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Sample records for acid-binding protein l-fabp

  1. Liver fatty acid-binding protein (L-FABP) promotes cellular angiogenesis and migration in hepatocellular carcinoma.

    PubMed

    Ku, Chung-Yu; Liu, Yu-Huei; Lin, Hsuan-Yuan; Lu, Shao-Chun; Lin, Jung-Yaw

    2016-04-01

    Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and that this expression was positively correlated with that of VEGF-A. Additionally, L-FABP significantly promoted tumor growth and metastasis in a xenograft mouse model. We also assessed the mechanisms of L-FABP activity in tumorigenesis; L-FABP was found to associate with VEGFR2 on membrane rafts and subsequently activate the Akt/mTOR/P70S6K/4EBP1 and Src/FAK/cdc42 pathways, which resulted in up-regulation of VEGF-A accompanied by an increase in both angiogenic potential and migration activity. Our results thus suggest that L-FABP could be a potential target for HCC chemotherapy. PMID:26919097

  2. Liver fatty acid-binding protein (L-FABP) promotes cellular angiogenesis and migration in hepatocellular carcinoma

    PubMed Central

    Ku, Chung-Yu; Liu, Yu-Huei; Lin, Hsuan-Yuan; Lu, Shao-Chun; Lin, Jung-Yaw

    2016-01-01

    Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and that this expression was positively correlated with that of VEGF-A. Additionally, L-FABP significantly promoted tumor growth and metastasis in a xenograft mouse model. We also assessed the mechanisms of L-FABP activity in tumorigenesis; L-FABP was found to associate with VEGFR2 on membrane rafts and subsequently activate the Akt/mTOR/P70S6K/4EBP1 and Src/FAK/cdc42 pathways, which resulted in up-regulation of VEGF-A accompanied by an increase in both angiogenic potential and migration activity. Our results thus suggest that L-FABP could be a potential target for HCC chemotherapy. PMID:26919097

  3. Liver Fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet induced nonalcoholic fatty liver disease

    PubMed Central

    Chen, Anping; Tang, Youcai; Davis, Victoria; Hsu, Fong-Fu; Kennedy, Susan M.; Song, Haowei; Turk, John; Brunt, Elizabeth M.; Newberry, Elizabeth P.; Davidson, Nicholas O.

    2013-01-01

    Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild type (WT) HSCs, and exhibit upregulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. PMID:23401290

  4. Liver fatty acid binding protein (L-Fabp) modifies intestinal fatty acid composition and adenoma formation in ApcMin/+ mice

    PubMed Central

    Dharmarajan, Sekhar; Newberry, Elizabeth P.; Montenegro, Grace; Nalbantoglu, ILKe; Davis, Victoria R.; Clanahan, Michael J.; Blanc, Valerie; Xie, Yan; Luo, Jianyang; Fleshman, James W.; Kennedy, Susan; Davidson, Nicholas O.

    2013-01-01

    Evidence suggests a relationship between dietary fat intake, obesity and colorectal cancer, implying a role for fatty acid (FA) metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid binding protein (L-Fabp), a dominant intestinal FA binding protein, regulates intestinal FA trafficking and metabolism and L-Fabp deletion attenuates diet-induced obesity. Here we examined whether changes in intestinal FA metabolism following L-Fabp deletion modify adenoma development in ApcMin/+ mice. Compound L-Fabp−/−ApcMin/+ mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated and polyunsaturated fat. L-Fabp−/−ApcMin/+ mice displayed significant reductions in adenoma number and total polyp area compared to ApcMin/+controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp−/−ApcMin/+ mice exhibited reductions in cellular proliferation, high-grade dysplasia and nuclear β-catenin translocation. Intestinal FA content was increased in L-Fabp−/−ApcMin/+ mice and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated FA species with reduced saturated FA species. L-Fabp−/−ApcMin/+mice also demonstrated corresponding changes in mRNA expression of enzymes involved in FA elongation and desaturation. Furthermore, adenomas from L-Fabp−/−ApcMin/+mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis and identify FA trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity and intestinal tumor formation. PMID:23921281

  5. Urinary liver-type fatty acid-binding protein change in gestational diabetes mellitus.

    PubMed

    Fu, Wen-Jin; Wang, Du-Juan; Deng, Ren-Tang; Huang, Zhi-Hong; Chen, Mei-Lian; Jang, You-Ming; Wen, Shu; Yang, Hong-Ling; Huang, Xian-zhang

    2015-09-01

    We compared urinary liver-type fatty acid-binding protein (L-FABP) among non-pregnant and pregnant women with and without gestational diabetes mellitus (GDM). Higher urinary L-FABP was found in pregnant with and without GDM, and considerably higher urinary L-FABP was found in the GDM group compared with the non-GDM group. Hyperglycemia and anemia were related with high urinary L-FABP expression. PMID:26254248

  6. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    PubMed

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes. PMID:25966259

  7. L-FABP: A novel biomarker of kidney disease.

    PubMed

    Xu, Yao; Xie, Yuanyuan; Shao, Xinghua; Ni, Zhaohui; Mou, Shan

    2015-05-20

    Human liver-type fatty acid-binding protein (hL-FABP), which is found in both the normal and the diseased human kidney, has been observed to bind free fatty acids. Recently, the predictive and prognostic value of L-FABP in kidney diseases has attracted considerable attention. Numerous studies have demonstrated that L-FABP is a promising biomarker of several kidney diseases, and it has also been shown to attenuate renal injury. We performed a literature review regarding the ability of L-FABP to identify patients at risk of developing kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD) and to protect the kidneys in the course of kidney disease. PMID:25797895

  8. Bile salt recognition by human liver fatty acid binding protein.

    PubMed

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  9. Elevation of urinary liver-type fatty acid binding protein after cardiac catheterization related to cardiovascular events

    PubMed Central

    Kamijo-Ikemori, Atsuko; Hashimoto, Nobuyuki; Sugaya, Takeshi; Matsui, Katsuomi; Hisamichi, Mikako; Shibagaki, Yugo; Miyake, Fumihiko; Kimura, Kenjiro

    2015-01-01

    Purpose Contrast medium (CM) induces tubular hypoxia via endothelial damage due to direct cytotoxicity or viscosity. Urinary liver-type fatty acid binding protein (L-FABP) increases along with tubular hypoxia and may be a detector of systemic circulation injury. The aim of this study was to evaluate the clinical usefulness of detecting increases in urinary L-FABP levels due to administration of CM, as a prognostic biomarker for cardiovascular disease in patients without occurrence of CM-induced nephropathy undergoing cardiac catheterization procedure (CCP). Methods Retrospective longitudinal analyses of the relationship between urinary L-FABP levels and occurrence of cardiovascular events were performed (n=29). Urinary L-FABP was measured by ELISA before CCP, and at 6, 12, 24, and 48 hours after CCP. Results Urinary L-FABP levels were significantly higher at 12 hours (P<0.05) and 24 hours (P<0.005) after CCP compared with before CCP, only in the patients with occurrence of cardiovascular events (n=17), but not in those without cardiovascular events (n=12). The parameter with the largest area under the curve (0.816) for predicting the occurrence of cardiovascular events was the change in urinary L-FABP at 24 hours after CCP. The difference in urinary L-FABP levels (ΔL-FABP ≥11.0 μg/g creatinine) between before CCP and at 24 hours after CCP was a risk factor for the occurrence of cardiovascular events (hazard ratio, 4.93; 95% confidence interval, 1.27–19.13; P=0.021). Conclusion Measurement of urinary L-FABP before CCP and at 24 hours after CCP in patients with mild to moderate renal dysfunction may be an important indicator for risk stratification of onset of cardiovascular events. PMID:26316797

  10. Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice.

    PubMed

    Martin, Gregory G; Atshaves, Barbara P; McIntosh, Avery L; Mackie, John T; Kier, Ann B; Schroeder, Friedhelm

    2006-01-01

    Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity. PMID:16123197

  11. Molecular characterization, tissue expression, and polymorphism analysis of liver-type fatty acid binding protein in Landes geese.

    PubMed

    Song, Z; Shao, D; Sun, X X; Niu, J W; Gong, D Q

    2015-01-01

    Liver weight is an important economic trait in the fatty goose liver industry. Liver-type fatty acid binding protein (L-FABP) is involved in the formation and metabolism of fatty acids. Thus, we hypothesized that sequence polymorphisms in L-FABP were associated with fatty liver weight in goose. We first isolated, sequenced, and characterized the goose L-FABP gene, which had not been previously reported. The goose L-FABP gene was 2490 bp and included 4 exons coding for a 126-amino acid protein. Analysis of expression levels of the goose L-FABP gene in different tissues showed that the expression level in the liver tissue was higher than in other tissues, and was significantly higher in the liver tissue of overfed geese than in control geese. Moreover, a single nucleotide polymorphism located at 774 bp in the gene was identified in a Landes goose population. To test whether this single nucleotide polymorphism was associated with fatty liver production, liver weight and the ratio of liver to carcass weights were determined for the 3 genotypes with this single nucleotide polymorphism (TT, TG, GG) in overfed Landes geese. Our data indicate that individuals with the GG genotype had higher values for the variables measured than those with the other 2 genotypes, suggesting that L-FABP can be a selection marker for the trait of fatty liver production in goose. PMID:25729971

  12. Liver fatty acid-binding protein: specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators.

    PubMed Central

    Khan, S H; Sorof, S

    1994-01-01

    Peroxisome proliferators (PP) are a diverse group of chemicals that induce dramatic increases in peroxisomes in rodent hepatocytes, followed by hypertrophy, hepatomegaly, alterations in lipid metabolism, mitogenesis, and finally hepatocarcinomas. Termed nongenotoxic carcinogens, they do not interact with DNA, are not mutagenic in bacterial assays, and fail to elicit many of the phenotypes associated with classic genotoxic carcinogens. We report here that the mitogenesis induced by the major PP class, the amphipathic carboxylates, and by the tetrazole-substituted acetophenones specifically requires liver fatty acid-binding protein (L-FABP) in cultured rat hepatoma cells transfected with the sense cDNA of L-FABP, in contrast to L-FABP-nonexpressing cells transfected with its antisense cDNA. The mitogenic actions of L-FABP were protein-specific, inasmuch as no other protein in the nonexpressing cells could act like L-FABP. L-FABP was previously shown not only (i) to interact covalently with metabolites of the two genotoxic carcinogens 2-acetylaminofluorene and aminoazo dyes during liver carcinogenesis, but also (ii) to bind noncovalently the two classes of PP in vitro with avidities that correlate with their abilities to elicit peroxisomal enzymatic responses, and (iii) together with unsaturated fatty acids, especially linoleic acid, to promote multiplication of the transfected hepatoma cells in culture. The convergence of the two types of genotoxic carcinogens with the two classes of PP nongenotoxic carcinogens, and also with unsaturated fatty acids, at L-FABP actions in inducing mitogenesis allows the following hypothesis. During tumor promotion of carcinogenesis in vivo, these groups of genotoxic and nongenotoxic carcinogens act on the normal process by which L-FABP, functioning as a specific receptor of unsaturated fatty acids or their metabolites, promotes hepatocyte proliferation. Images PMID:8302856

  13. Liver Fatty Acid Binding Protein Gene-ablation Exacerbates Weight Gain in High-Fat Fed Female Mice

    PubMed Central

    McIntosh, Avery L.; Atshaves, Barbara P.; Landrock, Danilo; Landrock, Kerstin K.; Martin, Gregory G.; Storey, Stephen M.; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Loss of liver fatty acid binding protein (L-FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L-FABP gene ablation would potentiate high-fat diet-induced weight gain and weight gain/energy intake. While this was indeed the case when L-FABP null (−/−) mice on the C57BL/6NCr background were pair-fed high fat diet, whether this would also be observed under high-fat diet fed ad libitum was not known. Therefore, this possibility was examined in female L-FABP (−/−) mice on the same background. L-FABP (−/−) mice consumed equal amounts of defined high-fat or isocaloric control diets fed ad libitum. However, on the ad libitum fed high-fat diet the L-FABP (−/−) mice exhibited: 1) Decreased hepatic long chain fatty acid (LCFA) β-oxidation as indicated by lower serum β–hydroxybutyrate level; 2) Decreased hepatic protein levels of key enzymes mitochondrial (rate limiting carnitine palmitoyl acyltransferase A1, CPT1A; HMG-CoA synthase) and peroxisomal (acyl CoA oxidase 1, ACOX1) LCFA β-oxidation; 3) Increased fat tissue mass (FTM) and FTM/energy intake to the greatest extent; and 4) Exacerbated body weight gain, weight gain/energy intake, liver weight, and liver weight/body weight to the greatest extent. Taken together, these findings showed that L-FABP gene-ablation exacerbated diet-induced weight gain and fat tissue mass gain in mice fed high-fat diet ad libitum—consistent with the known biochemistry and cell biology of L-FABP. PMID:23539345

  14. Human liver-type fatty acid-binding protein protects against tubulointerstitial injury in aldosterone-induced renal injury.

