)...

  1. [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

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

    PubMed

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

    2015-09-01

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

  4. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

    PubMed Central

    2012-01-01

    Background In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates

  5. Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP).

    PubMed

    Gross, Alec W; Lodish, Harvey F

    2006-01-27

    Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.

  6. 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

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

    SciTech Connect

    Wielbold, J.L.

    1982-01-01

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

  8. 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

  9. Is adiponectin a risk factor for transient ischaemic attacks?

    PubMed

    Sener, Ufuk; Uludag, Irem Fatma; Kose, Sukran; Ozcelik, Murat; Zorlu, Yasar

    2015-01-01

    Adiponectin is an adipocytokine, and it plays a role in atherosclerosis. The role of adiponectin in the development of ischaemic stroke is controversial. Up to now, adiponectin was not evaluated in transient ischaemic stroke. In this study, we investigated the relationship between adiponectin and transient ischaemic attack. Forty patients with transient ischaemic attack were included into the study. In all patients, traditional risk factors of ischaemic stroke and intima-media thickness of carotid arteries were determined. Also, the relationship between these parameters and adiponectin levels were examined. No difference was found in terms of adiponectin levels between patients and healthy subjects. In addition, there was no association between adiponectin levels and traditional risk factors. Our results suggest that adiponectin may not be a predictive risk factor of transient ischaemic attack.

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

    PubMed

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

    2014-10-16

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

  11. Disturbed adiponectin – AMPK system in skeletal muscle of patients with metabolic syndrome.

    PubMed

    Van Berendoncks, An M; Stensvold, Dorthe; Garnier, Anne; Fortin, Dominique; Sente, Tahnee; Vrints, Christiaan J; Arild, Slørdahl Stig; Ventura-Clapier, Renee; Wisløff, Ulrik; Conraads, Viviane M

    2015-02-01

    Patients with metabolic syndrome are characterized by low circulating adiponectin levels and reduced adiponectin sensitivity in skeletal muscles. Through binding on its main skeletal muscle receptor AdipoR1, adiponectin activates AMP-activated protein kinase (AMPK), a key player in energy homeostasis. Fourteen metabolic syndrome patients and seven healthy control subjects were included. Blood samples were taken to determine insulin resistance, adiponectin, lipoproteins, and C-reactive protein. Muscle biopsies (m. vastus lateralis) were obtained to assess mRNA expression of AdipoR1 and both AMPKα1 and AMPKα2 subunits, as well as downstream targets in lipid and glucose metabolism. Skeletal muscle mRNA expression of AMPKα1 and AMPKα2 was lower in metabolic syndrome patients (100 ± 6 vs. 122 ± 8 AU, p = 0.030 and 64 ± 4 vs. 85 ± 9 AU, p = 0.044, respectively), whereas the expression of AdipoR1 was upregulated (138 ± 9 vs. 105 ± 7, p = 0.012). AMPKα1 and AdipoR1 correlated positively in both the control (r = 0.964, p < 0.001) and the metabolic syndrome group (r = 0.600, p = 0.023). However, this relation was shifted upwards in metabolic syndrome patients, indicating increased AdipoR1mRNA expression for a similar AMPKα1 expression. Previously, a blunted stimulatory effect of adiponectin on AMPK activation has been shown in metabolic syndrome patients. The present data suggest that the disturbed interaction of adiponectin with AMPK is located downstream of the AdipoR1 receptor.

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

    PubMed

    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.

  13. 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.

  14. 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.

  15. The Senescence-Induced Staygreen Protein Regulates Chlorophyll Degradation[W

    PubMed Central

    Park, So-Yon; Yu, Jae-Woong; Park, Jong-Sung; Li, Jinjie; Yoo, Soo-Cheul; Lee, Na-Yeoun; Lee, Sang-Kyu; Jeong, Seok-Won; Seo, Hak Soo; Koh, Hee-Jong; Jeon, Jong-Seong; Park, Youn-Il; Paek, Nam-Chon

    2007-01-01

    Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase–impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively. PMID:17513504

  16. Argon Laser Welding Induces Degradation And Crosslinking Of Extracellular Matrix Protein: A Preliminary Report

    NASA Astrophysics Data System (ADS)

    Su, Lyndon; Murray, Louann W.; White, Rodney A.; Kopchok, George E.; Guthrie, Carol; White, Geoffrey H.

    1989-09-01

    Extracellular matrix components from untreated and laser-welded skin and blood vessels were extracted with guanidine hydrochloride and separated by SDS polyacrylamide gel electrophoresis. When compared to matched, untreated tissues, protein electrophoretic profiles from laser-treated samples showed several changes. In both tissue types, the concentration of a protein normally migrating between alpha and beta chains of type I collagen decreased in laser-treated samples. However, laser-treated blood vessels showed significantly more low molecular weight protein, whereas significantly more high molecular weight protein appeared in laser-treated skin samples when compared to untreated tissue. These results suggest that the argon laser either degrades or crosslinks proteins in-vivo. Laser induced protein crosslinks may be the biochemical basis of laser welding.

  17. 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

  18. 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.

  19. 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

  20. Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics

    PubMed Central

    Valliere-Douglass, John F; Connell-Crowley, Lisa; Jensen, Randy; Schnier, Paul D; Trilisky, Egor; Leith, Matt; Follstad, Brian D; Kerr, Jennifer; Lewis, Nathan; Vunnum, Suresh; Treuheit, Michael J; Balland, Alain; Wallace, Alison

    2010-01-01

    Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled 13C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified–free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from 13C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO2 through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers. PMID:20836085

  1. Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.

    PubMed

    Raule, Mary; Cerruti, Fulvia; Cascio, Paolo

    2014-09-01

    Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit β2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.

  2. 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…

  3. 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

  4. 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.; Bartz, Jason C.

    2011-01-01

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

  5. 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.

  6. 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.

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

    PubMed

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

    2011-05-11

    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.

  8. 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

  9. Backmasking in the yeast genome: encoding overlapping information for protein-coding and RNA degradation

    PubMed Central

    Cakiroglu, S. Aylin; Zaugg, Judith B.; Luscombe, Nicholas M.

    2016-01-01

    Backmasking is a recording technique used to hide a sound or message in a music track in reverse, meaning that it is only audible when the record is played backwards. Analogously, the compact yeast genome encodes for diverse sources of information such as overlapping coding and non-coding transcripts, and protein-binding sites on the two complementary DNA strands. Examples are the consensus binding site sequences of the RNA-binding proteins Nrd1 and Nab3 that target non-coding transcripts for degradation. Here, by examining the overlap of stable (SUTs, stable unannotated transcripts) and unstable (CUTs, cryptic unstable transcripts) transcripts with protein-coding genes, we show that the predicted Nrd1 and Nab3-binding site sequences occur at differing frequencies. They are always depleted in the sense direction of protein-coding genes, thus avoiding degradation of the transcript. However in the antisense direction, predicted binding sites occur at high frequencies in genes with overlapping unstable ncRNAs (CUTs), so limiting the availability of non-functional transcripts. In contrast they are depleted in genes with overlapping stable ncRNAs (SUTs), presumably to avoid degrading the non-coding transcript. The protein-coding genes maintain similar amino-acid contents, but they display distinct codon usages so that Nrd1 and Nab3-binding sites can arise at differing frequencies in antisense depending on the overlapping transcript type. Our study demonstrates how yeast has evolved to encode multiple layers of information—protein-coding genes in one strand and the relative chance of degrading antisense RNA in the other strand—in the same regions of a compact genome. PMID:27492286

  10. 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

  11. 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

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

    2012-04-01

    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.

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Ilalov, Kiril; Yu, Shuang; Xu, Ke; Prazak, Lisa; Fajardo, Marc; Sehgal, Bantoo; Di Cesare, Paul E.

    2006-01-01

    Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.—Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J. 20, E129 -E140 (2006) PMID:16585064

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

    PubMed

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

    2014-08-01

    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.

  16. 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

  17. Insulin-degrading enzyme is exported via an unconventional protein secretion pathway

    PubMed Central

    Zhao, Ji; Li, Lilin; Leissring, Malcolm A

    2009-01-01

    Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid β-protein (Aβ), and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD). Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s) remain poorly understood. To address this topic, we investigated the effects of known inhibitors or stimulators of protein secretion on the secretion of IDE from murine hepatocytes and HeLa cells. IDE secretion was found to be unaffected by the classical secretion inhibitors brefeldin A (BFA), monensin, or nocodazole, treatments that readily inhibited the secretion of α1-antitrypsin (AAT) overexpressed in the same cells. Using a novel cell-based Aβ-degradation assay, we show further that IDE secretion was similarly unaffected by multiple stimulators of protein secretion, including glyburide and 3'-O-(4-benzoyl)benzoyl-ATP (Bz-ATP). The calcium ionophore, A23187, increased extracellular IDE activity, but only under conditions that also elicited cytotoxicity. Our results provide the first biochemical evidence that IDE export is not dependent upon the classical secretion pathway, thereby identifying IDE as a novel member of the select class of unconventionally secreted proteins. Further elucidation of the mechanisms underlying IDE secretion, which would be facilitated by the assays described herein, promises to uncover processes that might be defective in disease or manipulated for therapeutic benefit. PMID:19144176

  18. Mitochondria contain a proteolytic system which can recognize and degrade oxidatively-denatured proteins.

    PubMed Central

    Marcillat, O; Zhang, Y; Lin, S W; Davies, K J

    1988-01-01

    When incubated with mitochondria in an air atmosphere, menadione and doxorubicin (which redox cycle with the respiratory chain to produce oxygen radicals), as well as xanthine oxidase plus xanthine (which generate superoxide and H2O2), stimulated the degradation of newly-synthesized [( 3H]leucine-labelled) mitochondrial polypeptides. No stimulation was observed in an N2 atmosphere, ATP was not required, and xanthine oxidase was not effective without xanthine. Various forms of oxidative stress induced varying degrees of protein cross-linking, protein fragmentation and proteolysis, as judged by gel electrophoresis and amino acid analysis. To learn more about the proteolytic enzymes involved in degradation, we undertook studies with purified protein substrates which had been exposed to oxidative stress (OH or H2O2) in vitro. Despite mitochondrial contamination with acid proteases of lysosomal (and other) origin, pH profiles revealed distinct proteolytic activities at both pH 4 and pH 8. The pH 8 activity preferentially degraded the oxidatively-denatured forms of haemoglobin, albumin and superoxide dismutase; was unaffected by digitonin; and exhibited a several-fold increase in activity upon mitochondrial disruption (highest activity being found in the matrix). In contrast, the pH 4 activity was dramatically decreased by digitonin treatment (to reduce lysosomal contamination); was unaffected by mitochondrial disruption; and showed no preference for oxidatively-denatured proteins. The pH 8 activity was not stimulated by ATP, but was inhibited by EDTA, haemin and phenylmethylsulphonyl fluoride. In contrast, the contaminating pH 4 activity was only inhibited by pepstatin and leupeptin. Thus, our experiments reveal a distinct mitochondrial (matrix) proteolytic pathway which can preferentially degrade oxidatively-denatured proteins. PMID:3196285

  19. Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms

    PubMed Central

    Locatelli, Denise; Terao, Mineko; Kurosaki, Mami; Zanellati, Maria Clara; Pletto, Daniela Rita; Finardi, Adele; Colciaghi, Francesca; Garattini, Enrico; Battaglia, Giorgio Stefano

    2015-01-01

    The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN. PMID:26214005

  20. Pepsin degradation of Cry1A(b) protein purified from genetically modified maize (Zea mays).

    PubMed

    de Luis, Ruth; Lavilla, María; Sánchez, Lourdes; Calvo, Miguel; Pérez, María D

    2010-02-24

    The aim of this work was to study the in vitro digestion of Cry1A(b) protein by pepsin. To perform this work, a protein fraction purified from transgenic maize by immunoadsorption was employed. The undigested fraction showed several bands of molecular weight ranging between 14 and 70 kDa when assayed by SDS-PAGE. These bands were identified as corresponding to Cry1A(b) protein by immunochemical techniques and mass spectrometry. The rate of degradation of the purified fraction by pepsin estimated by ELISA was found to be about 75% within 30 min, and the protein concentration remained constant up to 4 h. In all treated samples, the full-length protein and fragments present in Cry1A(b) fraction were absent and peptides of less than 8.5 kDa were mainly found by SDS-PAGE and mass spectrometry. These peptides did not react with antiserum against Cry1A(b) protein by Western blotting. These results suggest that Cry1A(b) fraction purified from transgenic maize is rapidly and extensively degraded by pepsin, giving peptides of low molecular mass.