    PubMed

    Ichikawa, Daisuke; Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Shibagaki, Yugo; Yasuda, Takashi; Hoshino, Seiko; Katayama, Kimie; Igarashi-Migitaka, Junko; Hirata, Kazuaki; Kimura, Kenjiro

    2015-01-15

    To demonstrate the renoprotective function of human liver-type fatty acid-binding protein (hL-FABP) expressed in proximal tubules in aldosterone (Aldo)-induced renal injury, hL-FABP chromosomal transgenic (Tg) and wild-type (WT) mice received systemic Aldo infusions (Tg-Aldo and WT-Aldo, respectively) were given 1% NaCl water for 28 days. In this model, elevation of systolic blood pressure, monocyte chemoattractant protein-1 expression, macrophage infiltration in the interstitium, tubulointerstitial damage, and depositions of type I and III collagens were observed. Elevation of systolic blood pressure did not differ in WT-Aldo vs. Tg-Aldo animals, however, renal injury was suppressed in Tg-Aldo compared with WT-Aldo mice. Dihydroethidium fluorescence was used to evaluate reactive oxidative stress, which was suppressed in Tg-Aldo compared with WT-Aldo mice. Gene expression of angiotensinogen in the kidney was upregulated, and excretion of urinary angiotensinogen was increased in WT-Aldo mice. This exacerbation was suppressed in Tg-Aldo mice. Expression of hL-FABP was upregulated in proximal tubules of Tg-Aldo mice. Urinary excretion of hL-FABP was significantly greater in Tg-Aldo than in Tg-control mice. In conclusion, hL-FABP ameliorated the tubulointerstitial damage in Aldo-induced renal injury via reducing oxidative stress and suppressing activation of the intrarenal renin-angiotensin system. PMID:25339700

  15. Impact of clinical context on acute kidney injury biomarker performances: differences between neutrophil gelatinase-associated lipocalin and L-type fatty acid-binding protein

    PubMed Central

    Asada, Toshifumi; Isshiki, Rei; Hayase, Naoki; Sumida, Maki; Inokuchi, Ryota; Noiri, Eisei; Nangaku, Masaomi; Yahagi, Naoki; Doi, Kent

    2016-01-01

    Application of acute kidney injury (AKI) biomarkers with consideration of nonrenal conditions and systemic severity has not been sufficiently determined. Herein, urinary neutrophil gelatinase-associated lipocalin (NGAL), L-type fatty acid-binding protein (L-FABP) and nonrenal disorders, including inflammation, hypoperfusion and liver dysfunction, were evaluated in 249 critically ill patients treated at our intensive care unit. Distinct characteristics of NGAL and L-FABP were revealed using principal component analysis: NGAL showed linear correlations with inflammatory markers (white blood cell count and C-reactive protein), whereas L-FABP showed linear correlations with hypoperfusion and hepatic injury markers (lactate, liver transaminases and bilirubin). We thus developed a new algorithm by combining urinary NGAL and L-FABP with stratification by the Acute Physiology and Chronic Health Evaluation score, presence of sepsis and blood lactate levels to improve their AKI predictive performance, which showed a significantly better area under the receiver operating characteristic curve [AUC-ROC 0.940; 95% confidential interval (CI) 0.793–0.985] than that under NGAL alone (AUC-ROC 0.858, 95% CI 0.741–0.927, P = 0.03) or L-FABP alone (AUC-ROC 0.837, 95% CI 0.697–0.920, P = 0.007) and indicated that nonrenal conditions and systemic severity should be considered for improved AKI prediction by NGAL and L-FABP as biomarkers. PMID:27605390

  16. Impact of clinical context on acute kidney injury biomarker performances: differences between neutrophil gelatinase-associated lipocalin and L-type fatty acid-binding protein.

    PubMed

    Asada, Toshifumi; Isshiki, Rei; Hayase, Naoki; Sumida, Maki; Inokuchi, Ryota; Noiri, Eisei; Nangaku, Masaomi; Yahagi, Naoki; Doi, Kent

    2016-01-01

    Application of acute kidney injury (AKI) biomarkers with consideration of nonrenal conditions and systemic severity has not been sufficiently determined. Herein, urinary neutrophil gelatinase-associated lipocalin (NGAL), L-type fatty acid-binding protein (L-FABP) and nonrenal disorders, including inflammation, hypoperfusion and liver dysfunction, were evaluated in 249 critically ill patients treated at our intensive care unit. Distinct characteristics of NGAL and L-FABP were revealed using principal component analysis: NGAL showed linear correlations with inflammatory markers (white blood cell count and C-reactive protein), whereas L-FABP showed linear correlations with hypoperfusion and hepatic injury markers (lactate, liver transaminases and bilirubin). We thus developed a new algorithm by combining urinary NGAL and L-FABP with stratification by the Acute Physiology and Chronic Health Evaluation score, presence of sepsis and blood lactate levels to improve their AKI predictive performance, which showed a significantly better area under the receiver operating characteristic curve [AUC-ROC 0.940; 95% confidential interval (CI) 0.793-0.985] than that under NGAL alone (AUC-ROC 0.858, 95% CI 0.741-0.927, P = 0.03) or L-FABP alone (AUC-ROC 0.837, 95% CI 0.697-0.920, P = 0.007) and indicated that nonrenal conditions and systemic severity should be considered for improved AKI prediction by NGAL and L-FABP as biomarkers. PMID:27605390

  17. Loss of L-FABP, SCP-2/SCP-x, or both induces hepatic lipid accumulation in female mice.

    PubMed

    Martin, Gregory G; Atshaves, Barbara P; Landrock, Kerstin K; Landrock, Danilo; Schroeder, Friedhelm; Kier, Ann B

    2015-08-15

    Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals-suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO- and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD). PMID:26116377

  18. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

    PubMed

    Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

    2016-01-01

    Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans. PMID:25370009

  19. Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α- and γ-mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus

    PubMed Central

    Wolfrum, Christian; Borrmann, Carola M.; Börchers, Torsten; Spener, Friedrich

    2001-01-01

    Peroxisome proliferator-activated receptor α (PPARα) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARα. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARα and PPARγ but not with PPARβ and retinoid X receptor-α by protein–protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARα and PPARγ transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARα and PPARγ agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes. PMID:11226238

  20. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  1. Urinary L-FABP predicts poor outcomes in critically ill patients with early acute kidney injury.

    PubMed

    Parr, Sharidan K; Clark, Amanda J; Bian, Aihua; Shintani, Ayumi K; Wickersham, Nancy E; Ware, Lorraine B; Ikizler, T Alp; Siew, Edward D

    2015-03-01

    Biomarker studies for early detection of acute kidney injury (AKI) have been limited by nonselective testing and uncertainties in using small changes in serum creatinine as a reference standard. Here we examine the ability of urine L-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), and kidney injury molecule-1 (KIM-1) to predict injury progression, dialysis, or death within 7 days in critically ill adults with early AKI. Of 152 patients with known baseline creatinine examined, 36 experienced the composite outcome. Urine L-FABP demonstrated an area under the receiver-operating characteristic curve (AUC-ROC) of 0.79 (95% confidence interval 0.70-0.86), which improved to 0.82 (95% confidence interval 0.75-0.90) when added to the clinical model (AUC-ROC of 0.74). Urine NGAL, IL-18, and KIM-1 had AUC-ROCs of 0.65, 0.64, and 0.62, respectively, but did not significantly improve discrimination of the clinical model. The category-free net reclassification index improved with urine L-FABP (total net reclassification index for nonevents 31.0%) and urine NGAL (total net reclassification index for events 33.3%). However, only urine L-FABP significantly improved the integrated discrimination index. Thus, modest early changes in serum creatinine can help target biomarker measurement for determining prognosis with urine L-FABP, providing independent and additive prognostic information when combined with clinical predictors. PMID:25229339

  2. Urine Liver-Type Fatty Acid-Binding Protein and Kidney Injury Molecule-1 in HIV-Infected Patients Receiving Combined Antiretroviral Treatment Based on Tenofovir

    PubMed Central

    Wójcik, Kamila; Piekarska, Anna

    2014-01-01

    Abstract The aim of this study was to determine the presence of kidney tubular damage in the absence of overt evidence of glomerular dysfunction (GFR>60 ml/min without proteinuria) in HIV-infected patients receiving antiretroviral therapy. Urine kidney injury molecule-1 (KIM-1) and liver-type fatty acid-binding protein (L-FABP) levels were measured by ELISA and expressed as a ratio to creatinine. Sixty-six patients (median age 38 years) and 10 healthy controls (median age 35.5 years) were included in the study. Patients with chronic diseases such as diabetes, hypertension, heart disease, or kidney disease were excluded from the study. All patients received tenofovir/emtricitabine combined with one of three other components, namely efavirenz, atazanavir/norvir, or lopinavir/norvir. A lower concentration of L-FABP/creatinine was observed in HIV-infected as compared to healthy individuals (p=0.0353); KIM-1/creatinine was also lower in comparison with healthy controls but not statistically significantly. Patients receiving efavirenz had higher levels of L-FABP/creatinine in comparison to healthy controls (p=0.0039). Patients with anti-HCV had higher concentrations of L-FABP/creatinine as compared to the HIV-monoinfected individuals (not statistically significant) and to healthy subjects (p=0.0356). All four patients with L-FABP>17.5 μg/g creatinine were HIV/HCV coinfected. On multivariate logistic regression urine L-FABP above 5.5 μg/g creatinine was independently associated with body weight (OR=0.93 p=0.039). This study suggests that HIV/HCV-coinfected patients with lower body weight treated with tenofovir may be at an increased risk of tubular dysfunction and should be monitored more closely. The use of protease inhibitors was not associated with an increased risk of tubular disorders. PMID:24164392

  3. Relative contributions of L-FABP, SCP-2/SCP-x, or both to hepatic biliary phenotype of female mice.

    PubMed

    Martin, Gregory G; Landrock, Danilo; Landrock, Kerstin K; Howles, Philip N; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2015-12-15

    Both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed to function in hepatobiliary bile acid metabolism/accumulation. To begin to address this issue, the impact of ablating L-FABP (LKO) or SCP-2/SCP-x (DKO) individually or both together (TKO) was examined in female mice. Biliary bile acid levels were decreased in LKO, DKO, and TKO mice; however, hepatic bile acid concentration was decreased in LKO mice only. In contrast, biliary phospholipid level was decreased only in TKO mice, while biliary cholesterol levels were unaltered regardless of phenotype. The loss of either or both genes increased hepatic expression of the major bile acid synthetic enzymes (CYP7A1 and/or CYP27A1). Loss of L-FABP and/or SCP-2/SCP-x genes significantly altered the molecular composition of biliary bile acids, but not the proportion of conjugated/unconjugated bile acids or overall bile acid hydrophobicity index. These data suggested that L-FABP was more important in hepatic retention of bile acids, while SCP-2/SCP-x more broadly affected biliary bile acid and phospholipid levels. PMID:26541319

  4. A newly developed kit for the measurement of urinary liver-type fatty acid-binding protein as a biomarker for acute kidney injury in patients with critical care.

    PubMed

    Sato, Ryo; Suzuki, Yasushi; Takahashi, Gaku; Kojika, Masahiro; Inoue, Yoshihiro; Endo, Shigeatsu

    2015-03-01

    In recent years, it has been reported that the urinary level of Liver-type fatty acid-binding protein (L-FABP) serves as a useful biomarker for diagnosing acute kidney injury (AKI) or sepsis complicated by AKI. However, because the urinary level of L-FABP is currently measured by enzyme-linked immunosorbent assay (ELISA), several days may elapse before the results of the measurement become available. We have newly developed a simplified kit, the Dip-test, for measuring the urinary level of L-FABP. The Dip-test was measured at 80 measurement points (22 points in noninfectious disease, 13 points in SIRS, 20 points in infectious disease, and 25 points in sepsis) in 20 patients. The urinary L-FABP levels as determined by ELISA in relation to the results of the Dip-test were as follows: 10.10 ± 12.85 ng/ml in patients with a negative Dip-test ([-] group), 41.93 ± 50.51 ng/ml in patients with a ± test ([±] group), 70.36 ± 73.70 ng/ml in patients with a positive test ([+] group), 1048.96 ± 2117.68 ng/ml in patients with a 2 + test ([2+] group), and 23,571.55 ± 21,737.45 ng/ml in patients with a 3 + test ([3+] group). The following tendency was noted: the stronger the positive Dip-test reaction, the higher the urinary L-FABP level. Multigroup comparison revealed a significant differences in the urinary L-FABP levels between the Dip-test (-) group and each of the other groups. In this study, the usefulness of the Dip-test, our newly developed simplified kit for measuring the urinary L-FABP level, is suggested. PMID:25499195

  5. Level of urinary liver-type fatty acid-binding protein is associated with cardiac markers and electrocardiographic abnormalities in type-2 diabetes with chronic kidney disease stage G1 and G2.

    PubMed

    Maeda, Yoshiteru; Suzuki, Atsushi; Ishii, Junnichi; Sekiguchi-Ueda, Sahoko; Shibata, Megumi; Yoshino, Yasumasa; Asano, Shogo; Hayakawa, Nobuki; Nakamura, Kazuhiro; Akiyama, Yasukazu; Kitagawa, Fumihiko; Sakuishi, Toshiaki; Fujita, Takashi; Hashimoto, Shuji; Ozaki, Yukio; Itoh, Mitsuyasu

    2015-05-01

    Urinary liver-type fatty acid-binding protein (L-FABP) reflects the degree of stress in proximal tubules of the kidney. We examined the level of L-FABP in type-2 diabetes mellitus (T2DM) patients with chronic kidney disease (CKD) stage G1 and G2, and its relationship with cardiac markers and electrocardiographic (ECG) abnormalities. T2DM patients whose estimated glomerular filtration rate (eGFR) was ≥60 mL/min/1.73 m(2) were recruited [n = 276 (165 males), mean age 64 years]. The median level of urinary L-FABP was 6.6 μg/gCr. Urinary L-FABP showed significant correlation with urinary albumin-to-creatinine ratio (ACR) (r = 0.51, p < 0.0001). Median (25th-75th percentile) eGFR was 82 (72-95) mL/min/1.73 m2. We divided patients into four subgroups (group 1, L-FABP ≤8.4 μg/gCr and ACR ≤30 mg/gCr; group 2, L-FABP ≤8.4 μg/gCr and ACR >30 mg/gCr; group 3, L-FABP >8.4 μg/gCr and ACR ≤30 mg/gCr; group 4, L-FABP >8.4 μg/gCr and ACR >30 mg/gCr). Compared with group 1, group 4 was significantly higher in systolic blood pressure, and eGFR using standardized serum cystatin C, high-sensitivity troponin T, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Group 4 had significantly higher level of NT-proBNP than group 3. Groups 2, 3 and 4 showed more ECG abnormalities than group 1. These findings suggest that simultaneous measurement of urinary L-FABP and ACR should be useful to assess cardiovascular damage reflecting on the elevation of cardiac markers and ECG abnormalities in T2DM with CKD G1 and G2. PMID:24626813

  6. Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion

    PubMed Central

    Martin, Gregory G.; Atshaves, Barbara P.; Landrock, Kerstin K.; Landrock, Danilo; Storey, Stephen M.; Howles, Philip N.; Kier, Ann B.

    2014-01-01

    On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol. PMID:25277800

  7. Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion.

    PubMed

    Martin, Gregory G; Atshaves, Barbara P; Landrock, Kerstin K; Landrock, Danilo; Storey, Stephen M; Howles, Philip N; Kier, Ann B; Schroeder, Friedhelm

    2014-12-01

    On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol. PMID:25277800

  8. Association of urinary KIM-1, L-FABP, NAG and NGAL with incident end-stage renal disease and mortality in American Indians with type 2 diabetes mellitus

    PubMed Central

    Fufaa, Gudeta D.; Weil, E. Jennifer; Nelson, Robert G.; Hanson, Robert L.; Bonventre, Joseph V.; Sabbisetti, Venkata; Waikar, Sushrut S.; Mifflin, Theodore E.; Zhang, Xiaoming; Xie, Dawei; Hsu, Chi-yuan; Feldman, Harold I.; Coresh, Josef; Vasan, Ramachandran S.; Kimmel, Paul L.; Liu, Kathleen D.

    2014-01-01

    Aims/hypothesis Kidney injury molecule 1 (KIM-1), liver fatty acid-binding protein (L-FABP), N-acetyl-β-D-glucosaminidase (NAG) and neutrophil gelatinase-associated lipocalin (NGAL) are urinary biomarkers of renal tubular injury. We examined their association with incident end-stage renal disease (ESRD) and all-cause mortality in American Indians with type 2 diabetes. Methods Biomarker concentrations were measured in baseline urine samples in 260 Pima Indians who were followed for a median of 14 years. HRs were reported per SD of creatinine (Cr)-normalised log-transformed KIM-1, NAG and NGAL, and for three categories of L-FABP. Results During follow-up, 74 participants developed ESRD and 101 died. Median concentrations of KIM-1/Cr, NAG/Cr and NGAL/Cr and the proportion of detectable L-FABP were highest in those with macroalbuminuria (p < 0.001 for KIM-1/Cr, NAG/Cr and L-FABP; p = 0.006 for NGAL/Cr). After multivariable adjustment, NGAL/Cr was positively associated with ESRD (HR 1.59, 95% CI 1.20, 2.11) and mortality (HR 1.39, 95% CI 1.06, 1.82); L-FABP/Cr was inversely associated with ESRD (HR [for highest vs lowest tertile] 0.40, 95% CI 0.19, 0.83). Addition of NGAL/Cr to models that included albuminuria and glomerular filtration rate increased the c-statistic for predicting ESRD from 0.828 to 0.833 (p = 0.001) and for death from 0.710 to 0.722 (p = 0.018). Addition of L-FABP/Cr increased the c-statistic for ESRD from 0.828 to 0.832 (p = 0.042). Conclusions/interpretation In Pima Indians with type 2 diabetes, urinary concentrations of NGAL and L-FABP are associated with important health outcomes, but they are unlikely to add to risk prediction with standard markers in a clinically meaningful way given the small increase in the c-statistic. PMID:25316431

  9. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. PMID:26873273

  10. Urinary KIM-1, NGAL and L-FABP for the diagnosis of AKI in patients with acute coronary syndrome or heart failure undergoing coronary angiography.

    PubMed

    Torregrosa, Isidro; Montoliu, Carmina; Urios, Amparo; Andrés-Costa, María Jesús; Giménez-Garzó, Carla; Juan, Isabel; Puchades, María Jesús; Blasco, María Luisa; Carratalá, Arturo; Sanjuán, Rafael; Miguel, Alfonso

    2015-11-01

    Acute kidney injury (AKI) is a common complication after coronary angiography. Early biomarkers of this disease are needed since increase in serum creatinine levels is a late marker. To assess the usefulness of urinary kidney injury molecule-1 (uKIM-1), neutrophil gelatinase-associated lipocalin (uNGAL) and liver-type fatty acid-binding protein (uL-FABP) for early detection of AKI in these patients, comparing their performance with another group of cardiac surgery patients. Biomarkers were measured in 193 patients, 12 h after intervention. In the ROC analysis, AUC for KIM-1, NGAL and L-FABP was 0.713, 0.958 and 0.642, respectively, in the coronary angiography group, and 0.716, 0.916 and 0.743 in the cardiac surgery group. Urinary KIM-1 12 h after intervention is predictive of AKI in adult patients undergoing coronary angiography, but NGAL shows higher sensitivity and specificity. L-FABP provides inferior discrimination for AKI than KIM-1 or NGAL in contrast to its performance after cardiac surgery. This is the first study showing the predictive capacity of KIM-1 for AKI after coronary angiography. Further studies are still needed to answer relevant questions about the clinical utility of biomarkers for AKI in different clinical settings. PMID:24989970

  11. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  12. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation. PMID:6283503

  13. Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1987-01-01

    The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991]. Images Fig. 1. Fig. 3. PMID:3593284

  14. Cold stress initiates the Nrf2/UGT1A1/L-FABP signaling pathway in chickens.

    PubMed

    Chen, X Y; Li, R; Geng, Z Y

    2015-11-01

    Cold stress triggers an anti-oxidative response in animals regulated by Nrf2 (nuclear factor 2-like, NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. To identify chicken Nrf2-regulated genes, 3 genetically related experimental groups (EG) with 40 Huainan partridge chickens in each group were chosen. The chickens were maintained at 20°C environmental temperature from 5 wk of age. At 6 wk of age, 10 chickens from each EG were still maintained at 20°C as control, and the other 30 chickens from each EG were exposed to 6 ± 2°C. Liver samples were collected from the control and from chickens exposed to 6 ± 2°C for 12, 24, and 72 h for co-immuno-precipitation (CoIP) analysis. Chromatin immunoprecipitation (ChIP)-sequencing experiment in liver cells treated with Dimethyl fumarate (DMF) were carried out. A de novo motif was discovered which closely matched the core Nrf2 consensus binding motif. Genes involved in de novo motif discovery were further analyzed for their enrichment in the anti-oxidative response pathway and the lipid anabolism pathway. There were 14 genes found which are related to oxidative stress. To examine the downstream factors of the 14 responsive genes, one of them, UGT1A1 (UDP glucuronosyltransferase), was further analyzed by CoIP experiment and nano LC-ESI-MS/MS analysis. It was detected that fatty acid-binding protein (L-FABP, 127 AA) might be the potential UGT1A1 downstream interaction proteins. In conclusion, it is proposed that chickens under cold stress generate anti-oxidative stress and thus trigger the Nrf2/ARE signaling pathway, which further up-regulates the expression of L-FABP to inactivate lipid peroxidation of the cell membrane and promote fatty acid storage against the cold environment. PMID:26453599

  15. Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism.

    PubMed

    Ong, Kuok Teong; Mashek, Mara T; Davidson, Nicholas O; Mashek, Douglas G

    2014-05-01

    Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation. PMID:24610891

  16. Retinoic acid-binding protein, rhombomeres and the neural crest.

    PubMed

    Maden, M; Hunt, P; Eriksson, U; Kuroiwa, A; Krumlauf, R; Summerbell, D

    1991-01-01

    We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed. PMID:1707786

  17. Expression of liver fatty acid binding protein in hepatocellular carcinoma.

    PubMed

    Cho, Soo-Jin; Ferrell, Linda D; Gill, Ryan M

    2016-04-01

    Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  18. Distinct Roles of Urinary Liver-Type Fatty Acid-Binding Protein in Non-Diabetic Patients with Anemia

    PubMed Central

    Imai, Naohiko; Yasuda, Takashi; Kamijo-Ikemori, Atsuko; Shibagaki, Yugo; Kimura, Kenjiro

    2015-01-01

    Background Various stresses including ischemia are known to up-regulate renal L-FABP gene expression and increase the urinary excretion of L-FABP. In diabetic patients with anemia, the urinary excretion of L-FABP is significantly increased. We studied the clinical significance of urinary L-FABP and its relationship with anemia in non-diabetic patients. Subjects and Methods A total of 156 patients were studied in this retrospective cross-sectional analysis. The associations between anemia and urinary L-FABP levels, and the predictors of urinary L-FABP levels in non-diabetic patients were evaluated. Results Urinary L-FABP levels were significantly higher in patients with anemia compared to those in patients without anemia. Similarly, the urinary L-FABP levels were significantly higher in patients with albuminuria compared to those in patients without albuminuria. Urinary L-FABP levels correlated with urinary albumin-to-creatinine ratios, estimated glomerular filtration rates, body mass index, and hemoglobin levels. Multivariate linear regression analysis determined that hemoglobin levels (β = -0.249, P = 0.001) and urinary albumin-to-creatinine ratios (β = 0.349, P < 0.001) were significant predictors of urinary L-FABP levels. Conclusions Urinary L-FABP is strongly associated with anemia in non-diabetic patients. PMID:26010898

  19. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  20. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  1. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    PubMed

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  2. Fatty Acid Binding Protein-1 (FABP1) and the Human FABP1 T94A Variant: Roles in the Endocannabinoid System and Dyslipidemias.