  1. 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

  2. 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

  3. Mechanisms for the anti-inflammatory effects of adiponectin in macrophages.

    PubMed

    Huang, Honglian; Park, Pil-Hoon; McMullen, Megan R; Nagy, Laura E

    2008-03-01

    Adiponectin is an adipokine with potent anti-inflammatory properties. The development of alcoholic liver disease is thought to involve increased pro-inflammatory activity, mediated in part by the activation of hepatic macrophages (Kupffer cells). Chronic ethanol feeding sensitizes hepatic macrophages to activation by lipopolysaccharide (LPS), leading to increased production of reactive oxygen species and tumor necrosis factor-alpha (TNF-alpha). Adiponectin can normalize Toll-like receptor-4 (TLR-4) mediated signaling in hepatic macrophages after ethanol feeding, likely contributing to the hepatoprotective effect of adiponectin in the progression of alcoholic liver disease. However, the mechanisms by which adiponectin suppress TLR-4 mediated responses are not well understood. Using the macrophage-like cell line, RAW264.7, we have investigated the molecular mechanisms by which adiponectin suppresses LPS-stimulated TNF-alpha production. Globular adiponectin (gAcrp)-mediated desensitization of LPS-stimulated responses in RAW264.7 macrophages was dependent on the production of the anti-inflammatory cytokine interleukin (IL)-10. gAcrp initially increased TNF-alpha expression in RAW264.7 macrophages; this TNF-alpha then contributed to increased expression of IL-10. This initial gAcrp-mediated increase in TNF-alpha production by macrophages was mediated via activation of ERK1/2-->Egr-1 and nuclear factor (NF)-kappaB-dependent mechanisms. gAcrp-stimulated IL-10 expression was also dependent on the phosphorylation of cAMP response element-binding protein and the cAMP response element in the IL-10 promoter. In summary, these studies reveal a complex, integrated response of macrophages to gAcrp. gAcrp initially activated signaling pathways considered to be pro-inflammatory, with a subsequent increase in the expression of the potent, anti-inflammatory cytokine, IL-10. Increased IL-10 expression was ultimately required for the suppression of TLR4-mediated signaling by g

  4. HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

    PubMed Central

    Buckley, Dennis L; Raina, Kanak; Darricarrere, Nicole; Hines, John; Gustafson, Jeffrey L; Smith, Ian E.; Miah, Afjal H; Harling, John D; Crews, Craig M

    2015-01-01

    Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knockdown widely used HaloTag7 fusion proteins in a general fashion. PMID:26070106

  5. Deltex1 promotes protein kinase Cθ degradation and sustains Casitas B-lineage lymphoma expression.

    PubMed

    Hsu, Tzu-Sheng; Hsiao, Huey-Wen; Wu, Pei-Jung; Liu, Wen-Hsien; Lai, Ming-Zong

    2014-08-15

    The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b.

  6. Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle.

    PubMed

    Scherbel, Christina; Pichner, Rohtraud; Groschup, Martin H; Mueller-Hellwig, Simone; Scherer, Siegfried; Dietrich, Richard; Maertlbauer, Erwin; Gareis, Manfred

    2006-01-01

    A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion. PMID:16820134

  7. Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression

    PubMed Central

    Lu, Min; Staszewski, Lena; Echeverri, Fernando; Xu, Hong; Moyer, Bryan D

    2004-01-01

    Background Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells. Results To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway. Conclusions These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination. PMID:15369603

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

    PubMed

    Krapp, Andrea; Simanis, Viesturs

    2014-07-15

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

  9. Control of Death-associated Protein Kinase (DAPK) Activity by Phosphorylation and Proteasomal Degradation*

    PubMed Central

    Jin, Yijun; Blue, Emily K.; Gallagher, Patricia J.

    2010-01-01

    Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-β, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability. PMID:17056602

  10. 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

  11. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

    PubMed

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

    2014-06-20

    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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. A Chaperone-Assisted Degradation Pathway Targets Kinetochore Proteins to Ensure Genome Stability

    PubMed Central

    Kriegenburg, Franziska; Jakopec, Visnja; Poulsen, Esben G.; Nielsen, Sofie Vincents; Roguev, Assen; Krogan, Nevan; Gordon, Colin; Fleig, Ursula; Hartmann-Petersen, Rasmus

    2014-01-01

    Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast Schizosaccharomyces pombe. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of spc7 mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the spc7-23 phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity. PMID:24497846

  17. Plasmolysis, red blood cell partitioning, and plasma protein binding of etofibrate, clofibrate, and their degradation products.

    PubMed

    Altmayer, P; Garrett, E R

    1983-11-01

    Etofibrate (I), the ethylene glycol diester of clofibric and nicotinic acids, degrades almost equally through both half-esters with half-lives of approximately 10 and 1 min in fresh dog and human plasma, respectively. The nicotinate V degrades with half-lives of approximately 12 hr and 50 min in fresh dog and human plasma, respectively. Ester III and clofibrate VI degrade by saturable Michaelis-Menten kinetics in fresh human plasma, with similar maximum initial rates and respective terminal first-order half-lives of 12 and 26 min. Tetraethyl pyrophosphate at 100 micrograms/ml inhibited human plasma and red blood cell esterases permitting plasma protein binding and red blood cell partitioning studies. The red blood cell-plasma water partition coefficient was 5.4 for 0.2-80 micrograms/ml of I. Clofibrate (VI) showed a saturable erythrocyte partitioning that decreased from 7.8 (10 micrograms/ml) to 1 (50 micrograms/ml). The strong binding of I and VI to ultrafiltration membranes necessitated the determination of their plasma protein binding by the method of variable plasma concentrations of erythrocyte suspensions to give 96.6% (0.2-80 micrograms/ml) and 98.2% (13.6-108.4 micrograms/ml) binding, respectively. Methods for the determination of the parameters of saturable and nonsaturable plasma protein binding for unstable and membrane-binding drugs by the method of variable plasma concentrations in partitioning erythrocyte suspensions are presented.

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

    PubMed Central

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

    2009-01-01

    Summary Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation. PMID:19596797

  19. High-Risk Human Papillomavirus E7 Proteins Target PTPN14 for Degradation

    PubMed Central

    Münger, Karl; Howley, Peter M.

    2016-01-01

    ABSTRACT The major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7. PMID:27651363

  20. Hawthorn leaf flavonoids alleviate nonalcoholic fatty liver disease by enhancing the adiponectin/AMPK pathway.

    PubMed

    Li, Zhongping; Xu, Jiaoya; Zheng, Peiyong; Xing, Lianjun; Shen, Hongyi; Yang, Lili; Zhang, Li; Ji, Guang

    2015-01-01

    Hawthorn (Crataeguspinnatifida) belongs to the genus Rosaceae family of plants. The hawthorn leaf, Crataeguspinnatifida Bunge, is used for both condiment and medicinal purposes to prevent and treat metabolic dysfunctions, such as hyperlipidemia, hypertension, and cardiovascular disease in traditional Chinese medicine. However, its effects on nonalcoholic fatty liver disease (NAFLD) remain obscure. The purpose of the present study was to investigate the protective effect of hawthorn leaf flavonoids (HLF), the dominant bioactive extracts of hawthorn leaves, on high fat diet (HFD)-induced hepatic steatosis and to elucidate its underlying mechanisms. HLF supplementation significantly lowered body weight, liver weight, liver/body weight ratio, improved serum parameters and liver dysfunction and markedly decreased hepatic lipid accumulation in HFD-fed rats. In addition, HLF intervention dramatically increased circulating adiponectin levels and up-regulated the expression of adiponectin receptors, particularly adiponectin receptor 2 (AdipoR2) in the liver. Moreover, adenosine monophosphate (AMP)-activated protein kinase (AMPK) was also activated, as well as AMPK-mediated alteration of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor α (PPARα) and their downstream targets. Taken together, our data suggest that HLF ameliorates hepatic steatosis by enhancing the adiponectin/AMPK pathway in the liver of HFD-induced NAFLD rats. PMID:26770322

  1. Effect of a nutritional shift on the degradation of abnormal proteins in the mouse liver. Decreased degradation during rapid liver growth.

    PubMed Central

    Amils, R; Conde, R D; Scornik, O A

    1977-01-01

    1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins. PMID:880243

  2. 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

  3. 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

  4. 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

  5. The Unfolded Protein Response, Degradation from the Endoplasmic Reticulum, and Cancer

    PubMed Central

    Tsai, Yien Che; Weissman, Allan M.

    2010-01-01

    The endoplasmic reticulum (ER) is an essential organelle involved in many cellular functions including protein folding and secretion, lipid biosynthesis, and calcium homeostasis. Proteins destined for the cell surface or for secretion are made in the ER, where they are folded and assembled into multi-subunit complexes. The ER plays a vital role in cellular protein quality control by extracting and degrading proteins that are not correctly folded or assembled into native complexes. This process, known as ER-associated degradation (ERAD), ensures that only properly folded and assembled proteins are transported to their final destinations. Besides its role in protein folding and transport in the secretory pathway, the ER regulates the biosynthesis of cholesterol and other membrane lipids. ERAD is an important means to ensure that levels of the responsible enzymes are appropriately maintained. The ER is also a major organelle for oxygen and nutrient sensing as cells adapt to their microenvironment. Stresses that disrupt ER function lead to accumulation of unfolded proteins in the ER, a condition known as ER stress. Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response (UPR). The UPR represents a survival response by the cells to restore ER homeostasis. If ER stress persists, cells activate mechanisms that result in cell death. Chronic ER stress is increasingly being recognized as a factor in many human diseases such as diabetes, neurodegenerative disorders, and cancer. In this review, we discuss the roles of the UPR and ERAD in cancer and suggest directions for future research. PMID:21331300

  6. 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.

  7. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein.

    PubMed

    Taylor, J Paul; Tanaka, Fumiaki; Robitschek, Jon; Sandoval, C Miguel; Taye, Addis; Markovic-Plese, Silva; Fischbeck, Kenneth H

    2003-04-01

    Expression of misfolded protein in cultured cells frequently leads to the formation of juxtanuclear inclusions that have been termed 'aggresomes'. Aggresome formation is an active cellular response that involves trafficking of the offending protein along microtubules, reorganization of intermediate filaments and recruitment of components of the ubiquitin proteasome system. Whether aggresomes are benevolent or noxious is unknown, but they are of particular interest because of the appearance of similar inclusions in protein deposition diseases. Here we present evidence that aggresomes serve a cytoprotective function and are associated with accelerated turnover of mutant proteins. We show that mutant androgen receptor (AR), the protein responsible for X-linked spinobulbar muscular atrophy, forms insoluble aggregates and is toxic to cultured cells. Mutant AR was also found to form aggresomes in a process distinct from aggregation. Molecular and pharmacological interventions were used to disrupt aggresome formation, revealing their cytoprotective function. Aggresome-forming proteins were found to have an accelerated rate of turnover, and this turnover was slowed by inhibition of aggresome formation. Finally, we show that aggresome-forming proteins become membrane-bound and associate with lysosomal structures. Together, these findings suggest that aggresomes are cytoprotective, serving as cytoplasmic recruitment centers to facilitate degradation of toxic proteins.