    PubMed

    Schroeder, Friedhelm; McIntosh, Avery L; Martin, Gregory G; Huang, Huan; Landrock, Danilo; Chung, Sarah; Landrock, Kerstin K; Dangott, Lawrence J; Li, Shengrong; Kaczocha, Martin; Murphy, Eric J; Atshaves, Barbara P; Kier, Ann B

    2016-06-01

    The first discovered member of the mammalian FABP family, liver fatty acid binding protein (FABP1, L-FABP), occurs at high cytosolic concentration in liver, intestine, and in the case of humans also in kidney. While the rat FABP1 is well studied, the extent these findings translate to human FABP1 is not clear-especially in view of recent studies showing that endocannabinoids and cannabinoids represent novel rat FABP1 ligands and FABP1 gene ablation impacts the hepatic endocannabinoid system, known to be involved in non-alcoholic fatty liver (NAFLD) development. Although not detectable in brain, FABP1 ablation nevertheless also impacts brain endocannabinoids. Despite overall tertiary structure similarity, human FABP1 differs significantly from rat FABP1 in secondary structure, much larger ligand binding cavity, and affinities/specificities for some ligands. Moreover, while both mouse and human FABP1 mediate ligand induction of peroxisome proliferator activated receptor-α (PPARα), they differ markedly in pattern of genes induced. This is critically important because a highly prevalent human single nucleotide polymorphism (SNP) (26-38 % minor allele frequency and 8.3 ± 1.9 % homozygous) results in a FABP1 T94A substitution that further accentuates these species differences. The human FABP1 T94A variant is associated with altered body mass index (BMI), clinical dyslipidemias (elevated plasma triglycerides and LDL cholesterol), atherothrombotic cerebral infarction, and non-alcoholic fatty liver disease (NAFLD). Resolving human FABP1 and the T94A variant's impact on the endocannabinoid and cannabinoid system is an exciting challenge due to the importance of this system in hepatic lipid accumulation as well as behavior, pain, inflammation, and satiety. PMID:27117865

  3. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  4. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    PubMed Central

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    SUMMARY Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have difference functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  5. Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

    PubMed Central

    Keler, T; Barker, C S; Sorof, S

    1992-01-01

    The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen

  6. Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein.

    PubMed

    Long, Dong; Yang, Daiwen

    2009-02-18

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands. PMID:19217864

  7. Fatty acid induced remodeling within the human liver fatty acid-binding protein.

    PubMed

    Sharma, Ashwani; Sharma, Amit

    2011-09-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP. PMID:21757748

  8. The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

    PubMed

    Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

    1992-02-01

    The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

  9. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-05-15

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  10. Phenotypic divergence in two lines of L-Fabp-/- mice reflects substrain differences and environmental modifiers.

    PubMed

    Newberry, Elizabeth P; Kennedy, Susan; Xie, Yan; Luo, Jianyang; Jiang, Hui; Ory, Daniel S; Davidson, Nicholas O

    2015-10-15

    Phenotypic divergence in diet-induced obesity (DIO) and hepatic steatosis has been reported in two independently generated lines of L-Fabp(-/-) mice [New Jersey (NJ) L-Fabp(-/-) vs. Washington University (WU) L-Fabp(-/-) mice]. We performed side-by-side studies to examine differences between the lines and investigate the role of genetic background, intestinal microbiota, sex, and diet in the divergent phenotypes. Fasting-induced steatosis was attenuated in both L-Fabp(-/-) lines compared with C57BL/6J controls, with restoration of hepatic triglyceride levels following adenoviral L-Fabp rescue. Both lines were protected against DIO after high-saturated-fat diet feeding. Hepatic steatosis was attenuated in WU but not NJ L-Fabp(-/-) mice, although this difference between the lines disappeared upon antibiotic treatment and cohousing. In contrast, there was phenotypic divergence in L-Fabp(-/-) mice fed a high cocoa butter fat diet, with WU L-Fabp(-/-) mice, but not NJ L-Fabp(-/-) mice, showing protection against both DIO and hepatic steatosis, with some sex-dependent (female > male) differences. Dense mapping revealed no evidence of unintended targeting, duplications, or deletions surrounding the Fabp1 locus in either line and only minor differences in mRNA expression of genes located near the targeted allele. However, a C57BL/6 substrain screen showed that the NJ L-Fabp(-/-) line contains ∼40% C57BL/6N genomic DNA, despite reports that these mice were backcrossed six generations. Overall, these findings suggest that some of the phenotypic divergence between the two L-Fabp(-/-) lines may reflect unanticipated differences in genetic background, underscoring the importance of genetic background in phenotypic characterization. PMID:26251469

  11. Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: the key role of the neutral species.

    PubMed

    Woodcroft, Mark W; Ellis, David A; Rafferty, Steven P; Burns, Darcy C; March, Raymond E; Stock, Naomi L; Trumpour, Kyle S; Yee, Janet; Munro, Kim

    2010-08-01

    Perfluorocarboxylic acids (PFCAs) of chain length greater than seven carbon atoms bioconcentrate in the livers of fish. However, a mechanistic cause for the empirically observed increase in the bioconcentration potential of PFCAs as a function of chain length has yet to be determined. To this end, recombinant rat liver fatty acid-binding protein (L-FABP) was purified, and its interaction with PFCAs was characterized in an aqueous system at pH 7.4. Relative binding affinities of L-FABP with PFCAs of carbon chain lengths of five to nine were established fluorimetrically. The energetics, mechanism, and stoichiometry of the interaction of perfluorooctanoic acid (PFOA) with L-FABP were examined further by isothermal titration calorimetry (ITC) and electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS). Perfluorooctanoic acid was shown to bind to L-FABP with an affinity approximately an order of magnitude less than the natural ligand, oleic acid, and to have at least 3:1 PFOA:L-FABP stoichiometry. Two distinct modes of PFOA binding to L-FABP were observed by ESI-MS/MS analysis; in both cases, PFOA binds solely as the neutral species under typical physiological pH and aqueous concentrations of the anion. A comparison of their chemical and physical properties with other well-studied biologically relevant chemicals showed that accumulation of PFCAs in proteins as the neutral species is predictable. For example, the interaction of PFOA with L-FABP is almost identical to that of the acidic ionizing drugs ketolac, ibuprofen, and warfarin that show specificity to protein partitioning with a magnitude that is proportional to the K(OW) (octanol-water partitioning) of the neutral species. The experimental results suggest that routine pharmacochemical models may be applicable to predicting the protein-based bioaccumulation of long-chain PFCAs. PMID:20821618

  12. Bile acid binding capacity of fish protein hydrolysates from discard species of the West Mediterranean Sea.

    PubMed

    Pérez-Gálvez, Raúl; García-Moreno, Pedro J; Morales-Medina, Rocío; Guadix, Antonio; Guadix, Emilia M

    2015-04-01

    Fish protein hydrolysates (FPH), produced from the six main discard species from the West Mediterranean Sea (sardine, horse mackerel, axillary seabream, bogue, small-spotted catshark and blue whiting) were tested for their bile acid binding capacity. This capacity is directly linked to the ability to inhibit bile reabsorption in the ileum and therefore to lower cholesterol levels in the bloodstream. From each species, FPH were obtained by three different enzymatic treatments employing two serine endoproteases (subtilisin and trypsin) sequentially or in combination. The results show statistically significant differences among the fish species, attaining interesting average values of bile acid binding capacity for blue whiting (27.32% relative to cholestyramine on an equal protein basis) and horse mackerel (27.42% relative to cholestyramine on an equal protein basis). The enzymatic treatments did not significantly affect the ability of a given species to bind bile acids. These results are similar to other protein sources, such as soy protein or casein, of proven hypocholesterolemic effect. It can be concluded that fish protein hydrolysates from these discard species are suitable as ingredients in the formulation of cholesterol-lowering supplements. PMID:25756593

  13. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    PubMed

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  14. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  15. Intramuscular fat content and genetic variants at fatty acid-binding protein loci in Austrian pigs.

    PubMed

    Nechtelberger, D; Pires, V; Söolknet, J; Stur; Brem, G; Mueller, M; Mueller, S

    2001-11-01

    Intramuscular fat is an important meat quality trait in pig production. Previously, genetic variants of the heart fatty acid-binding protein (H-FABP) gene and the adipocyte fatty acid-binding protein (A-FABP) gene were suggested to be associated with intramuscular fat content. The objective of this investigation was to study these associations in the three most important Austrian breeding populations (Piétrain, Large White, and Landrace). Restriction fragment length polymorphism analysis of the H-FABP gene revealed a new MspI polymorphic site and genetic variation in all three breeds. Microsatellite analysis of the A-FABP locus showed up to nine different microsatellite alleles segregating. In Austrian breeds, no significant influence of the A-FABP and H-FABP gene polymorphisms on intramuscular fat could be detected. We also evaluated possible associations between the genetic variations at the H-FABP and A-FABP loci and other growth and carcass traits (average daily gain, feed conversion ratio, lean meat content, pH values, meat color, and drip loss). With regard to the extent of the effects, these genetic markers cannot be recommended for selection on growth and carcass traits in Austrian breeding populations. PMID:11768107

  16. Molecular dynamics simulation of ligand dissociation from liver fatty acid binding protein.

    PubMed

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-01-01

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized "portal region" could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques. PMID:19564911

  17. Liver fatty acid binding protein: species variation and the accommodation of different ligands.

    PubMed

    Thompson, J; Reese-Wagoner, A; Banaszak, L

    1999-11-23

    The crystal structure of rat liver fatty acid binding protein (LFABP) and an alignment of amino acid sequences of all known species have been used to demonstrate two groups or sub-classes. Based on estimates at neutral pH and the electrostatic field calculated using the crystal coordinates, some evidence of changes that occur in going from holo- to apo-forms has been obtained. LFABP belongs to a large family frequently referred to as the intracellular lipid binding proteins or iLBPs. LFABP, unlike other family members, has two fatty acid binding sites. The two cavity sites have been reviewed and arguments for interactions between the sites are presented. Based on the crystal structure of rat LFABP, differences between the A and B groups have been postulated. Last of all, hypothetical models have been built of complexes of LFABP and heme, and LFABP and oleoyl CoA. In both cases, the stoichiometry is one to one and the models show why this is likely. PMID:10570240

  18. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

  19. Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology

    PubMed Central

    Kizilbash, Nadeem A; Hai, Abdul; Alruwaili, Jamal

    2013-01-01

    The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A “gap” is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein. PMID:24497726

  20. Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1986-01-01

    Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

  1. Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.

    PubMed Central

    Stremmel, W; Strohmeyer, G; Borchard, F; Kochwa, S; Berk, P D

    1985-01-01

    When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids. Images PMID:3881757

  2. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  3. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4)

    PubMed Central

    González, Javier M.; Fisher, S. Zoë

    2015-01-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments. PMID:25664790

  4. Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

    PubMed

    Uehara, Hisanori; Takahashi, Tetsuyuki; Oha, Mina; Ogawa, Hirohisa; Izumi, Keisuke

    2014-12-01

    Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link. PMID:24740818

  5. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  6. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM. PMID:19911253

  7. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  8. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy.

    PubMed

    Bottasso Arias, Natalia M; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  9. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

    PubMed Central

    Krempl, C; Schultze, B; Laude, H; Herrler, G

    1997-01-01

    Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV. PMID:9060696

  10. BILE ACIDS REGULATE THE ONTOGENIC EXPRESSION OF ILEAL BILE ACID BINDING PROTEIN IN THE RAT VIA THE FARNESOID X RECEPTOR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the rat, an increase in ileal bile acid binding protein (IBABP) expression occurs during the third postnatal week. In vitro studies suggest that bile acids (BAs) increase IBABP transcription by activating the BA receptor, farnesoid X receptor (FXR). Thus, we investigated the role of BAs on the on...

  11. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  12. Nucleic acid-binding properties of the RRM-containing protein RDM1

    SciTech Connect

    Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric . E-mail: Vandyck@iarc.fr

    2006-05-26

    RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L{sub 119}GF {sup {yields}} AAA mutation affects the mode of RDM1 binding to single-stranded DNA.

  13. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  14. Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

    PubMed Central

    Simões, Ana Leda Bertoncini; Figueira, Rebeca Lopes; Gonçalves, Frances Lilian Lanhellas; Mitidiero, Luís Felipe Tsuyoshi; Silva, Orlando Castro e; Peiró, José Luis; Sbragia, Lourenço

    2016-01-01

    OBJECTIVES: Necrotizing enterocolitis is a severe multifactorial intestinal disorder that primarily affects preterm newborns, causing 20-40% mortality and morbidity. Intestinal fatty acid-binding protein has been reported to be a biomarker for the detection of intestinal injuries. Our aim was to assess intestinal tissue injury and the molecular expression of intestinal fatty acid-binding protein over time in a necrotizing enterocolitis model. METHODS: A total of 144 Newborn rats were divided into two groups: 1) Control, which received breastfeeding (n=72) and 2) Necrotizing Enterocolitis, which received formula feeding and underwent hypoxia and hypothermia (n=72). A total of six time points of ischemia (2 times a day for 3 days; 12 pups for each time point) were examined. Samples were collected for analysis of body weight, morphological and histological characteristics, intestinal weight, intestinal weight/body weight ratio, injury grade, and intestinal fatty acid-binding protein levels. RESULTS: Body and intestinal weights were lower in the Necrotizing Enterocolitis group than in the Control group (p<0.005 and p<0.0005, respectively). The intestinal weight/body weight ratio was higher in the Necrotizing Enterocolitis group than in the Control group (p<0.005) only at the sixth ischemia time point. The Necrotizing Enterocolitis group displayed higher expression of intestinal fatty acid-binding protein (p<0.0005) and showed greater tissue damage than the Control group. CONCLUSION: Intestinal fatty acid-binding protein was an efficient marker of ischemic injury to the intestine and a good correlation was demonstrated between the time of ischemic injury and the grade of intestinal injury. PMID:27464299

  15. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  16. Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

    PubMed

    Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

    2016-09-01

    The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets. PMID:27495901

  17. Plasma Fatty Acid Binding Protein 4 and Risk of Sudden Cardiac Death in Older Adults

    PubMed Central

    Djoussé, Luc; Maziarz, Marlena; Biggs, Mary L.; Ix, Joachim H.; Zieman, Susan J.; Kizer, Jorge R.; Lemaitre, Rozenn N.; Mozaffarian, Dariush; Tracy, Russell P.; Mukamal, Kenneth J.; Siscovick, David S.; Sotoodehnia, Nona

    2013-01-01

    Although fatty acid binding protein 4 (FABP4) may increase risk of diabetes and exert negative cardiac inotropy, it is unknown whether plasma concentrations of FABP4 are associated with incidence of sudden cardiac death (SCD). We prospectively analyzed data on 4,560 participants of the Cardiovascular Health Study. FABP4 was measured at baseline using ELISA, and SCD events were adjudicated through review of medical records. We used Cox proportional hazards to estimate effect measures. During a median followup of 11.8 years, 146 SCD cases occurred. In a multivariable model adjusting for demographic, lifestyle, and metabolic factors, relative risk of SCD associated with each higher standard deviation (SD) of plasma FABP4 was 1.15 (95% CI: 0.95–1.38), P = 0.15. In a secondary analysis stratified by prevalent diabetes status, FABP4 was associated with higher risk of SCD in nondiabetic participants, (RR per SD higher FABP4: 1.33 (95% CI: 1.07–1.65), P = 0.009) but not in diabetic participants (RR per SD higher FABP4: 0.88 (95% CI: 0.62–1.27), P = 0.50), P for diabetes-FABP4 interaction 0.049. In summary, a single measure of plasma FABP4 obtained later in life was not associated with the risk of SCD in older adults overall. Confirmation of our post-hoc results in nondiabetic people in other studies is warranted. PMID:24455402

  18. Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

    SciTech Connect

    Montoudis, Alain; Delvin, Edgard; Menard, Daniel

    2006-01-06

    Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.

  19. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  20. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels.

    PubMed

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  1. Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia

    PubMed Central

    Kaczocha, Martin; Rebecchi, Mario J.; Ralph, Brian P.; Teng, Yu-Han Gary; Berger, William T.; Galbavy, William; Elmes, Matthew W.; Glaser, Sherrye T.; Wang, Liqun; Rizzo, Robert C.; Deutsch, Dale G.; Ojima, Iwao

    2014-01-01

    The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics. PMID:24705380

  2. Serologic Intestinal-Fatty Acid Binding Protein in Necrotizing Enterocolitis Diagnosis: A Meta-Analysis

    PubMed Central

    Cheng, Shupeng; Yu, Jialin; Zhou, Min; Tu, Yan; Lu, Qi

    2015-01-01

    Background. Previous studies showed that intestinal-fatty acid binding protein (I-FABP) may be a valid and promising serologic biomarker for early diagnosis of necrotizing enterocolitis (NEC). Objective. To investigate the early diagnostic value of serologic I-FABP in NEC for the premature neonates. Methods. All major databases were searched from January 1, 1990, to May 1, 2015. We used Meta-Disc 1.4 and Revman5.0 software to calculate the diagnostic accuracy. Results. Seven studies with 444 subjects were identified. The pooled sensitivity of I-FABP was 0.67 for NEC I, 0.74 for NEC II, and 0.83 for NEC III, and the pooled specificity was 0.84, respectively, which showed a moderate diagnostic accuracy. The area under curve (AUC) for each stage was 0.75 (Q⁎ = 0.69), 0.82 (Q⁎ = 0.76), and 0.91 (Q⁎ = 0.84). The diagnostic threshold analysis showed no significant difference in threshold effect. The metaregression showed that the cut-off value has the largest effect on heterogeneity. The funnel plots indicated the existence of publication bias. Conclusion. I-FABP is a valid serologic biomarker for early diagnosis in NEC for the premature neonates with a moderate accuracy. PMID:26798632

  3. Polymorphisms in Fatty Acid Binding Protein 5 Show Association with Type 2 Diabetes

    PubMed Central

    Bu, Liming; Salto, Lorena M.; De Leon, Kevin J; De Leon, Marino

    2011-01-01

    Genes for the fatty acid binding protein (FABP) family encode small 14–15 kDa cytosolic proteins and can be regulated during type 2 diabetes mellitus (T2DM) and obesity. This study compared association of single nucleotide polymorphisms (SNPs) in FABP1-5 with T2DM in different ethnic groups. Associations with T2DM of SNPs in these proteins were assessed in African American (AA), non-Hispanic White (NHW), and Hispanic American (HA) individuals. A total of 650 DNA samples were genotyped; control samples were obtained from Coriell’s North American Human Variation Panel Repository (NAVP) of apparently healthy individuals and T2DM cases were taken from the American Diabetes Association GENNID Study. The rs454550 SNP of FABP5 showed a significant association with T2DM in NHW (OR: 9.03, 95% CI: 1.13–71.73, p=0.014). Our analysis also identified a new FABP5 SNP (nSNP) that showed a significant association with T2DM in NHW (OR: 0.44, 95% CI: 0.19–0.99, p=0.045) and AA (OR: 0.17, 95% CI: 0.03–0.80, p=0.016). The Ala54Thr FABP2 polymorphism was significantly associated with T2DM in HA individuals only (OR: 1.85, 95% CI: 1.05–3.27, p=0.032). All other FABP SNPs did not show association with T2DM. These findings suggest a potential distinct role of SNPs in FABP5, 2 genes in T2DM in different populations. PMID:21288588

  4. Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

    PubMed

    Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

    2009-11-15

    Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. PMID:19603488

  5. Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

    PubMed

    Sani, B P; Dawson, M I; Hobbs, P D; Chan, R L; Schiff, L J

    1984-01-01

    Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed. PMID:6317169

  6. Fatty acid-binding proteins and peribronchial angiogenesis in bronchopulmonary dysplasia.

    PubMed

    Ghelfi, Elisa; Karaaslan, Cagatay; Berkelhamer, Sara; Akar, Serra; Kozakewich, Harry; Cataltepe, Sule

    2011-09-01

    Inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD). Fatty acid-binding proteins (FABPs) 4 and 5 regulate the inflammatory activity of macrophages. Whether FABPs 4 and 5 could play a role in the pathogenesis of BPD via the promotion of macrophage inflammatory activity is unknown. This study sought to examine whether the expression levels of FABP4 and FABP5 were altered in bronchoalveolar lavage fluid and lung tissue in a baboon model of BPD. This study also sought to characterize the cell types that express these proteins. Real-time PCR, immunoblotting, immunohistochemistry, and double immunofluorescence were used to examine the expression of FABPs in samples of BPD. Morphometric analysis was used to quantify FABP4-positive peribronchial blood vessels in lung sections. FABP4 was primarily expressed in macrophages in samples of BPD. In addition, FABP4 was expressed in the endothelial cells of blood vessels in peribronchial areas and the vasa vasorum, but not in the alveolar vasculature in samples of BPD. FABP4 concentrations were significantly increased in lungs and bronchoalveolar lavage fluid samples with BPD. An increased density of FABP4-positive peribronchial blood vessels was evident in both baboon and human BPD sections. FABP5 was expressed in several cell types, including alveolar epithelial cells and macrophages. FABP5 concentrations did not show any significant alterations in BPD. In conclusion, FABP4 but not FABP5 levels are increased in BPD. FABP4 is differentially expressed in endothelial cells of the bronchial microvasculature, which demonstrates a previously unrecognized expansion in BPD. PMID:21177979

  7. Clinical significance of urinary liver-type fatty acid binding protein at various stages of nephropathy.

    PubMed

    Viswanathan, V; Sivakumar, S; Sekar, V; Umapathy, D; Kumpatla, S

    2015-01-01

    This cross-sectional study was to evaluate the levels of urinary liver-type fatty acid binding protein (u-LFABP pg/mg urine creatinine ratio) at different stages of diabetic nephropathy and to see its correlation with other clinical parameters in South Indian patients with type 2 diabetes mellitus (T2DM). A total of 65 (M: F; 42:23) T2DM subjects were divided into three groups, and were compared with 13 (M: F; 3:10) nondiabetic controls. The study groups were as follows: normoalbuminuric (n = 22), microalbuminuric (n = 22) and macroalbuminuric (n = 21). Estimated glomerular filtration rate (eGFR) was calculated using Cockcroft and Gault formula. u-LFABP levels in spot urine samples were measured with a solid phase enzyme linked immunosorbent assay. This study showed that u-LFABP levels were undetectable in healthy controls and was very low in the normoalbuminuric subjects. Elevated levels of u-LFABP are evident from the microalbuminuric stage indicating tubular damage. The levels of u-LFABP increased gradually with declining renal function. Geometric mean (95% confidence interval) for normoalbuminuria was 0.65 (0.47-0.97), microalbuminuria was 0.99 (0.55-1.97) and macroalbuminuria was 5.16 (1.8-14.5), (P = 0.005). In conclusion, u-LFABP levels were elevated in patients with reduced eGFR and showed a positive correlation with systolic blood pressure and protein to creatinine ratio in the total study subjects. PMID:26628791

  8. Clinical significance of urinary liver-type fatty acid binding protein at various stages of nephropathy

    PubMed Central

    Viswanathan, V.; Sivakumar, S.; Sekar, V.; Umapathy, D.; Kumpatla, S.