  8. 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.

  9. Low-molecular-weight adiponectin is more closely associated with disease activity of rheumatoid arthritis than other adiponectin multimeric forms.

    PubMed

    Li, Ping; Yang, Li; Ma, Cui-Li; Liu, Bo; Zhang, Xin; Ding, Rui; Bi, Li-qi

    2015-06-01

    Adiponectin is divided into high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW) forms. These forms differ not only in the number of adiponectin molecules but also in their biological activity. There are conflicting findings regarding the role of adiponectin in rheumatoid arthritis (RA). Moreover, few reports have described the relationships between serum adiponectin multimers levels and RA. Therefore, we examined the association of total adiponectin and its multimers with RA. Two study groups were examined: 180 recently diagnosed untreated RA patients with disease duration less than 1 year (RA group) and 160 age- and sex-matched control subjects (control group). RA-related factors, blood pressure, body mass index, glucose, complete lipid profile, and adiponectin multimers were measured. The levels of total adiponectin and each multimer of adiponectin were significantly lower in the RA than in the control (P < 0.01). Serum levels of total, HMW, MMW, and LMW were positively correlated with triglycerides levels and negatively correlated with the Disease Activity Score for 28 joints (DAS28). Multivariate regression analysis showed that total, HMW, and MMW adiponectin were independently associated with serum triglycerides level. LMW adiponectin was independently correlated with serum triglycerides level and DAS28. The decreased LMW adiponectin levels may be associated with disease activity of RA.

  10. Effect of temperature and pH on the post-mortem degradation of myofibrillar proteins.

    PubMed

    Yates, L D; Dutson, T R; Caldwell, J; Carpenter, Z L

    1983-01-01

    Incubation of bovine muscle at 37°C promoted a more drastic proteolytic change in myofibrillar proteins, as determined from sodium dodecyl sulphate polyacrylamide gels of isolated myofibrils, than incubation at 4°C. Degradation of myosin and troponin-T were the most noticeable changes at 37°C. Loss of α-actinin was observed in the 4°C incubated muscle. Ground bovine muscle incubated at pH 5·4 and 7 revealed that alterations in myosin and troponin-T were the most noticeable changes at ph 5·4 while troponin-T and α-actinin were altered at pH7. More troponin-T degradation occurred at pH 5·4 and 37°C than at pH7 and 4°C (similar to the degradation of myosin), indicating that troponin-T degradation in post-mortem muscle may be an indicator of overall myofibrillar proteolysis and not responsible for post-mortem tenderisation per se. Myosin degradation in the ground samples incubated at pH 5·4 and in whole samples incubated at 37°C was compared with the digestion of myofibrillar myosin by papain. Both pyrophosphate and Guba-Straub extracts of the 37°C and pH 5·4 treated samples confirmed that myosin degradation did occur to a much greater extent at this temperature and pH than at 4°C and pH7, and, in addition, at pH 5·4 frequent cleavage occurred near the papain sensitive site of myosin. PMID:22055822

  11. Leptin and adiponectin levels in middle-aged postmenopausal women: associations with lifestyle habits, hormones, and inflammatory markers--a cross-sectional study.

    PubMed

    Rolland, Yves M; Perry, Horace M; Patrick, Ping; Banks, William A; Morley, John E

    2006-12-01

    To investigate the relationships between blood levels of leptin or adiponectin and lifestyle habits, hormones, and inflammatory markers, we measured parameters of alcohol intake, smoking, physical activity, and blood levels of leptin, adiponectin, testosterone, estrone, estradiol, cortisol, dihydroepiandrostenedione, luteinizing hormone, thyroxin, C-reactive protein (CRP), and interleukin 6 and interleukin 2 receptor in 76 healthy middle-aged postmenopausal women. Anthropometric measures and body composition (evaluated by dual-energy x-ray absorptiometry) and lipid profiles were also assessed. By simple regression, leptin correlated positively with fat and lean masses, glucose, triglycerides, low-density lipoprotein cholesterol, and total cholesterol, and negatively with high-density lipoprotein cholesterol. Adioponectin correlated negatively with fat and lean masses and low-density lipoprotein cholesterol, and positively with high-density lipoprotein cholesterol. Leptin concentration was correlated inversely with adiponectin (r = -0.26, P < .05) and positively with CRP (r = 0.56, P < .01). Adiponectin concentration was negatively correlated with time since last alcoholic drink (r = -0.24, P < .05) and CRP (r = -0.27, P < .05) and positively with testosterone level (r = 0.23, P < .05). By multiple regression analysis, leptin concentration was predicted by age (P < .05), testosterone (P < .05), adiponectin (P < .05), CRP (P < .01), and interleukin 6 receptor (P < .01). Adiponectin concentration was predicted by the time since last alcoholic drink (P < .05), testosterone (P < .05), leptin (P < .05), and C-reactive protein (P = .05). Similar results were found when leptin or adiponectin concentration was adjusted for fat mass. These results suggested that levels of leptin and adiponectin in middle-aged postmenopausal women are partially determined by sexual hormones and inflammatory marker levels, and both predicted one another. Moreover, adiponectin level may be

  12. 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

  13. 5-Aza-2'-deoxycytidine reactivates gene expression via degradation of pRb pocket proteins.

    PubMed

    Zheng, Zhixing; Li, Lian; Liu, Xiangyu; Wang, Donglai; Tu, Bo; Wang, Lina; Wang, Haiying; Zhu, Wei-Guo

    2012-01-01

    Not only does 5-aza-2'-deoxycytidine (5-aza-CdR) induce the reexpression of silenced genes through the demethylation of CpG islands, but it increases the expression of unmethylated genes. However, the mechanism by which 5-aza-CdR activates the expression of genes is not completely understood. Here, we report that the pRb pocket proteins pRb, p107, and p130 were degraded in various cancer cell lines in response to 5-aza-CdR treatment, and this effect was dependent on the proteasome pathway. Mouse double minute 2 (MDM2) played a critical role in this 5-aza-CdR-induced degradation of pRb. Furthermore, PP2A phosphatase-induced MDM2 dephosphorylation at S260 was found to be essential for MDM2 binding to pRb in the presence of 5-aza-CdR. pRb degradation resulted in the significant reexpression of several genes, including methylated CDKN2A, RASFF1A, and unmethylated CDKN2D. Finally, knockdown of pRb pocket proteins by either RNAi or 5-aza-CdR treatment induced a significant decrease in the recruitment of SUV39H1 and an increase in the enrichment of KDM3B and KDM4A to histones around the promoter of RASFF1A and thus reduced H3K9 di- and trimethylation, by which RASFF1A expression is activated. Our data reveal a novel mechanism by which 5-aza-CdR induces the expression of both methylated and unmethylated genes by degrading pRb pocket proteins.

  14. 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

  15. 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

    2016-06-09

    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.

  16. 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

  17. 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

  18. 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

  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. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells.

    PubMed

    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

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2015-05-01

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

  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. 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

  6. 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

  7. A parasite cysteine protease is key to host protein degradation and iron acquisition.

    PubMed

    O'Brien, Theresa C; Mackey, Zachary B; Fetter, Richard D; Choe, Youngchool; O'Donoghue, Anthony J; Zhou, Min; Craik, Charles S; Caffrey, Conor R; McKerrow, James H

    2008-10-24

    Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

  8. Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

    PubMed Central

    Grotzke, Jeff E.; Lu, Qiao; Cresswell, Peter

    2013-01-01

    Endoplasmic reticulum–associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome. The accumulation of misfolded proteins can be catastrophic at both the cellular and organismal level. Although the players involved and mechanistic details of ERAD are being characterized, much remains to be learned. Because of the complexity of the pathway, experimental studies generally require labor-intensive biochemical techniques. Here, we report the development of a system to analyze ERAD based on mutants of split or intact Venus fluorescent protein for which fluorescence depends on enzymatic deglycosylation. We have generated variants that only become fluorescent when they are first glycosylated in the ER and subsequently deglycosylated after retrotranslocation into the cytosol. The E3 ubiquitin ligase HMG-coA reductase degradation 1 homolog (Hrd1) and, consistent with the demonstrated glycosylation/deglycosylation requirement, the cytosolic deglycosylating enzyme peptide:N′glycanase are both required for fluorescence. Furthermore, although these deglycosylation-dependent fluorescent proteins are themselves ERAD substrates, they can also be fused to additional ERAD substrates to interrogate substrate-specific pathways. To validate the system we performed a genomewide siRNA screen that successfully identified known ERAD factors such as Hrd1; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP); sel-1 suppressor of lin-12-like (SEL1L); and p97. These tools should greatly facilitate the identification of ERAD components and investigation of the mechanisms involved in this critical pathway. PMID:23401531

  9. 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

    2016-07-22

    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.

  10. [Intracellular Protein Degradation in Growth of Atlantic Salmon, Salmo salar L].

    PubMed

    Lysenko, L A; Kantserova, N P; Krupnova, M Yu; Veselov, A E; Nemova, N N

    2015-01-01

    A brief review on the common characteristics and specific features of proteolytic machinery in fish skeletal muscles (based on Atlantic salmon, Salmo salar L., Salmonidae) has been given. Among a variety of proteases in the muscle tissue, those determining protein degradation level in developing and intensively growing muscles in salmon young and by this way regulating protein retention intensity and growth at all namely lysosomal cathepsins B and D and calcium-dependent proteases (calpains) were comprehensively studied. Revealed age-related differences in intracellular protease activity in salmon skeletal muscles indicate the role of proteolysis regulation in growth in general and a specific role of the individual proteolytic enzymes in particular. The data on negative correlation of cathepsin D and calpain activity levels in muscles and the rate of weight increase in juvenile salmon were obtained. A revealed positive correlation of cathepsin B activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover.

  11. Age-dependent degradation of the protein adsorption capacity of titanium.

    PubMed

    Hori, N; Att, W; Ueno, T; Sato, N; Yamada, M; Saruwatari, L; Suzuki, T; Ogawa, T

    2009-07-01

    Reported bone-implant contact percentages are far below the ideal 100%. We tested a hypothesis that the protein adsorption capability of titanium, which is critical to the process of osseointegration, changes over time before its use. Machined, acid-etched, and sandblasted surfaces were prepared and stored under dark ambient conditions for 3 days, 1 week, or 4 weeks. For all surfaces, protein adsorption decreased as the storage time increased, and their decreasing rates were dependent on titanium topography. After 4 weeks, the amounts of albumin and fibronectin adsorbed by the acid-etched surface were only 20% and 35%, respectively, of that adsorbed by the fresh surface after 2 hours of incubation, and remained substantially low even after 24 hours. This time-dependent degradation in protein adsorption of titanium correlated with its naturally decreasing hydrophilicity, which was not observed for the nickel and chromium surfaces, indicating a titanium-specific biological aging.

  12. 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

  13. Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation.

    PubMed

    Waites, Clarissa L; Leal-Ortiz, Sergio A; Okerlund, Nathan; Dalke, Hannah; Fejtova, Anna; Altrock, Wilko D; Gundelfinger, Eckart D; Garner, Craig C

    2013-04-01

    The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.

  14. 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

  15. 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

  16. Isolation and Quantitation of Adiponectin Higher Order Complexes

    PubMed Central

    Rutkowski, Joseph M.; Scherer, Philipp E.