    2015-01-01

    This cross-sectional study was to evaluate the levels of urinary liver-type fatty acid binding protein (u-LFABP pg/mg urine creatinine ratio) at different stages of diabetic nephropathy and to see its correlation with other clinical parameters in South Indian patients with type 2 diabetes mellitus (T2DM). A total of 65 (M: F; 42:23) T2DM subjects were divided into three groups, and were compared with 13 (M: F; 3:10) nondiabetic controls. The study groups were as follows: normoalbuminuric (n = 22), microalbuminuric (n = 22) and macroalbuminuric (n = 21). Estimated glomerular filtration rate (eGFR) was calculated using Cockcroft and Gault formula. u-LFABP levels in spot urine samples were measured with a solid phase enzyme linked immunosorbent assay. This study showed that u-LFABP levels were undetectable in healthy controls and was very low in the normoalbuminuric subjects. Elevated levels of u-LFABP are evident from the microalbuminuric stage indicating tubular damage. The levels of u-LFABP increased gradually with declining renal function. Geometric mean (95% confidence interval) for normoalbuminuria was 0.65 (0.47–0.97), microalbuminuria was 0.99 (0.55–1.97) and macroalbuminuria was 5.16 (1.8–14.5), (P = 0.005). In conclusion, u-LFABP levels were elevated in patients with reduced eGFR and showed a positive correlation with systolic blood pressure and protein to creatinine ratio in the total study subjects. PMID:26628791

  9. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-01

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  10. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  11. Fatty Acid Binding Protein 4 Deficiency Protects against Oxygen-Induced Retinopathy in Mice

    PubMed Central

    Saint-Geniez, Magali; Ghelfi, Elisa; Liang, Xiaoliang; Yu, Chenwei; Spencer, Carrie; Abend, Stephanie; Hotamisligil, Gokhan; Cataltepe, Sule

    2014-01-01

    Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4−/− mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4−/− OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies. PMID:24802082

  12. Fatty acid binding protein 4 deficiency protects against oxygen-induced retinopathy in mice.

    PubMed

    Saint-Geniez, Magali; Ghelfi, Elisa; Liang, Xiaoliang; Yu, Chenwei; Spencer, Carrie; Abend, Stephanie; Hotamisligil, Gokhan; Cataltepe, Sule

    2014-01-01

    Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4-/- mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4-/- OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies. PMID:24802082

  13. Examination of the Addictive and Behavioral Properties of Fatty Acid-Binding Protein Inhibitor SBFI26.

    PubMed

    Thanos, Panayotis K; Clavin, Brendan H; Hamilton, John; O'Rourke, Joseph R; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested. PMID:27092087

  14. Examination of the Addictive and Behavioral Properties of Fatty Acid-Binding Protein Inhibitor SBFI26

    PubMed Central

    Thanos, Panayotis K.; Clavin, Brendan H.; Hamilton, John; O’Rourke, Joseph R.; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested. PMID:27092087

  15. Fatty acid-binding protein 5 limits the anti-inflammatory response in murine macrophages.

    PubMed

    Moore, Sherri M; Holt, Vivian V; Malpass, Lillie R; Hines, Ian N; Wheeler, Michael D

    2015-10-01

    The beginning stages of liver damage induced by various etiologies (i.e. high fat diet, alcohol consumption, toxin exposure) are characterized by abnormal accumulation of lipid in liver. Alterations in intracellular lipid transport, storage, and metabolism accompanied by cellular insult within the liver play an important role in the pathogenesis of liver disease, often involving a sustained inflammatory response. The intracellular lipid transporter, fatty acid binding protein 5 (FABP5), is highly expressed in macrophages and may play an important role in the hepatic inflammatory response after endotoxin exposure in mice. This study tested the hypothesis that FABP5 regulates macrophage response to LPS in male C57bl/6 (wild type) and FABP5 knockout mice, both in vitro and in vivo. Treatment with LPS revealed that loss of FABP5 enhances the number of hepatic F4/80(+) macrophages in the liver despite limited liver injury. Conversely, FABP5 knock out mice display higher mRNA levels of anti-inflammatory cytokines IL-10, arginase, YM-1, and Fizz-1 in liver compared to wild type mice. Bone marrow derived macrophages stimulated with inflammatory (LPS and IFN-γ) or anti-inflammatory (IL-4) mediators also showed significantly higher expression of anti-inflammatory/regulatory factors. These findings reveal a regulatory role of FABP5 in the acute inflammatory response to LPS-induced liver injury, which is consistent with the principle finding that FABP5 is a regulator of macrophage phenotype. Specifically, these findings demonstrate that loss of FABP5 promotes a more anti-inflammatory response. PMID:26105806

  16. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    PubMed

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  17. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    PubMed Central

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  18. Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

    PubMed

    Espino, A M; Rodríguez Medina, J R; Hillyer, G V

    2001-10-01

    A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex. PMID:11695360

  19. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes

    SciTech Connect

    Bassuk, J.A.; Tsichlis, P.N.; Sorof, S.

    1987-11-01

    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). The authors report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambdagt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO/sub 4/ gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens.

  20. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  1. Serum complements and heart fatty acid binding protein in Bangladeshi patients with acute myocardial infarction

    PubMed Central

    Akhtar, Nayareen; Taher, Abu; Rahman, Rezwanur; Chowdhury, Ashesh Kumar

    2012-01-01

    The complement system is activated following acute myocardial infarction (AMI). Heart fatty acid binding protein (H-FABP) is a sensitive early biomarker of myocardial necrosis that can be used to confirm or exclude a diagnosis of AMI and to monitor recurrent infarction. This study was designed to detect changes in C3, C4 and H-FABP after AMI. Forty patients with AMI and a control group of 40 apparently healthy people were included. Selections were based on inclusion and exclusion criteria. The baseline characteristics were not significantly different between the groups. Patients’ blood samples were collected within 12 h of admission. Significant increases in C3 (AMI group 1.4260+0.04, healthy group 1.26040+0.04; p<0.05), C4 (AMI group 0.29305±0.013, healthy group 0.20860±0.012; p<0.05) and H-FABP (AMI group 12.3±1.69, healthy group 0.16±0.057; p<0.001) were seen in patients with AMI. The correlation between serum C3 and body mass index (BMI, r=0.33; p<0.05), serum C4 and BMI(r=0.313; p<0.05), serum C3 and total cholesterol high density lipoprotein (HDL, r=0.32; p<0.05), serum C4 and HbA1C (r=0.335; p<0.05) and serum C3 and troponin I (r= 0.325p<0.05) was found to be significant. But the correlation between serum C3 and waist:hip ratio (p=0.56), serum C4 and waist:hip ratio (p=0.83), serum C4 and total cholesterol HDL (p=0.993), serum C3 and HbA1C (p=0.440), serum C3 and random blood sugar (p=0.563), serum C4 and random blood sugar (p=0.828) and serum C4 and troponin I (p=0.373) was not significant. The significant complement activation detected in the plasma of patients with AMI indicated that complement plays a part in the pathogenesis of myocardial infarction. A significant increase of H-FABP improves the diagnosis of AMI.

  2. Association Between Serum Levels of Adipocyte Fatty Acid-binding Protein and Free Thyroxine

    PubMed Central

    Tseng, Fen-Yu; Chen, Pei-Lung; Chen, Yen-Ting; Chi, Yu-Chao; Shih, Shyang-Ron; Wang, Chih-Yuan; Chen, Chi-Ling; Yang, Wei-Shiung

    2015-01-01

    Abstract Adipocyte fatty acid-binding protein (AFABP) has been shown to be a biomarker of body weight change and atherosclerosis. Changes in thyroid function are associated with changes in body weight and risks of cardiovascular diseases. The association between AFABP and thyroid function status has been seldom evaluated. The aim of this study was to compare the serum AFABP concentrations in hyperthyroid patients and those in euthyroid individuals, and to evaluate the associations between serum AFABP and free thyroxine (fT4) levels. For this study, 30 hyperthyroid patients and 30 euthyroid individuals at a referral medical center were recruited. The patients with hyperthyroidism were treated with antithyroid regimens as clinically indicated. No medication was given to the euthyroid individuals. The body weight, body mass index, thyroid function, serum levels of AFABP, and biochemical data of both groups at baseline and at the 6th month were compared. Associations between AFABP and fT4 levels were also analyzed. At the baseline, the hyperthyroid patients had significantly higher serum AFABP levels than the euthyroid individuals (median [Q1, Q3]: 22.8 [19.4, 30.6] ng/mL vs 18.6 [15.3, 23.2] ng/mL; P = 0.038). With the antithyroid regimens, the AFABP serum levels of the hyperthyroid patients decreased to 16.6 (15.0, 23.9) ng/mL at the 6th month. No difference in the AFABP level was found between the hyperthyroid and the euthyroid groups at the 6th month. At baseline, sex (female vs male, ß = 7.65, P = 0.022) and fT4 level (ß = 2.51, P = 0.018) were significantly associated with AFABP levels in the univariate regression analysis. At the 6th month, sex and fT4 level (ß = 8.09, P < 0.001 and ß = 3.61, P = 0.005, respectively) were also significantly associated with AFABP levels. The associations between sex and fT4 level with AFABP levels remained significant in the stepwise multivariate regression analysis, both at baseline and at

  3. Concomitant increase in hepatic triacylglycerol biosynthesis and cytosolic fatty-acid-binding-protein content after feeding rats with a cholestyramine-containing diet.

    PubMed Central

    Kempen, H J; Glatz, J F; de Lange, J; Veerkamp, J H

    1983-01-01

    Cholestyramine feeding of rats increased the rate of palmitate and glycerol incorporation into triacylglycerols of isolated hepatocytes. Concomitantly an increase of fatty-acid binding by hepatic cytosolic proteins was observed, which could be attributed to an elevation of the content of the fatty-acid-binding protein (Mr 12000). The involvement of this protein in cholesterol, bile-acid and triacylglycerol metabolism is discussed. PMID:6661214

  4. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  5. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

    PubMed Central

    Bassuk, J A; Tsichlis, P N; Sorof, S

    1987-01-01

    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens. Images PMID:3478711

  6. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    PubMed

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  7. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

    PubMed Central

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-01-01

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  8. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    PubMed

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  9. Enterocyte fatty acid-binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine.

    PubMed

    Gajda, Angela M; Storch, Judith

    2015-02-01

    Fatty acid-binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both liver- (LFABP; FABP1) and intestinal FABPs (IFABP; FABP2) are expressed. These proteins display high-affinity binding for long-chain fatty acids (FA) and other hydrophobic ligands; thus, they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand-binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have different functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  10. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    PubMed

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  11. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients

    PubMed Central

    2012-01-01

    Background Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. Methods This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Results Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR), triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT) levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Conclusions Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients. PMID:23249450

  12. Water-mediated recognition of simple alkyl chains by heart-type fatty-acid-binding protein.

    PubMed

    Matsuoka, Shigeru; Sugiyama, Shigeru; Matsuoka, Daisuke; Hirose, Mika; Lethu, Sébastien; Ano, Hikaru; Hara, Toshiaki; Ichihara, Osamu; Kimura, S Roy; Murakami, Satoshi; Ishida, Hanako; Mizohata, Eiichi; Inoue, Tsuyoshi; Murata, Michio

    2015-01-26

    Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10-C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths. PMID:25491543

  13. Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein

    PubMed Central

    Wu, Yun-li; Peng, Xian-e; Zhu, Yi-bing; Yan, Xiao-li; Chen, Wan-nan

    2015-01-01

    ABSTRACT Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3β-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. IMPORTANCE Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism

  14. Fatty Acid Binding Proteins FABP9 and FABP10 Participate in Antibacterial Responses in Chinese Mitten Crab, Eriocheir sinensis

    PubMed Central

    Li, Shuang; Guo, Xiao-Nv; Wang, Juan; Gong, Ya-Nan; He, Lin; Wang, Qun

    2013-01-01

    Invertebrates rely solely on the innate immune system for defense against pathogens and other stimuli. Fatty acid binding proteins (FABP), members of the lipid binding proteins superfamily, play a crucial role in fatty acid transport and lipid metabolism and are also involved in gene expression induced by fatty acids. In the vertebrate immune system, FABP is involved in inflammation regulated by fatty acids through its interaction with peroxidase proliferator activate receptors (PPARs). However, the immune functions of FABP in invertebrates are not well characterized. For this reason, we investigated the immune functionality of two fatty acid binding proteins, Es-FABP9 and Es-FABP10, following lipopolysaccharide (LPS) challenge in the Chinese mitten crab (Eriocheir sinensis). An obvious variation in the expression of Es-FABP9 and Es-FABP10 mRNA in E. sinensis was observed in hepatopancreas, gills, and hemocytes post-LPS challenge. Recombinant proteins rEs-FABP9 and rEs-FABP10 exhibited distinct bacterial binding activity and bacterial agglutination activity against Escherichia coli and Staphylococcus aureus. Furthermore, bacterial growth inhibition assays demonstrated that rEs-FABP9 responds positively to the growth inhibition of Vibrio parahaemolyticuss and S. aureus, while rEs-FABP10 responds positively to the growth inhibition of Aeromonas hydrophila and Bacillus subtilis. Coating of agarose beads with recombinant rEs-FABP9 and rEs-FABP10 dramatically enhanced encapsulation of the beads by crab hemocytes in vitro. In conclusion, the data presented here demonstrate the participation of these two lipid metabolism-related proteins in the innate immune system of E. sinensis. PMID:23365646

  15. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells

    PubMed Central

    Elmasri, Harun; Karaaslan, Cagatay; Teper, Yaroslav; Ghelfi, Elisa; Weng, MeiQian; Ince, Tan A.; Kozakewich, Harry; Bischoff, Joyce; Cataltepe, Sule

    2009-01-01

    Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs.—Elmasri, H., Karaaslan, C., Teper, Y., Ghelfi, E., Weng, M., Ince, T. A., Kozakewich, H., Bischoff, J., Cataltepe, S. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. PMID:19625659

  16. Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues

    PubMed Central

    Shan, Tizhong; Liu, Weiyi; Kuang, Shihuan

    2013-01-01

    Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP-based cell lineage tracing in mice, we have identified a population of aP2-expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2-lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFRα. When isolated and grown in culture, the aP2-expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild-type mice, the aP2-lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.—Shan, T., Liu, W., Kuang, S. Fatty acid-binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. PMID:23047894

  17. Peri-operative heart-type fatty acid binding protein is associated with acute kidney injury after cardiac surgery

    PubMed Central

    Schaub, Jennifer A.; Garg, Amit X.; Coca, Steven G.; Testani, Jeffrey M.; Shlipak, Michael G.; Eikelboom, John; Kavsak, Peter; McArthur, Eric; Shortt, Colleen; Whitlock, Richard; Parikh, Chirag R.

    2015-01-01

    Acute Kidney Injury (AKI) is a common complication after cardiac surgery and is associated with worse outcomes. Since heart fatty acid binding protein (H-FABP) is a myocardial protein that detects cardiac injury, we sought to determine if plasma H-FABP was associated with AKI in the TRIBE-AKI cohort; a multi-center cohort of 1219 patients at high risk for AKI who underwent cardiac surgery. The primary outcomes of interest were any AKI (Acute Kidney Injury Network (AKIN) stage 1 or higher) and severe AKI (AKIN stage 2 or higher). The secondary outcome was long-term mortality after discharge. Patients who developed AKI had higher levels of H-FABP pre- and post-operatively than patients who did not have AKI. In analyses adjusted for known AKI risk factors, first post-operative log(H-FABP) was associated with severe AKI (adjusted OR 5.39 [95% CI, 2.87-10.11] per unit increase), while pre-operative log(H-FABP) was associated with any AKI (2.07 [1.48-2.89]) and mortality (1.67 [1.17-2.37]). These relationships persisted after adjustment for change in serum creatinine (for first postoperative log(H-FABP)) and biomarkers of cardiac and kidney injury, including brain natriuretic peptide, cardiac troponin-I, interleukin-18, liver fatty acid binding protein, kidney injury molecule-1, and neutrophil gelatinase associated lipocalin. Thus, peri-operative plasma H-FABP levels may be used for risk-stratification of AKI and mortality following cardiac surgery. PMID:25830762

  18. The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation.

    PubMed

    Zhou, Yang; Nie, Tao; Zhang, Yan; Song, Ming; Li, Kuai; Ding, Mengxiao; Ding, Ke; Wu, Donghai; Xu, Yong

    2016-09-15

    Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (Ki=0.29±0.07μM, ΔTm=8.5°C). This compound's effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs. PMID:27460668

  19. Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

    PubMed Central

    Cannon, J R; Eacho, P I

    1991-01-01

    Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation. Images Fig. 2. PMID:1747111

  20. Identification of a functional polymorphism at the Adipose Fatty Acid Binding protein gene (FABP4) and demonstration of its association with cardiovascular disease: A path to follow

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty acid binding proteins (FABPs) are proteins that reversibly bind fatty acids and other lipids. So far, 9 tissue-specific cytoplasmic FABPs have been identified. Adipose tissue FABP (FABP4) has been suggested to be a bridge between inflammation and other pathways related to the metabolic syndrom...

  1. Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein.

    PubMed

    He, Yan; Yang, Xiaomin; Wang, Hsin; Estephan, Rima; Francis, Fouad; Kodukula, Sarala; Storch, Judith; Stark, Ruth E

    2007-11-01

    Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single

  2. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. PMID:26037116

  3. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.

    PubMed

    Elmasri, Harun; Karaaslan, Cagatay; Teper, Yaroslav; Ghelfi, Elisa; Weng, Meiqian; Ince, Tan A; Kozakewich, Harry; Bischoff, Joyce; Cataltepe, Sule

    2009-11-01

    Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs. PMID:19625659

  4. Fatty acid-binding protein (fabp) genes of spotted green pufferfish (Tetraodon nigroviridis): comparative genomics and spatial transcriptional regulation.

    PubMed

    Thirumaran, Aruloli; Wright, Jonathan M

    2014-05-01

    The fatty acid-binding protein (fabp) genes belong to the multigene family of intracellular lipid-binding proteins. To date, 12 different FABPs have been identified in vertebrate genomes. Owing to the teleost-specific genome duplication event, many fishes have duplicated copies of the fabp genes. Here, we identified and characterized the fabp genes of spotted green pufferfish (Tetraodon nigroviridis). Seven fabp genes were identified, out of which, two were retained in the pufferfish genome as duplicated copies. Each putative pufferfish Fabp protein shares greatest sequence identity and similarity with their teleost and tetrapod orthologs, and clustered together as a distinct clade in phylogenetic analysis. Conserved gene synteny was evident between the pufferfish fabp genes and the orthologs of human, zebrafish, three-spined stickleback, and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the teleost-specific genome duplication event. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes. PMID:25153522

  5. Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

    PubMed

    Kagawa, Yoshiteru; Yasumoto, Yuki; Sharifi, Kazem; Ebrahimi, Majid; Islam, Ariful; Miyazaki, Hirofumi; Yamamoto, Yui; Sawada, Tomoo; Kishi, Hiroko; Kobayashi, Sei; Maekawa, Motoko; Yoshikawa, Takeo; Takaki, Eiichi; Nakai, Akira; Kogo, Hiroshi; Fujimoto, Toyoshi; Owada, Yuji

    2015-05-01

    Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794. PMID:25601031

  6. Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.

    PubMed

    Herr, F M; Matarese, V; Bernlohr, D A; Storch, J

    1995-09-19

    The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7547918

  7. Low abdominal NIRS values and elevated plasma intestinal fatty acid-binding protein in a premature piglet model of necrotizing enterocolitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC...

  8. Identification of a Soluble, High-Affinity Salicylic Acid-Binding Protein in Tobacco.

    PubMed Central

    Du, H.; Klessig, D. F.

    1997-01-01

    Salicylic acid (SA) is a key component in the signal transduction pathway(s), leading to the activation of certain defense responses in plants after pathogen attack. Previous studies have identified several proteins, including catalase and ascorbate peroxidase, through which the SA signal might act. Here we describe a new SA-binding protein. This soluble protein is present in low abundance in tobacco (Nicotiana tabacum) leaves and has an apparent molecular weight of approximately 25,000. It reversibly binds SA with an apparent dissociation constant of 90 nM, an affinity that is 150-fold higher than that between SA and catalase. The ability of most analogs of SA to compete with labeled SA for binding to this protein correlated with their ability to induce defense gene expression and enhanced resistance. Strikingly, benzothiadiazole, a recently described chemical activator that induces plant defenses and disease resistance at very low rates of application, was the strongest competitor, being much more effective than unlabeled SA. The possible role of this SA-binding protein in defense signal transduction is discussed. PMID:12223676

  9. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  10. A Photocytes-Associated Fatty Acid-Binding Protein from the Light Organ of Adult Taiwanese Firefly, Luciola cerata

    PubMed Central

    Goh, King-Siang; Li, Chia-Wei

    2011-01-01

    Background Intracellular fatty acid-binding proteins (FABPs) are considered to be an important energy source supplier in lipid metabolism; however, they have never been reported in any bioluminescent tissue before. In this study, we determined the structural and functional characteristics of a novel FABP (lcFABP) from the light organ of adult Taiwanese firefly, Luciola cerata, and showed anatomical association of lcFABP with photocytes. Principal Findings Our results demonstrated the primary structure of lcFABP deduced from the cDNA clone of light organ shares structural homologies with other insect and human FABPs. In vitro binding assay indicated the recombinant lcFABP binds saturated long chain fatty acids (C14-C18) more strongly than other fatty acids and firefly luciferin. In addition, tissue distribution screening assay using a rabbit antiserum specifically against the N-terminal sequence of lcFABP confirmed the light organ-specific expression of lcFABP. In the light organ, the lcFABP constituted about 15% of total soluble proteins, and was detected in both cytosol and nucleus of photocytes. Conclusions The specific localization of abundant lcFABP in the light organ suggests that sustained bioluminescent flashes in the light organ might be a high energy demanding process. In photocytes, lcFABP might play a key role in providing long chain fatty acids to peroxisomes for the luciferase-catalyzed long chain acyl-CoA synthetic reaction. PMID:22242133

  11. A Novel Fatty Acid-Binding Protein-Like Carotenoid-Binding Protein from the Gonad of the New Zealand Sea Urchin Evechinus chloroticus

    PubMed Central

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9′-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

  12. A novel fatty acid-binding protein-like carotenoid-binding protein from the gonad of the New Zealand sea urchin Evechinus chloroticus.

    PubMed

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9'-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

  13. Adenovirus coded deoxyribonucleic acid binding protein. Isolation, physical properties, and effects of proteolytic digestion

    SciTech Connect

    Schechter, N.M.; Davies, W.; Anderson, C.W.

    1980-01-01

    A procedure has been developed for the purification of adenovirus type 2 DNA-binding protein (DBP) from nuclei of infected HeLa cells. This procedure routinely yields 0.2 to 0.6 mg of protein per 10/sup 9/ cells that is greater than 98% DBP. Binding protein so prepared does not precipitate at low ionic strength, interacts with both single- and double-stranded DNA, and complements Ad5 ts125 function in an in vitro DNA synthesizing system dependent upon exogenous DBP. An examination of the hydrodynamic properties of Ad2 DBP indicated that DBP undergoes a concentration-dependent self-association process. In high ionic strength solutions (1.0 M NaCl), self-association is a limited process observed at DBP concentrations above about 0.1 mg/mL; the product is a unit having a molecular weight of a trimer. At low ionic strengths (0.1 M NaCl), self-association is more extensive and is observed at lower protein concentrations. Our findings suggest that units other than the 72,000 molecular weight monomer may interact with DNA in the cell. Purified Ad2 DBP was digested with several proteolytic enzymes to determine if smaller DNA-binding products could be generated that resemble the 48,000 molecular weight species observed in extracts of infected cells. Digestion of purified DBP with Pronase or chymotrypsin produced relatively stable fragments with molecular weights of 45,000 and 53,000, respectively. Trypsin cleavage produced a 51,000 molecular weight fragment that upon continued incubation was further digested to produce a 35,000-M/sub r/ peptide. The production of the 35,000-M/sub r/ peptide by trypsin cleavage of the 51,000-M/sub r/ fragment was not observed if a sufficient amount of DNA was added to the DBP solution prior to trypsin digestion. This result indicates that bound DNA protects a trypsin-sensitive site(s) in the 51,000-M/sub r/ fragment, and it suggests that the 51,000-M/sub r/ fragment contains at least a part of the binding site for single-stranded DNA.