    2014-01-01

    Adiponectin is a circulating bioactive hormone secreted by adipocytes as oligomers ranging in size from 90 kDa trimers and 180 kDa hexamers to larger high molecular weight oligomers that may reach 18- or 36-mers in size. While total circulating adiponectin levels correlate well with metabolic health, it is the relative distribution of adiponectin complexes that is most clinically relevant to glucose sensitivity and inflammation. High molecular weight adiponectin best mirrors insulin sensitivity, while trimeric adiponectin dominates with insulin resistance and adipose tissue inflammation. Experimental animal and in vitro models have also linked the relative fraction of high molecular weight adiponectin to its positive effects. Quantitating adiponectin size distribution thus provides a window into metabolic health and can serve as a surrogate marker for adipose tissue fitness. Here, we present a detailed protocol for isolating and quantitating adiponectin complexes in serum or plasma that has been extensively utilized for both human clinical samples and numerous animal models under various experimental conditions. Examples are presented of different adiponectin distributions and tips are provided for optimization using available equipment. Comparison of this rigorous approach to other available methods is also discussed. In total, this summary is a blueprint for the expanded quantitation and study of adiponectin complexes. PMID:24480350

  17. 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

  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. 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

  20. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy.

    PubMed

    Zhao, Jinghui; Zhai, Bo; Gygi, Steven P; Goldberg, Alfred Lewis

    2015-12-29

    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

  1. Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone

    PubMed Central

    Wadsworth, Caroline; Buckley, Mike

    2014-01-01

    RATIONALE We report the use of proteomics techniques to study how the fossil bone proteome changes in complexity over one million years. METHODS We include the attempted use of a previously unreported methodology in proteome research, to remove the dominant bone collagens using bacterial collagenase as well as conventional shotgun proteomics methodology following digestion with the protease trypsin. In this study we expand upon a set of 19 bovine sub-fossil specimens ranging over one and a half million years that had previously been shown to possess collagen, using a total of 46 LTQ-Orbitrap liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses containing 462,186 precursor ion analyses. RESULTS Although many types of proteins can typically be identified in recent bone, in degraded bone we observe a rapid loss of lower abundance proteins. Abundant serum proteins such as serum albumin and alpha-2-HS-glycoprotein appear to be more easily recovered in ancient bone, both being identified in specimens dating to the Early Pleistocene, the earliest period tested in this study. Proteins belonging to the leucine-rich repeat family such as lumican, biglycan and chondroadherin also survive well, possibly because of their interactions with bone collagen. CONCLUSIONS Of these 'survivor proteins' A2HSG shows a remarkable amount of sequence variation, making it potentially one of the most useful proteins to study for species identification and phylogenetic inference in archaeological and palaeontological bone. PMID:24519823

  2. 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.

  3. Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability

    PubMed Central

    Stas, Jelle; Nguyen, Nhu H.; Kuyper, Thomas W.; Carleer, Robert; Vangronsveld, Jaco; Colpaert, Jan V.; Kennedy, Peter G.

    2015-01-01

    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

  4. 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

  5. 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

  6. Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction.

    PubMed

    Sugimoto, Shinya; Iwamoto, Takeo; Takada, Koji; Okuda, Ken-Ichi; Tajima, Akiko; Iwase, Tadayuki; Mizunoe, Yoshimitsu

    2013-04-01

    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 (Esp(S235A)) 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.

  7. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    PubMed Central

    Kanazawa, Ippei; Yamaguchi, Toru; Yano, Shozo; Yamauchi, Mika; Yamamoto, Masahiro; Sugimoto, Toshitsugu

    2007-01-01

    Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. PMID:18047638

  8. 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

  9. 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.

  10. The effect of electrical stimulation on post mortem myofibrillar protein degradation and small heat shock protein kinetics in bull beef.

    PubMed

    Contreras-Castillo, C J; Lomiwes, D; Wu, G; Frost, D; Farouk, M M

    2016-03-01

    This study aimed to determine the effect of electrical stimulation and ultimate pH (pHu) on shear force, myofibrillar protein degradation and small heat shock protein (sHSP) concentrations in M. longissimus lumborum (LL). The LL from both sides of carcasses (n=15) was excised with low voltage electrical stimulation (ES) applied to an LL muscle from one side, while the opposing LL muscle was not stimulated (NS). Muscles were categorised into low (pHu<5.8), intermediate (5.8≤pHu<6.2) and high pHu (pHu≥6.2) and aged for up to 28days post mortem at -1.5°C. High pHu meat tenderised faster which corresponded with the faster degradation of titin and desmin in this group compared with low and intermediate pHu meat. Electrical stimulation significantly affected the variable levels of αβ-crystallin and HSP20 with higher concentrations of these sHSP in ES muscles at later ageing timepoints compared with NS muscles.

  11. Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

    PubMed Central

    Rothbarth, Karsten; Stammer, Hermann; Werner, Dieter

    2002-01-01

    The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells. PMID:12379155

  12. Back to the heart: the protective role of adiponectin.

    PubMed

    Caselli, C; D'Amico, A; Cabiati, M; Prescimone, T; Del Ry, S; Giannessi, D

    2014-04-01

    Cardiovascular disease (CVD) is the leading cause of death worldwide and the prevalence of obesity and diabetes are increasing. In obesity, adipose tissue increases the secretion of bioactive mediators (adipokines) that may represent a key mechanism linking obesity to CVD. Adiponectin, extensively studied in metabolic diseases, exerts anti-diabetic, anti-atherogenic and anti-inflammatory activities. Due to these positive actions, the role of adiponectin in cardiovascular protection has been evaluated in recent years. In particular, for its potential therapeutic benefits in humans, adiponectin has become the subject of intense preclinical research. In the cardiovascular context, understanding of the cellular and molecular mechanisms underlying the adiponectin system, throughout its secretion, regulation and signaling, is critical for designing new drugs that target adiponectin system molecules. This review focused on recent advances regarding molecular mechanisms related to protective effects of the adiponectin system on both cardiac and vascular compartments and its potential use as a target for therapeutic intervention of CVD.

  13. Mitochondrial Quality Control in the Myocardium: Cooperation between Protein Degradation and Mitophagy

    PubMed Central

    Hammerling, Babette C.; Gustafsson, Åsa B.

    2014-01-01

    Mitochondria are critical for cardiomyocyte survival and maintenance of normal cardiac function. However, changes in the extra- or intracellular environments during stress can cause excessive damage to mitochondria and lead to activation of cell death. In fact, there is evidence that mitochondrial dysfunction is an important contributor to both development of heart failure and the aging process. To counteract the adverse effects resulting from mitochondrial damage, cells have evolved mitochondrial quality control pathways that act at both the protein and organelle levels. Quality control of proteins in the outer mitochondrial membrane is monitored by the ubiquitin-protease system, whereas chaperons and proteases act in the various compartments of the mitochondria. When the damage is too excessive and the degradation machinery is overwhelmed, the entire mitochondrion is eliminated by an autophagosome. Together, these pathways ensure that myocytes maintain a functional network of mitochondria which provides ATP for contraction. Unfortunately, chronic stress and aging can negatively affects proteins that are involved in the mitochondrial quality control pathways which leads to accumulation of dysfunctional mitochondria and loss of myocytes. In this review, we provide an overview of the proteins and pathways that regulate mitochondrial quality control in the cell with an emphasis on pathways involved in maintaining protein and organelle homeostasis. We also delve into the effects of reduced mitochondrial quality control on aging and cardiovascular disease. PMID:25086292

  14. 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.

  15. Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation.

    PubMed

    Villar-Piqué, Anna; Ventura, Salvador

    2013-12-01

    Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid β peptide (Aβ42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these Aβ42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.

  16. 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

  17. ORMDL orosomucoid-like proteins are degraded by free-cholesterol-loading-induced autophagy.

    PubMed

    Wang, Shuhui; Robinet, Peggy; Smith, Jonathan D; Gulshan, Kailash

    2015-03-24

    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

  18. Globular adiponectin enhances invasion in human breast cancer cells

    PubMed Central

    FALK LIBBY, EMILY; LIU, JIANZHONG; LI, YI; LEWIS, MONICA J.; DEMARK-WAHNEFRIED, WENDY; HURST, DOUGLAS R.

    2016-01-01

    Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer

  19. 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

  20. The key to unlock the Hsp100/Clp protein degradation machines of Mycobacterium.

    PubMed

    Molière, Noël; Turgay, Kürşad

    2014-08-01

    Hsp100/Clp protease complexes are molecular machines important for cellular protein homeostasis and are concurrently embedded in the control of various signal transduction networks by regulatory proteolysis. In Mycobacteria, the genes encoding the components of these Hsp100/Clp protease complexes are essential for growth and were identified as targets for antibiotics, with a new antimicrobial mechanism, that are active on slow growing or even dormant cells. Schmitz and Sauer (2014) report the biochemical characterization of mycobacterial Hsp100/Clp protease complexes actively degrading folded substrate proteins. Their results suggest an unusual activation mechanism for this protease complex and will set the stage for further mechanistic studies of antibiotics acting on this new cellular target.

  1. 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.

  2. 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

  3. Adiponectin as a potential biomarker of vascular disease

    PubMed Central

    Ebrahimi-Mamaeghani, Mehrangiz; Mohammadi, Somayeh; Arefhosseini, Seyed Rafie; Fallah, Parviz; Bazi, Zahra

    2015-01-01

    The increasing prevalence of diabetes and its complications heralds an alarming situation worldwide. Obesity-associated changes in circulating adiponectin concentrations have the capacity to predict insulin sensitivity and are a link between obesity and a number of vascular diseases. One obvious consequence of obesity is a decrease in circulating levels of adiponectin, which are associated with cardiovascular disorders and associated vascular comorbidities. Human and animal studies have demonstrated decreased adiponectin to be an independent risk factor for cardiovascular disease. However, in animal studies, increased circulating adiponectin alleviates obesity-induced endothelial dysfunction and hypertension, and also prevents atherosclerosis, myocardial infarction, and diabetic cardiac tissue disorders. Further, metabolism of a number of foods and medications are affected by induction of adiponectin. Adiponectin has beneficial effects on cardiovascular cells via its antidiabetic, anti-inflammatory, antioxidant, antiapoptotic, antiatherogenic, vasodilatory, and antithrombotic activity, and consequently has a favorable effect on cardiac and vascular health. Understanding the molecular mechanisms underlying the regulation of adiponectin secretion and signaling is critical for designing new therapeutic strategies. This review summarizes the recent evidence for the physiological role and clinical significance of adiponectin in vascular health, identification of the receptor and post-receptor signaling events related to the protective effects of the adiponectin system on vascular compartments, and its potential use as a target for therapeutic intervention in vascular disease. PMID:25653535

  4. Dietary protein level and ruminal degradability for mohair production in Angora goats.

    PubMed

    Sahlu, T; Fernandez, J M; Lu, C D; Manning, R

    1992-05-01

    Twenty-eight Angora goat doelings (average BW 22.1 kg) were used in a 150-d study to examine the effects of dietary CP level and degradability on mohair fiber production. A 2 x 2 factorial arrangement was instituted using conventional, solvent-extracted soybean meal (high degradability) or expelled, heat-treated soybean meal (low degradability) incorporated into low- (12%) or high- (19%) CP diets. Grease and clean mohair weights were greater (P less than .05) in goats fed the diets containing 19% CP. Mohair fiber diameter was not affected (P greater than .10) by dietary CP level. Clean mohair weight tended (P less than .08) to be higher in the goats fed diets containing expelled, heat-treated soybean meal. Body weight gains were not affected (P greater than .10) by CP level or degradability, whereas DMI increased (P less than .01) with increasing CP level. Ruminal fluid pH and total VFA concentrations were not affected (P greater than .10) by diet. Ruminal ammonia N concentration increased (P less than .05) as CP level in the diet increased, and postprandial changes in concentrations were less noticeable in the group fed expelled, heat-treated soybean meal. Plasma urea N (P less than .001) and total protein (P less than .01) concentration increased as dietary CP level increased. Plasma glucose was elevated (P less than .001) 2 h after feeding in the goats fed conventional, solvent-extracted soybean meal, whereas glucagon concentrations were greater at 0 and 4 h in the group fed expelled, heat-treated soybean meal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1526921

  5. 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

  6. Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation.

    PubMed

    Nighot, Prashant K; Hu, Chien-An Andy; Ma, Thomas Y

    2015-03-13

    Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.

  7. Autophagy Enhances Intestinal Epithelial Tight Junction Barrier Function by Targeting Claudin-2 Protein Degradation*

    PubMed Central

    Nighot, Prashant K.; Hu, Chien-An Andy; Ma, Thomas Y.

    2015-01-01

    Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2. PMID:25616664

  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. Effect of degradable intake protein level on finishing cattle performance and ruminal metabolism.

    PubMed

    Shain, D H; Stock, R A; Klopfenstein, T J; Herold, D W

    1998-01-01

    Two finishing trials and a metabolism trial were conducted to evaluate level of supplemental degradable intake (crude) protein (DIP) in finishing diets on cattle performance, carcass characteristics, and ruminal metabolism. Finishing trials were conducted in two consecutive years using 128 crossbred yearling steers (BW = 343+/-5 kg, Trial 1) and 176 crossbred yearling steers (BW = 375+/-4 kg, Trial 2) in a randomized complete block design. Steers were fed dry-rolled corn diets containing urea at 0, .88, 1.34, or 1.96% (DM basis). No differences in DMI, daily gain, or feed efficiency were noted among steers receiving diets containing supplemental urea. However, steers fed diets supplemented with urea were 5.4% more efficient (P < .01) and gained 6.6% faster (P < .01) than steers receiving no supplemental urea. Metabolizable protein (MP) content of all diets exceeded the steers' requirements. However, diets containing no urea were deficient in DIP. In the metabolism trial, four ruminally fistulated steers (BW = 380+/-22 kg) were used in a 4 x 4 Latin square design and fed (ad libitum) diets similar to those used in the finishing trials. Nitrogen intake and ruminal ammonia N concentration increased linearly (P < .05) with increasing level of urea supplementation. Diets containing no supplemental urea were calculated to be deficient in DIP, resulting in reduced bacterial synthesis. Results indicate that dry-rolled corn finishing diets containing no supplemental N are deficient in ruminally degradable N. Supplementing these diets with an inexpensive source of ruminally degradable N improved animal performance. However, supplementation with urea above .88% was not beneficial. PMID:9464905

  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. 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.

  12. Peroxisome Proliferator-Activated Receptor α Reduces Endothelin-1-Caused Cardiomyocyte Hypertrophy by Inhibiting Nuclear Factor-κB and Adiponectin

    PubMed Central

    Jen, Hsu-Lung; Liu, Po-Len; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a role in the pathogenesis of cardiac hypertrophy, although its underlying mechanism remains unclear. The purpose of this study was to evaluate the effect of PPARα activation on endothelin-1- (ET-1-) caused cardiomyocyte hypertrophy and explore its underlying mechanisms. Human cardiomyocytes (HCMs) were cultured with or without ET-1, whereafter the inhibitory effects of fenofibrate, a PPARα activator, on cell size and adiponectin protein were tested. We examined the activation of extracellular signal-regulated kinase (ERK) and p38 proteins caused by ET-1 and the inhibition of the ERK and p38 pathways on ET-1-induced cell size and adiponectin expression. Moreover, we investigated the interaction of PPARα with adiponectin and nuclear factor-κB (NF-κB) by electrophoretic mobility shift assays and coimmunoprecipitation. ET-1 treatment significantly increased cell size, suppressed PPARα expression, and enhanced the expression of adiponectin. Pretreatment with fenofibrate inhibited the increase in cell size and enhancement of adiponectin expression. ET-1 significantly activated the ERK and p38 pathways, whereas PD98059 and SB205380, respectively, inhibited them. Our results suggest that activated PPARα can decrease activation of adiponectin and NF-κB and inhibit ET-1-induced cardiomyocyte hypertrophy. PMID:27807394

  13. Adiponectin Impairs Chicken Preadipocytes Differentiation through p38 MAPK/ATF-2 and TOR/p70 S6 Kinase Pathways

    PubMed Central

    Yan, Jun; Gan, Lu; Chen, Di; Sun, Chao

    2013-01-01

    Adiponectin is a protein hormone secreted exclusively by adipocytes that plays an important role in the modulation of glucose and lipid metabolism. In the present study, we investigated the ability of adiponectin to stimulate chicken preadipocyte differentiation and its effect on cellular signaling pathways associated with adipocyte differentiation. Data showed that over-expression of adiponectin inhibited adipocyte differentiation and the expression of adipogenic marker gene, while activated the expression of lipolytic marker gene. Meanwhile, adiponectin led to activation of p38 mitogen-activated protein kinase (p38 MAPK)/activating transcription factor 2 (ATF-2) signaling pathway and down-regulation of target of rapamycin (TOR)/p70 S6 Kinase signaling pathway. Furthermore, the activation of p38 MAPK/ATF-2 signaling pathway was blocked by the p38 MAPK inhibitor SB253580, whereas adiponectin had a synergistic effect on the suppression of TOR/p70 S6 Kinase signaling pathway with the TOR inhibitor rapamycin. In conclusion, the results demonstrate the ability of adiponectin to inhibit chicken preadipocyte differentiation, which depends on the p38 MAPK/ATF-2 and TOR/p70 S6 Kinase pathways. PMID:24194895

  14. 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.

  15. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  16. A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

    PubMed Central

    Sung, Min-Kyung; Porras-Yakushi, Tanya R; Reitsma, Justin M; Huber, Ferdinand M; Sweredoski, Michael J; Hoelz, André; Hess, Sonja; Deshaies, Raymond J

    2016-01-01

    Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001 PMID:27552055

  17. A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins.

    PubMed

    Sung, Min-Kyung; Porras-Yakushi, Tanya R; Reitsma, Justin M; Huber, Ferdinand M; Sweredoski, Michael J; Hoelz, André; Hess, Sonja; Deshaies, Raymond J

    2016-01-01

    Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. PMID:27552055

  18. 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.

  19. Bacterial proteasome and PafA, the pup ligase, interact to form a modular protein tagging and degradation machine.

    PubMed

    Forer, Nadav; Korman, Maayan; Elharar, Yifat; Vishkautzan, Marina; Gur, Eyal

    2013-12-17

    Proteasome-containing bacteria possess a tagging system that directs proteins to proteasomal degradation by conjugating them to a prokaryotic ubiquitin-like protein (Pup). A single ligating enzyme, PafA, is responsible for Pup conjugation to lysine side chains of protein substrates. As Pup is recognized by the regulatory subunit of the proteasome, Pup functions as a degradation tag. Pup presents overlapping regions for binding of the proteasome and PafA. It was, therefore, unclear whether Pup binding by the proteasome regulatory subunit, Mpa, and by PafA are mutually exclusive events. The work presented here provides evidence for the simultaneous interaction of Pup with both Mpa and PafA. Surprisingly, we found that PafA and Mpa can form a complex both in vitro and in vivo. Our results thus suggest that PafA and the proteasome can function as a modular machine for the tagging and degradation of cytoplasmic proteins. PMID:24228735

  20. Metabolomic profiling in liver of adiponectin-knockout mice uncovers lysophospholipid metabolism as an important target of adiponectin action

    PubMed Central

    Liu, Ying; Sen, Sanjana; Wannaiampikul, Sivaporn; Palanivel, Rengasamy; Hoo, Ruby L. C.; Isserlin, Ruth; Bader, Gary D.; Tungtrongchitr, Rungsunn; Deshaies, Yves; Xu, Aimin; Sweeney, Gary

    2016-01-01

    Adiponectin mediates anti-diabetic effects via increasing hepatic insulin sensitivity and direct metabolic effects. In the present study, we conducted a comprehensive and unbiased metabolomic profiling of liver tissue from AdKO (adiponectin-knockout) mice, with and without adiponectin supplementation, fed on an HFD (high-fat diet) to derive insight into the mechanisms and consequences of insulin resistance. Hepatic lipid accumulation and insulin resistance induced by the HFD were reduced by adiponectin. The HFD significantly altered levels of 147 metabolites, and bioinformatic analysis indicated that one of the most striking changes was the profile of increased lysophospholipids. These changes were largely corrected by adiponectin, at least in part via direct regulation of PLA2 (phospholipase A2) as palmitate-induced PLA2 activation was attenuated by adiponectin in primary hepatocytes. Notable decreases in several glycerolipids after the HFD were reversed by adiponectin, which also corrected elevations in several diacyglycerol and ceramide species. Our data also indicate that stimulation of ω-oxidation of fatty acids by the HFD is enhanced by adiponectin. In conclusion, this metabolomic profiling approach in AdKO mice identified important targets of adiponectin action, including PLA2, to regulate lysophospholipid metabolism and ω-oxidation of fatty acids. PMID:25915851

  1. The non-catalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation.

    PubMed

    Vaaje-Kolstad, Gustav; Horn, Svein J; van Aalten, Daan M F; Synstad, Bjørnar; Eijsink, Vincent G H

    2005-08-01

    The Gram-negative soil bacterium Serratia marcescens uses three different family 18 chitinases to degrade chitin, an abundant insoluble carbohydrate polymer composed of beta(1,4)-linked units of N-acetylglucosamine. We show that efficient chitin degradation additionally depends on the action of a small non-catalytic protein, CBP21, which binds to the insoluble crystalline substrate, leading to structural changes in the substrate and increased substrate accessibility. CBP21 strongly promoted hydrolysis of crystalline beta-chitin by chitinases A and C, while it was essential for full degradation by chitinase B. CBP21 variants with single mutations on the largely polar binding surface lost their ability to promote chitin degradation, while retaining considerable affinity for the polymer. Thus, binding alone is not sufficient for CBP21 functionality, which seems to depend on specific, mostly polar interactions between the protein and crystalline chitin. This is the first time a secreted binding protein is shown to assist in the enzymatic degradation of an insoluble carbohydrate via non-hydrolytic disruption of the substrate. Interestingly, homologues of CBP21 occur in most chitin-degrading microorganisms, suggesting a general mechanism by which chitin-binding proteins enhance chitinolytic activity. Homologues also occur in chitinase-containing insect viruses, whose infectiousness is known to depend on chitinase efficiency.

  2. 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

  3. 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.

  4. Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response.

    PubMed

    van Stijn, Caroline M W; Kim, Jason; Lusis, Aldons J; Barish, Grant D; Tangirala, Rajendra K

    2015-02-01

    Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40-60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 µg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-α, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by up-regulating liver X receptor-α (LXRα) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.

  5. 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

  6. 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.

  7. 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.

  8. Quality control autophagy degrades soluble ERAD-resistant conformers of the misfolded membrane protein GnRHR

    PubMed Central

    Houck, Scott A.; Ren, Hong Yu; Madden, Victoria J.; Bonner, Jaclyn N.; Conlin, Michael P.; Janovick, Jo Ann; Conn, P. Michael; Cyr, Douglas M.

    2014-01-01

    Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER) associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of GnRHR, a G-protein coupled receptor, between ER-associated degradation (ERAD) and a novel ERQC-autophagy pathway for membrane proteins. ERQC-autophagy degrades E90K-GnRHR because pools of its partially folded and detergent soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ERassociated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC-autophagy. PMID:24685158

  9. Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo?

    PubMed

    Salvat, C; Aquaviva, C; Jariel-Encontre, I; Ferrara, P; Pariat, M; Steff, A M; Carillo, S; Piechaczyk, M

    1999-04-01

    The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanisms responsible for the selective destruction of proteins within cells.

  10. 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

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

    PubMed

    Toshima, Kazunobu

    2013-05-01

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

  12. Effect of ethephon on protein degradation and the accumulation of pathogensis-related (PR) proteins in tomato leaf discs. [Lycopersicon esculentum

    SciTech Connect

    Vera, P.; Conejero, V. )

    1990-01-01

    The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69. In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other {sup 35}S-labeled proteins as well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.

  13. 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.

  14. DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation.

    PubMed

    Collado, Denise; Yoshihara, Takashi; Hamaguchi, Masaaki

    2007-08-31

    Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.

  15. 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...

  16. 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

  17. 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

  18. 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.

  19. Extralysosomal turnover of cellular proteins: Targeting substrates in the ubiquitin, ATP-dependent degradation system

    SciTech Connect

    Marriott, D.

    1988-01-01

    Calmodulin derived from a cloned chicken gene can be ubiquitinated and degraded by an in vitro reticulocyte lysate system. The chemical reactivity and the surface accessibility of the {epsilon}-amino group on lysine 115 in the calmodulin polypeptide chain were studied by trace labeling with acetic anhydride and with a ubiquitin derivative containing an azido group at the C-terminal glycine residue. Fractionation of reticulocyte lysate proteins separated the activity which degrades the calmodulin moiety of ubiquitin-calmodulin conjugates from that which acts on the isopeptide linkage. Neither of these two activities act on a synthetic isopeptide, which mimics the junction of ubiquitin-calmodulin, indicating the importance of the folding of ubiquitin for recognition. Based on recent findings that the ubiquitin moieties linked to {beta}galactosidase exist as a single multiubiquitin chain, studies were carried out to determine the structure of the ubiquitin-ubiquitin linkage. Ubiquitin was in vivo labeled with ({sup 3}H) and conjugated to {beta}galactosidase. Individual conjugates were isolated and subjected to peptide mapping by trypsin digestion, and tryptic fragments were analyzed of HPLC. The results indicated that the ubiquitin-ubiquitin linkage involves lysine residue 48 in the ubiquitin sequence.

  20. 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.

  1. The HIV-1 Protein Vpr Targets the Endoribonuclease Dicer for Proteasomal Degradation to Boost Macrophage Infection

    PubMed Central

    Klockow, Laurieann Casey; Sharifi, Hamayun J.; Wen, Xiaoyun; Flagg, Meg; Furuya, Andrea K. M.; Nekorchuk, Michael; de Noronha, Carlos M.

    2013-01-01

    The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4DCAF1 ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4DCAF1. Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4DCAF1. We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4DCAF1 ubiquitin ligase complex and its subsequent degradation. PMID:23849790

  2. 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.

  3. 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

  4. A role of protein degradation in memory consolidation after initial learning and extinction learning in the honeybee (Apis mellifera).

    PubMed

    Felsenberg, Johannes; Dombrowski, Vincent; Eisenhardt, Dorothea

    2012-09-17

    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 applied after initial learning. This result supports the idea that MG132 targets an ongoing consolidation process. Furthermore, we demonstrate that the sensitivity of memory formation after initial learning and extinction learning to MG132 depends in the same way on the number of CS-US trials and the intertrial interval applied during initial learning. This supports the idea that the learning parameters during acquisition are critical for memory formation after extinction and that protein degradation in both learning processes might be functionally linked.

  5. Effects of different fatty acids and dietary lipids on adiponectin gene expression in 3T3-L1 cells and C57BL/6J mice adipose tissue.

    PubMed

    Bueno, Allain Amador; Oyama, Lila Missae; de Oliveira, Cristiane; Pisani, Luciana Pelegrini; Ribeiro, Eliane Beraldi; Silveira, Vera Lucia Flor; Oller do Nascimento, Cláudia Maria

    2008-01-01

    Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis.

  6. 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

  7. Identification and characterization of CTRP9, a novel secreted glycoprotein, from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin.

    PubMed

    Wong, G William; Krawczyk, Sarah A; Kitidis-Mitrokostas, Claire; Ge, Guangtao; Spooner, Eric; Hug, Christopher; Gimeno, Ruth; Lodish, Harvey F

    2009-01-01

    Adiponectin is a major insulin-sensitizing, multimeric hormone derived from adipose tissue that acts on muscle and liver to regulate whole-body glucose and lipid metabolism. Here, we describe a novel and highly conserved paralog of adiponectin designated as C1q/TNF-related protein (CTRP) 9. Of all the CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and females expresses higher levels of the transcript than males. Moreover, its expression levels in ob/ob mice changed in an age-dependent manner, with significant up-regulation in younger mice. CTRP9 is a secreted glycoprotein with multiple post-translational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum with levels varying according to sex and metabolic state of mice. Furthermore, CTRP9 and adiponectin can be secreted as heterooligomers when cotransfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally coimmunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, we show that adiponectin and CTRP9 form heterotrimers. In cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. Adenovirus-mediated overexpression of CTRP9 in obese (ob/ob) mice significantly lowered serum glucose levels. Collectively, these results suggest that CTRP9 is a novel adipokine, and further study of CTRP9 will yield novel mechanistic insights into its physiological and metabolic function.

  8. 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

  9. 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.

  10. 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.

  11. Physical exercise-induced hippocampal neurogenesis and antidepressant effects are mediated by the adipocyte hormone adiponectin.

    PubMed

    Yau, Suk Yu; Li, Ang; Hoo, Ruby L C; Ching, Yick Pang; Christie, Brian R; Lee, Tatia M C; Xu, Aimin; So, Kwok-Fai

    2014-11-01

    Adiponectin (ADN) is an adipocyte-secreted protein with insulin-sensitizing, antidiabetic, antiinflammatory, and antiatherogenic properties. Evidence is also accumulating that ADN has neuroprotective activities, yet the underlying mechanism remains elusive. Here we show that ADN could pass through the blood-brain barrier, and elevating its levels in the brain increased cell proliferation and decreased depression-like behaviors. ADN deficiency did not reduce the basal hippocampal neurogenesis or neuronal differentiation but diminished the effectiveness of exercise in increasing hippocampal neurogenesis. Furthermore, exercise-induced reduction in depression-like behaviors was abrogated in ADN-deficient mice, and this impairment in ADN-deficient mice was accompanied by defective running-induced phosphorylation of AMP-activated protein kinase (AMPK) in the hippocampal tissue. In vitro analyses indicated that ADN itself could increase cell proliferation of both hippocampal progenitor cells and Neuro2a neuroblastoma cells. The neurogenic effects of ADN were mediated by the ADN receptor 1 (ADNR1), because siRNA targeting ADNR1, but not ADNR2, inhibited the capacity of ADN to enhance cell proliferation. These data suggest that adiponectin may play a significant role in mediating the effects of exercise on hippocampal neurogenesis and depression, possibly by activation of the ADNR1/AMPK signaling pathways, and also raise the possibility that adiponectin and its agonists may represent a promising therapeutic treatment for depression.

  12. 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

  13. Niacin stimulates adiponectin secretion through the GPR109A receptor.

    PubMed

    Plaisance, Eric P; Lukasova, Martina; Offermanns, Stefan; Zhang, Youyan; Cao, Guoqing; Judd, Robert L

    2009-03-01

    Niacin (nicotinic acid) has recently been shown to increase serum adiponectin concentrations in men with the metabolic syndrome. However, little is known about the mechanism(s) by which niacin regulates the intracellular trafficking and secretion of adiponectin. Since niacin appears to exert its effects on lipolysis through receptor (GPR109A)-dependent and -independent pathways, the purpose of this investigation was to examine the role of the recently identified GPR109A receptor in adiponectin secretion. Initial in vivo studies in rats demonstrated that niacin (30 mg/kg po) acutely increases serum adiponectin concentrations, whereas it decreases NEFAs. Further in vitro studies demonstrated an increase in adiponectin secretion and a decrease in lipolysis in primary adipocytes following treatment with niacin or beta-hydroxybutyrate (an endogenous ligand of the GPR109A receptor), but these effects were blocked when adipocytes were pretreated with pertussis toxin. Niacin had no effect on adiponectin secretion or lipolysis in 3T3-L1 adipocytes, which have limited cell surface expression of the GPR109A receptor. To further substantiate these in vitro findings, wild-type and GPR109A receptor knockout mice were administered a single dose of niacin or placebo, and serum was obtained for the determination of adiponectin and NEFA concentrations. Serum adiponectin concentrations increased and serum NEFAs decreased in the wild-type mice within 10 min following niacin administration. However, niacin administration had no effect on adiponectin and NEFA concentrations in the GPR109A receptor knockout mice. These results demonstrate that the GPR109A receptor plays an important role in the dual regulation of adiponectin secretion and lipolysis.

  14. 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

  15. 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

  16. Construction of a green fluorescent protein (GFP)-marked multifunctional pesticide-degrading bacterium for simultaneous degradation of organophosphates and γ-hexachlorocyclohexane.

    PubMed

    Yang, Chao; Liu, Ruihua; Yuan, Yulan; Liu, Jianli; Cao, Xiangyu; Qiao, Chuanling; Song, Cunjiang

    2013-02-13

    An autofluorescent whole-cell biocatalyst capable of simultaneously degrading organophosphates (OPs) and γ-hexachlorocyclohexane (γ-HCH) was constructed by display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion on the cell surface of a γ-HCH-degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-OPH-GFP fusion was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibits cell growth nor affects cell viability. In sterile and nonsterile soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days when inoculated with the engineered UT26, and the strain could be easily monitored by fluorescence during bioremediation. These results indicate that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of environments contaminated with multiple pesticides.

  17. 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

  18. The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.

    PubMed

    Sendersky, Eleonora; Kozer, Noga; Levi, Mali; Garini, Yuval; Shav-Tal, Yaron; Schwarz, Rakefet

    2014-07-01

    Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

  19. 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.

  20. Disturbance of proteasomal and autophagic protein degradation pathways by amyotrophic lateral sclerosis-linked mutations in ubiquilin 2.

    PubMed

    Osaka, Mayuko; Ito, Daisuke; Suzuki, Norihiro

    2016-04-01

    Ubiquilin (UBQLN), a member of the ubiquitin-like (UBL)-ubiquitin-associated (UBA) family, is a dual regulator of both the proteasomal and autophagic branches of the cellular protein degradation system. Mutations in the UBQLN2 gene encoding ubiquilin 2 cause X-linked amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), and UBQLN2-positive inclusions have been identified in ALS patients with UBQLN2 mutations as well as in cases of both familial and sporadic ALS without UBQLN2 mutations. Compelling evidence links UBQLN2 to disturbance of the protein quality control network in neurons, but the pathomechanisms remain obscure. This study aimed to clarify how ALS-linked mutations in UBQLN2 affect the protein degradation system. Overexpression of a UBQLN2 with ALS-associated mutations resulted in the accumulation of polyubiquitinated proteins in neuronal cells, including the ALS-associated protein TDP-43. This effect was dependent on the UBA domain but not on inclusion formation. Immunocytochemistry and protein fractionation analysis of IVm-UBQLN2 cellular distribution indicated that it sequesters ubiquitinated substrates from both the proteasomal and autophagic branches of the protein degradation system, resulting in accumulation of polyubiquitinated substrates. These findings provide a molecular basis for the development of ALS/FTD-associated proteinopathy and establish novel therapeutic targets for ALS.

  1. 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

  2. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  3. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

    PubMed

    Belogurov, Alexey; Kuzina, Ekaterina; Kudriaeva, Anna; Kononikhin, Alexey; Kovalchuk, Sergey; Surina, Yelena; Smirnov, Ivan; Lomakin, Yakov; Bacheva, Anna; Stepanov, Alexey; Karpova, Yaroslava; Lyupina, Yulia; Kharybin, Oleg; Melamed, Dobroslav; Ponomarenko, Natalia; Sharova, Natalia; Nikolaev, Eugene; Gabibov, Alexander

    2015-05-01

    Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and β1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived β1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

  4. The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications.

    PubMed

    Burger, Angelika M; Seth, Arun K

    2004-10-01

    The highly conserved eukaryotic ubiquitin-proteasome system (UP-S) plays a pivotal role in protein homeostasis and is critical in regulating normal and cancer-related cellular processes. The hierarchical nature of the UP-S provides a rich source of molecular targets for specific intervention and has therefore arisen as a promising approach to innovative anticancer therapies. The first in class proteasome inhibitory agent Bortezomib (Velcade) has recently obtained regulatory approval for the treatment of multiple myeloma. Ubiquitin-mediated degradation is a complex process that is comprised of well defined steps involving ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Although a single E1 activates the ubiquitin conjugation machinery, a large number of E2 conjugating enzymes and E3 ligases are now known to exist. Proteins tagged with ubiquitin are subsequently recognised by the proteasome for digestion and fragmentation. The enzymatic nature, multitude of E3s and their specific substrate recognition predestines them as therapeutic targets. This article will review known inhibitors of the proteasome and their molecular mechanisms as well as ongoing developments and promising avenues for targeting substrate-specific E3 ligases that are likely to yield a new class of therapeutics that will serve and complement the armamentarium of anticancer drugs. PMID:15454246

  5. Similar Adiponectin Levels in Obese Normotensive and Obese Hypertensive Men and No Vasorelaxant Effect of Adiponectin on Human Arteries.

    PubMed

    Dreier, Rasmus; Asferg, Camilla; Berg, Jais O; Andersen, Ulrik B; Flyvbjerg, Allan; Frystyk, Jan; Linneberg, Allan; Jeppesen, Jørgen L; Edvinsson, Lars; Skovsted, Gry F

    2016-02-01

    Obesity is a strong risk factor for hypertension, but the mechanism linking obesity to hypertension is not fully elucidated. In obesity, circulating concentrations of adiponectin are decreased and hypoadiponectinaemia has in some but not all studies been associated with increased risk of hypertension. Due to this inconsistency, we decided to study adiponectin from two aspects in a cross-sectional in vivo study and in an experimental in vitro study. In the cross-sectional study, 103 men with body mass index (BMI) ≥ 30.0 kg/m(2) were studied; 63 had 24-hr ambulatory blood pressure (ABP) ≥ 130/80 mmHg (ObeseHT) and 40 had 24-hr ABP < 130/80 mmHg (ObeseNT). As controls, we studied 27 men with BMI between 20.0 and 24.9 kg/m(2) and 24-hr ABP < 130/80 mmHg (LeanNT). Serum concentrations of adiponectin and body composition using dual-energy X-ray absorptiometry scanning were determined. In vitro, the direct vasomotor response of adiponectin was tested on subcutaneous resistance arteries from human abdominal adipose tissue. The two obese groups had lower adiponectin concentrations compared with LeanNT (p < 0.01) [median (interquartile range)]: ObeseHT 6.5 (5.1-8.3) mg/L; ObeseNT 6.6 (5.2-7.8) mg/L; and LeanNT 9.4 (6.7-12.4) mg/L, with no significant difference in adiponectin concentrations (or body composition) between ObeseHT and ObeseNT (p = 0.67). In vitro, adiponectin did not have any direct vasodilatory effect and adiponectin did not affect angiotensin II-stimulated vasoconstriction. In conclusion, obese hypertensive men have similar serum concentrations of adiponectin as obese normotensive men. In combination with the in vitro data, these findings question a pathogenic role of adiponectin in human hypertension.

  6. Autophagy and SQSTM1 on the RHOA(d) again: emerging roles of autophagy in the degradation of signaling proteins.

    PubMed

    Belaid, Amine; Ndiaye, Papa Diogop; Cerezo, Michaël; Cailleteau, Laurence; Brest, Patrick; Klionsky, Daniel J; Carle, Georges F; Hofman, Paul; Mograbi, Baharia

    2014-02-01

    Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth, its survival, and its motility. Emerging evidence suggests that autophagy limits several signaling pathways by degrading kinases, downstream components, and transcription factors; however, this often occurs under stressful conditions. Our recent studies revealed that constitutive autophagy temporally and spatially controls the RHOA pathway. Specifically, inhibition of autophagosome degradation induces the accumulation of the GTP-bound form of RHOA. The active RHOA is sequestered via SQSTM1/p62 within autolysosomes, and accordingly fails to localize to the spindle midbody or to the cell surface, as we demonstrate herein. As a result, all RHOA-downstream responses are deregulated, thus driving cytokinesis failure, aneuploidy and motility, three processes that directly have an impact upon cancer progression. We therefore propose that autophagy acts as a degradative brake for RHOA signaling and thereby controls cell proliferation, migration, and genome stability.

  7. Effect of ruminal ammonia-nitrogen concentration on protein degradation in situ.

    PubMed

    Grummer, R R; Clark, J H; Davis, C L; Murphy, M R

    1984-10-01

    Two experiments were conducted to determine the effect of ruminal ammonia-nitrogen concentration on rate of ruminal protein degradation. In Experiment 1, four Holstein steers were fed a basal diet of corn grain and corn silage at hourly intervals. Continuous intraruminal infusions of solutions containing sodium bicarbonate and either sodium chloride or ammonium chloride resulted in ruminal ammonia-nitrogen concentrations that averaged 4.8 and 17.3 mg/dl. Ruminal fluid pH, fluid volume, and turnover rate of fluid and molar percentage of acetate, propionate, and butyrate were similar across treatments, reflecting steady state conditions. Rates of nitrogen and dry matter disappearance from polyester bags containing soybean protein supplements with 10.2 or 50.1% soluble nitrogen were not affected by increase of ruminal ammonia-nitrogen concentrations from 4.8 to 17.3 mg/dl. In Experiment 2, Holstein steers were fed twice daily a basal diet of urea-supplemented corn grain and corn silage. Polyester bags containing soybean protein supplements were placed in the rumen at -4, 0, or 4 h with reference to feeding and incubated from 1 to 12 h. Peak ruminal ammonia-nitrogen concentrations occurred during different periods of incubation for each treatment. Ruminal ammonia-nitrogen concentrations ranged from 3 mg/dl at 6 h postfeeding to 46 mg/dl at 1 h postfeeding. Nitrogen and dry matter disappearance rates during 0 to 1 and 1 to 12 h of incubation did not differ among treatments.

  8. Carnosine ameliorates lens protein turbidity formations by inhibiting calpain proteolysis and ultraviolet C-induced degradation.

    PubMed

    Liao, Jiahn-Haur; Lin, I-Lin; Huang, Kai-Fa; Kuo, Pei-Ting; Wu, Shih-Hsiung; Wu, Tzu-Hua

    2014-06-25

    Carnosine (CAR) is an endogenous peptide and present in lens, but there is little evidence for its effectiveness in calpain-induced proteolysis inhibition and its differential effects toward different wavelengths of ultraviolet (UV) irradiation. This study aimed to develop three in vitro cataract models to compare the mechanisms underlying the protective activities of CAR. Crude crystallins extracted from porcine lenses were used for antiproteolysis assays, and purified γ-crystallins were used for anti-UV assays. The turbidity in those in vitro models mimics cataract formation and was assayed by measuring optical density (OD) at 405 nm. The effectiveness of CAR on calpain-induced proteolysis was studied at 37 and 58 °C. Patterns of proteins were then analyzed by SDS-PAGE. The turbidity was reduced significantly (p<0.05) at 60 min measurements with the increased concentration of CAR (10-300 mM). SDS-PAGE showed that the decreased intensities at both ∼28 and ∼30 kDa protein bands in heat-enhanced assays were ameliorated by CAR at ≥10 mM concentrations. In UV-B studies, CAR (200, 300 mM) reduced the turbidity of γ-crystallin significantly (p<0.05) at 6 h observations. The turbidity of samples containing γ-crystallins was ameliorated while incubated with CAR (100, 300 mM) significantly (p<0.05) following 4 h of exposure to UV-C. SDS-PAGE showed that the presence of CAR reduced UV-B-induced aggregation of γ-crystallins at ∼44 kDa and resulted in less loss of γ-crystallin following UV-C exposure. The result of modeling also suggests that CAR acts as an inhibitor of calpain. In conclusion, CAR protects lens proteins more readily by inhibiting proteolysis and UV-C-induced degradation than aggregation induced by UV-B irradiation.

  9. Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the UL47 tegument protein.

    PubMed

    Shu, Minfeng; Taddeo, Brunella; Zhang, Weiran; Roizman, Bernard

    2013-04-30

    Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHS-RNase degrades stable cellular mRNAs and α (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral β or γ mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, UL47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral α mRNAs and effectively blocks the degradation of β and γ mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of UL47 suggest that it, along with the α protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and UL47 overexpress α genes and delay the expression of β and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.

  10. 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.

  11. Long-Term Controlled Protein Release from Poly(Ethylene Glycol) Hydrogels by Modulating Mesh Size and Degradation.

    PubMed

    Tong, Xinming; Lee, Soah; Bararpour, Layla; Yang, Fan

    2015-12-01

    Poly(ethylene glycol) (PEG)-based hydrogels are popular biomaterials for protein delivery to guide desirable cellular fates and tissue repair. However, long-term protein release from PEG-based hydrogels remains challenging. Here, we report a PEG-based hydrogel platform for long term protein release, which allows efficient loading of proteins via physical entrapment. Tuning hydrogel degradation led to increase in hydrogel mesh size and gradual release of protein over 60 days of with retained bioactivity. Importantly, this platform does not require the chemical modification of loaded proteins, and may serve as a versatile tool for long-term delivery of a wide range of proteins for drug-delivery and tissue-engineering applications.

  12. Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases.

    PubMed

    Victoria, Guiliana Soraya; Zurzolo, Chiara

    2015-09-01

    Several neurodegenerative diseases such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's diseases are caused by the conversion of cellular proteins to a pathogenic conformer. Despite differences in the primary structure and subcellular localization of these proteins, which include the prion protein, α-synuclein and amyloid precursor protein (APP), striking similarity has been observed in their ability to seed and convert naïve protein molecules as well as transfer between cells. This review aims to cover what is known about the intracellular trafficking of these proteins as well as their degradation mechanisms and highlight similarities in their movement through the endocytic pathway that could contribute to the pathogenic conversion and seeding of these proteins which underlies the basis of these diseases.

  13. Impact of Adiponectin Overexpression on Allergic Airways Responses in Mice

    PubMed Central

    Verbout, Norah G.; Williams, Alison S.; Kasahara, David I.; Wurmbrand, Allison P.; Halayko, Andrew J.; Shore, Stephanie A.

    2013-01-01

    Obesity is an important risk factor for asthma. Obese individuals have decreased circulating adiponectin, an adipose-derived hormone with anti-inflammatory properties. We hypothesized that transgenic overexpression of adiponectin would attenuate allergic airways inflammation and mucous hyperplasia in mice. To test this hypothesis, we used mice overexpressing adiponectin (Adipo Tg). Adipo Tg mice had marked increases in both serum adiponectin and bronchoalveolar lavage (BAL) fluid adiponectin. Both acute and chronic ovalbumin (OVA) sensitization and challenge protocols were used. In both protocols, OVA-induced increases in total BAL cells were attenuated in Adipo Tg versus WT mice. In the acute protocol, OVA-induced increases in several IL-13 dependent genes were attenuated in Adipo Tg versus WT mice, even though IL-13 per se was not affected. With chronic exposure, though OVA-induced increases in goblet cells numbers per millimeter of basement membrane were greater in Adipo Tg versus WT mice, mRNA abundance of mucous genes in lungs was not different. Also, adiponectin overexpression did not induce M2 polarization in alveolar macrophages. Our results indicate that adiponectin protects against allergen-induced inflammatory cell recruitment to the airspaces, but not development of goblet cell hyperplasia. PMID:23861690

  14. Expression of adiponectin receptors in pancreatic beta cells.

    PubMed

    Kharroubi, Ilham; Rasschaert, Joanne; Eizirik, Décio L; Cnop, Miriam

    2003-12-26

    Pancreatic beta cell dysfunction is an early and crucial pathogenic factor in the development of type 2 diabetes. Free fatty acids (FFA) and adipokines released from adipose tissues lead to both the development of insulin resistance and beta cell dysfunction. Adiponectin is a novel adipokine with antidiabetic properties. Its circulating concentrations are reduced in subjects with increased visceral adiposity, insulin resistance, or type 2 diabetes. Very recently, the cloning of two adiponectin receptors AdipoR1 and AdipoR2 was reported. AdipoR1 is abundantly expressed in muscle, while AdipoR2 is predominantly expressed in liver. Here we report the marked expression of mRNAs for the adiponectin receptors AdipoR1 and AdipoR2 in human and rat pancreatic beta cells, at levels similar to liver and greater than muscle. Adiponectin receptor expression is increased by beta cell exposure to the unsaturated FFA oleate, and treatment of insulin-producing cells with globular adiponectin induces lipoprotein lipase expression. Regulated adiponectin receptor expression on pancreatic beta cells might be a novel mechanism modulating the effects of circulating adiponectin. PMID:14651988

  15. 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

  16. Degradation of proteins by enzymes exuded by Allium porrum roots - a potentially important strategy for acquiring organic nitrogen by plants.

    PubMed

    Adamczyk, Bartosz; Godlewski, Mirosław; Smolander, Aino; Kitunen, Veikko

    2009-10-01

    Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography-mass spectrometry (LC-MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.

  17. 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...

  18. Attenuation of microtubule associated protein-2 degradation after mild head injury by mexiletine and calpain-2 inhibitor.

    PubMed

    Atalay, B; Caner, H; Can, A; Cekinmez, M

    2007-06-01

    The objective of the study was to address the early effects of mild, closed, head injuries on neuronal stability and the prevention of microtubule-associated protein-2 (MAP-2) degradation by mexiletine and calpain-2 inhibitor. Twenty-four rats were divided into four groups: control group (1); trauma group without treatment (2); mexiletine-pretreated and subjected to trauma group (3); trauma subjected and then calpain-2 inhibitor received group (4). All animals were subjected to mild, closed, head trauma. Frontal lobes were removed and processed for staining and immunofluorescent labelling of MAP-2 cytoskeletal proteins, which were evaluated by confocal microscopy in serial optical sections showing the three dimensional cytoarchitecture of affected areas. MAP-2 decoration in almost all neurons obtained from traumatized brain regions drastically diminished, while minute filamentous and granular profiles in axons and/or dendrites were retained together implying a massive degradation/depolymerization of microtubules. In contrast, in mexiletine-pretreated animals, MAP-2 positivity in axonal and perikaryonal profiles was fairly retained, which clearly depicts the protective role of mexiletine after trauma. Compared with mexiletine-pretreated group, calpain-2 inhibitor treated group displayed a less well-preserved MAP-2 expression. Mexiletine can prevent cytoskeletal structure and protein degradation after mild head trauma. Calpain-2 inhibitor prevents protein degradation, but cytoskeletal organization is better preserved with mexiletine. PMID:17612919

  19. 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.

  20. 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

  1. 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

  2. The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation.

    PubMed

    Dill, Alyssa; Thomas, Stephen G; Hu, Jianhong; Steber, Camille M; Sun, Tai-Ping

    2004-06-01

    The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins. PMID:15155881

  3. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    SciTech Connect

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na Guo, Qinglong

    2013-09-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  4. The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources.

    PubMed

    Soliva, Carla R; Amelchanka, Sergej L; Kreuzer, Michael

    2015-01-01

    Ruminant feed evaluation systems use constant minimum requirements of rumen-degradable protein (RDP) and often relate this to apparently degradable organic matter (OM). However, studies with tropical forages indicate that RDP: apparently degraded OM might not be constant across high-fiber diets. This was tested with semi-continuous ruminal cultures (Rusitec) using dried contrasting low-protein fiber sources: brachiaria hay (high in fiber, medium lignified), apple pomace (medium in fiber, highly lignified), and sugar beet pulp (medium in fiber and lignification). Each feed was incubated at 14 g dry matter day(-1) with 0, 0.85, 1.7, 3.4, 6.8, 13.6, or 27.2 mg g(-1) urea. The amount of urea needed to reach a similar basal concentration of ammonia in the incubation fluid was tested for each feed in advance. Apparent fiber and OM degradability were determined after 48 h of incubation. Data was evaluated by regressions and analysis of variance. The response curve of incubation fluid ammonia to urea supplementation was similar in slope in all feeds. Plateaus in apparent OM degradability in relation to ammonia concentration were determined. The ammonia concentration where apparent OM and fiber degradability reached 95% of maximum was approached in the order of pomace < pulp < hay. With regard to fiber degradability, a plateau was reached at ≥ 80 g kg(-1) crude protein only with hay and pomace, whilst a linear relationship existed between RDP and OM degradation for pulp. In hay the ratio RDP: OM degraded was equal to 1.6 but was only 1.0 in the other feeds. There was no obvious lack of branched short-chain fatty acids at low RDP. Thus, the hypothesis was confirmed but the demand for RDP seems even higher in tropical forage compared to food industrial byproducts. The efficiency of urea to promote apparent OM and fiber degradation was also variable. Thus, it seems that minimum thresholds of either RDP or ruminal ammonia concentration may not be reflected appropriately by

  5. The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources

    PubMed Central

    Soliva, Carla R.; Amelchanka, Sergej L.; Kreuzer, Michael

    2015-01-01

    Ruminant feed evaluation systems use constant minimum requirements of rumen-degradable protein (RDP) and often relate this to apparently degradable organic matter (OM). However, studies with tropical forages indicate that RDP: apparently degraded OM might not be constant across high-fiber diets. This was tested with semi-continuous ruminal cultures (Rusitec) using dried contrasting low-protein fiber sources: brachiaria hay (high in fiber, medium lignified), apple pomace (medium in fiber, highly lignified), and sugar beet pulp (medium in fiber and lignification). Each feed was incubated at 14 g dry matter day−1 with 0, 0.85, 1.7, 3.4, 6.8, 13.6, or 27.2 mg g−1 urea. The amount of urea needed to reach a similar basal concentration of ammonia in the incubation fluid was tested for each feed in advance. Apparent fiber and OM degradability were determined after 48 h of incubation. Data was evaluated by regressions and analysis of variance. The response curve of incubation fluid ammonia to urea supplementation was similar in slope in all feeds. Plateaus in apparent OM degradability in relation to ammonia concentration were determined. The ammonia concentration where apparent OM and fiber degradability reached 95% of maximum was approached in the order of pomace < pulp < hay. With regard to fiber degradability, a plateau was reached at ≥ 80 g kg−1 crude protein only with hay and pomace, whilst a linear relationship existed between RDP and OM degradation for pulp. In hay the ratio RDP: OM degraded was equal to 1.6 but was only 1.0 in the other feeds. There was no obvious lack of branched short-chain fatty acids at low RDP. Thus, the hypothesis was confirmed but the demand for RDP seems even higher in tropical forage compared to food industrial byproducts. The efficiency of urea to promote apparent OM and fiber degradation was also variable. Thus, it seems that minimum thresholds of either RDP or ruminal ammonia concentration may not be reflected appropriately by

  6. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    PubMed Central

    Sommers, Joshua A.; Suhasini, Avvaru N.; Brosh, Robert M.

    2015-01-01

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. PMID:25906194

  7. Protein degradation pathways regulate the functions of helicases in the DNA damage response and maintenance of genomic stability.

    PubMed

    Sommers, Joshua A; Suhasini, Avvaru N; Brosh, Robert M

    2015-04-21

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom's syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis.

  8. Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

    PubMed

    Whyteside, Graham; Alcocer, Marcos J C; Kumita, Janet R; Dobson, Christopher M; Lazarou, Maria; Pleass, Richard J; Archer, David B

    2011-01-01

    We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. PMID:21818368

  9. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

    PubMed

    Shibata, Norihito; Ohoka, Nobumichi; Sugaki, Yusuke; Onodera, Chiaki; Inoue, Mizuho; Sakuraba, Yoshiyuki; Takakura, Daisuke; Hashii, Noritaka; Kawasaki, Nana; Gondo, Yoichi; Naito, Mikihiko

    2015-11-20

    During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders. PMID:26442586

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

    PubMed

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

    2016-04-06

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

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

    PubMed

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

    2016-01-01

    Control of organ size by cell proliferation and growth is a fundamental process, but the mechanisms that determine the final size of organs are largely elusive in plants. We have previously revealed that the ubiquitin receptor DA1 regulates organ size by repressing cell proliferation in Arabidopsis. Here we report that a mutant allele of STERILE APETALA (SAP) suppresses the da1-1 mutant phenotype. We show that SAP is an F-box protein that forms part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex and controls organ size by promoting the proliferation of meristemoid cells. Genetic analyses suggest that SAP may act in the same pathway with PEAPOD1 and PEAPOD2, which are negative regulators of meristemoid proliferation, to control organ size, but does so independently of DA1. Further results reveal that SAP physically associates with PEAPOD1 and PEAPOD2, and targets them for degradation. These findings define a molecular mechanism by which SAP and PEAPOD control organ size. PMID:27048938

  12. Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

    PubMed

    Johnson, A B; Cammer, W

    1977-05-01

    Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.

  13. Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

    PubMed Central

    Lobbestael, E.; Civiero, L.; De Wit, T.; Taymans, J.-M.; Greggio, E.; Baekelandt, V.

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies. PMID:27658356

  14. Acetone Extract of Almond Hulls Provides Protection against Oxidative Damage and Membrane Protein Degradation.

    PubMed

    Meshkini, Azadeh

    2016-06-01

    Several studies have revealed that among foods, the consumption of edible nuts has beneficial effects on health which are attributed to their high content of potent antioxidants. Among nuts, the whole seed of the almond (Prunus dulcis) has been demonstrated to possess potent free radical scavenging activity, which is related to the presence of phenolic compounds. The aim of the current study is to evaluate the polyphenol content and the antioxidant ability of almond hull, which is an agriculture solid waste. The present results revealed that among different extraction methods, the acetone extract of almond hulls has a high content of phenolic and flavonoid compounds and a high antioxidant ability, which were determined by using the phosphomolybdenum method and by measuring the potency of the antioxidant, respectively. Moreover, the experimental data disclosed that the acetone extract of almond hulls provides protection against the oxidative damage and the membrane protein degradation that are caused in human erythrocytes by hydrogen peroxide. These phenomena may likely be due to the recruitment of antioxidants by cell membranes and/or translocation to cytosol. Overall, almond hull extract could be considered as a natural source of antioxidants, and its consumption could have a positive effect on human health. PMID:27342887

  15. SCFSAP controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana

    PubMed Central

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

    2016-01-01

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