  14. Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding.

    PubMed

    Krishnan, V V; Sukumar, M; Gierasch, L M; Cosman, M

    2000-08-01

    Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions. PMID:10924105

  15. Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases

    PubMed Central

    Furuhashi, Masato; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

    2014-01-01

    Over the past decade, evidences of an integration of metabolic and inflammatory pathways, referred to as metaflammation in several aspects of metabolic syndrome, have been accumulating. Fatty acid-binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP) or aP2, is mainly expressed in adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis in relation to metaflammation. Despite lack of a typical secretory signal peptide, FABP4 has been shown to be released from adipocytes in a non-classical pathway associated with lipolysis, possibly acting as an adipokine. Elevation of circulating FABP4 levels is associated with obesity, insulin resistance, diabetes mellitus, hypertension, cardiac dysfunction, atherosclerosis, and cardiovascular events. Furthermore, ectopic expression and function of FABP4 in several types of cells and tissues have been recently demonstrated. Here, we discuss both the significant role of FABP4 in pathophysiological insights and its usefulness as a biomarker of metabolic and cardiovascular diseases. PMID:25674026

  16. Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

    PubMed

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-09-01

    Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:27312522

  17. Adipocyte fatty acid-binding protein levels are associated with left ventricular diastolic dysfunction in morbidly obese subjects

    PubMed Central

    Baessler, A; Lamounier-Zepter, V; Fenk, S; Strack, C; Lahmann, C; Loew, T; Schmitz, G; Blüher, M; Bornstein, S R; Fischer, M

    2014-01-01

    Objectives: This study aimed to examine the association of adipocyte fatty acid-binding protein (FABP4) levels with left ventricular diastolic dysfunction (LVDD) in obese subjects with varying degrees of the metabolic syndrome (MetS). Methods: Fifty morbidly obese subjects with LVDD were selected at random and matched by age (±5 years) and sex with 50 morbidly obese with normal left ventricular (LV) function. In addition, 24 healthy lean subjects were included as controls. Results: Median FABP4 levels (interquartile range) in obese subjects with LVDD were significantly higher (42 ng ml−1 (32–53)) than in obese with normal LV function (24 ng ml−1 (36–43), P=0.036), and in normal weight controls (13 ng ml−1 (10–20), P<0.0001). Increasing FABP4 tertiles were significantly associated with parameters of LVDD, the number of LVDD components, physical performance and epicardial fat thickness. In multivariate regression analysis adjusting for age, sex and adiposity, FABP4 levels remained significantly associated with parameters of diastolic function. The association of FABP4 levels with LVDD was mainly observed in subjects with metabolic complications, but not in metabolically healthy obese. Conclusions: FABP4 levels are significantly associated with LVDD in obese subjects, when the MetS is present. Thus, FABP4 may be a link between obesity and cardiometabolic disorders. PMID:24513579

  18. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

  19. Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway.

    PubMed

    Elmasri, Harun; Ghelfi, Elisa; Yu, Chen-wei; Traphagen, Samantha; Cernadas, Manuela; Cao, Haiming; Shi, Guo-Ping; Plutzky, Jorge; Sahin, Mustafa; Hotamisligil, Gokhan; Cataltepe, Sule

    2012-09-01

    Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4(-/-) mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis. PMID:22562362

  20. Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway

    PubMed Central

    Elmasri, Harun; Ghelfi, Elisa; Yu, Chen-wei; Traphagen, Samantha; Cernadas, Manuela; Cao, Haiming; Shi, Guo-Ping; Plutzky, Jorge; Sahin, Mustafa; Hotamisligil, Gokhan; Cataltepe, Sule

    2013-01-01

    Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4−/− mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis. PMID:22562362

  1. Urinary Intestinal Fatty Acid-Binding Protein Can Distinguish Necrotizing Enterocolitis from Sepsis in Early Stage of the Disease

    PubMed Central

    Snajdauf, Jiri; Rygl, Michal

    2016-01-01

    Necrotizing enterocolitis (NEC) is severe disease of gastrointestinal tract, yet its early symptoms are nonspecific, easily interchangeable with sepsis. Therefore, reliable biomarkers for early diagnostics are needed in clinical practice. Here, we analyzed if markers of gut mucosa damage, caspase cleaved cytokeratin 18 (ccCK18) and intestinal fatty acid-binding protein (I-FABP), could be used for differential diagnostics of NEC at early stage of disease. We collected paired serum (at enrollment and week later) and urine (collected for two days in 6 h intervals) samples from 42 patients with suspected NEC. These patients were later divided into NEC (n = 24), including 13 after gastrointestinal surgery, and sepsis (n = 18) groups using standard criteria. Healthy infants (n = 12), without any previous gut surgery, served as controls. Both biomarkers were measured by a commercial ELISA assay. There were no statistically significant differences in serum ccCK18 between NEC and sepsis but NEC patients had significantly higher levels of serum and urinary I-FABP than either sepsis patients or healthy infants. Urinary I-FABP has high sensitivity (81%) and specificity (100%) and can even distinguish NEC from sepsis in patients after surgery. Urinary I-FABP can be used to distinguish NEC from neonatal sepsis, including postoperative one, better than abdominal X-ray. PMID:27110575

  2. Fasciola gigantica Fatty Acid Binding Protein (FABP) as a Prophylactic Agent against Schistosoma mansoni Infection in CD1 Mice

    PubMed Central

    Diab, M.; El-Amir, A. M.; Hendawy, M.; Kadry, S.

    2012-01-01

    Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG1/IgG2b immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection. PMID:22451732

  3. An Evidence-Based Approach to the Assessment of Heart-Type Fatty Acid Binding Protein in Acute Coronary Syndrome

    PubMed Central

    Viswanathan, Karthik; Hall, Alistair S; Barth, Julian H

    2012-01-01

    Cardiac troponins have been the biomarkers of choice for the diagnosis of acute coronary syndrome (ACS) for over a decade. There has, however, been considerable interest over the last two decades for newer biomarkers that would bring added value to the measurement of troponin such as the provision of prognosis and assistance in the choice of therapeutic interventions. In this manuscript, we review the development of heart-type fatty acid binding protein (H-FABP) in patients with ACS using the evidence-based laboratory medicine format. Phase I studies have established that H-FABP reference intervals and pre-analytical factors influencing H-FABP. Phase II studies have confirmed a) that H-FABP is elevated in patients with established myocardial infarction; b) that its serum concentration is related to the extent of infarction using survival as a surrogate; and c) that its use in chest pain patients can identify ACS patients and also provide prognostic information on survival. Furthermore, it is an independent prognostic marker for patients with suspected ACS who are troponin negative. Phase III studies involving randomised control trials for diagnosis and prognosis have not yet been performed and Phase IV studies await uptake of H-FABP in a routine service. PMID:22363093

  4. Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

    PubMed Central

    Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

    2014-01-01

    Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

  5. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    PubMed

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-01

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  6. Fatty Acid-binding Proteins (FABPs) Are Intracellular Carriers for Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)*

    PubMed Central

    Elmes, Matthew W.; Kaczocha, Martin; Berger, William T.; Leung, KwanNok; Ralph, Brian P.; Wang, Liqun; Sweeney, Joseph M.; Miyauchi, Jeremy T.; Tsirka, Stella E.; Ojima, Iwao; Deutsch, Dale G.

    2015-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  7. The cancer-promoting gene fatty acid-binding protein 5 (FABP5) is epigenetically regulated during human prostate carcinogenesis.

    PubMed

    Kawaguchi, Koichiro; Kinameri, Ayumi; Suzuki, Shunsuke; Senga, Shogo; Ke, Youqiang; Fujii, Hiroshi

    2016-02-15

    FABPs (fatty-acid-binding proteins) are a family of low-molecular-mass intracellular lipid-binding proteins consisting of ten isoforms. FABPs are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting these ligands to the appropriate compartments in the cell. FABP5 is overexpressed in multiple types of tumours. Furthermore, up-regulation of FABP5 is strongly associated with poor survival in triple-negative breast cancer. However, the mechanisms underlying the specific up-regulation of the FABP5 gene in these cancers remain poorly characterized. In the present study, we determined that FABP5 has a typical CpG island around its promoter region. The DNA methylation status of the CpG island in the FABP5 promoter of benign prostate cells (PNT2), prostate cancer cells (PC-3, DU-145, 22Rv1 and LNCaP) and human normal or tumour tissue was assessed by bisulfite sequencing analysis, and then confirmed by COBRA (combined bisulfite restriction analysis) and qAMP (quantitative analysis of DNA methylation using real-time PCR). These results demonstrated that overexpression of FABP5 in prostate cancer cells can be attributed to hypomethylation of the CpG island in its promoter region, along with up-regulation of the direct trans-acting factors Sp1 (specificity protein 1) and c-Myc. Together, these mechanisms result in the transcriptional activation of FABP5 expression during human prostate carcinogenesis. Importantly, silencing of Sp1, c-Myc or FABP5 expression led to a significant decrease in cell proliferation, indicating that up-regulation of FABP5 expression by Sp1 and c-Myc is critical for the proliferation of prostate cancer cells. PMID:26614767

  8. Effects of chronic ethanol consumption on sterol transfer proteins in mouse brain.

    PubMed

    Myers-Payne, S C; Fontaine, R N; Loeffler, A; Pu, L; Rao, A M; Kier, A B; Wood, W G; Schroeder, F

    1996-01-01

    Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross-reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein-2 (SCP-2) and the liver form of fatty acid-binding protein (L-FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP-2 and L-FABP were used to identify the lipid transfer proteins in the brains of alcohol-treated and control mice. L-FABP was not detectable in brain of either control or chronic ethanol-treated mice. In contrast, SCP-2 not only was present, but its level was significantly (p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP-2 reflected true levels of SCP-2 in brain, the cDNA sequence for brain SCP-2 was isolated from a brain cDNA library. The mouse brain SCP-2 sequence was 99.99% identical to the mouse liver SCP-2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP-2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl-CoA binding protein, a lipid-binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP-2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP-2 may contribute to membrane tolerance to ethanol. PMID:8522969

  9. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    PubMed Central

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  10. Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

    PubMed

    Liou, H L; Storch, J

    2001-05-29

    The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions. PMID:11371211

  11. Gender difference in plasma fatty-acid-binding protein 4 levels in patients with chronic obstructive pulmonary disease

    PubMed Central

    Zhang, Xue; Li, Diandian; Wang, Hao; Pang, Caishuang; Wu, Yanqiu; Wen, Fuqiang

    2016-01-01

    COPD (chronic obstructive pulmonary disease) is characterized by airway inflammation and increases the likelihood of the development of atherosclerosis. Recent studies have indicated that FABP4 (fatty-acid-binding protein 4), an intracellular lipid chaperone of low molecular mass, plays an important role in the regulation of inflammation and atherosclerosis. We carried out a preliminary clinical study aiming at investigating the relationships between circulating FABP4 levels in patients with COPD and inflammation and lung function. We enrolled 50 COPD patients and 39 healthy controls in the study. Lung function tests were performed in all subjects. Plasma levels of FABP4 and adiponectin, TNFα (tumour necrosis factor α) and CRP (C-reactive protein) were measured. The correlations between FABP4 and lung function, adipokine (adiponectin), inflammatory factors and BMI (body mass index) were analysed. Compared with both males with COPD and healthy females, plasma FABP4 levels in females with COPD were significantly increased. Adiponectin and CRP levels were significantly higher in patients with COPD. Furthermore, we found that FABP4 levels were inversely correlated with FEV1% predicted (FEV1 is forced expiratory volume in 1 s) and positively correlated with adiponectin and TNFα in COPD patients. In addition, a positive correlation between plasma FABP4 and CRP was found in females with COPD. However, FABP4 levels were not correlated with BMI. Our results underline a gender difference in FABP4 secretion in stable COPD patients. Further studies are warranted to clarify the exact role of FABP4 in the pathogenesis of COPD. PMID:26823558

  12. Brief Report: Differential Associations of Interleukin 6 and Intestinal Fatty Acid-Binding Protein With Progressive Untreated HIV-1 Infection in Rakai, Uganda.

    PubMed

    Olwenyi, Omalla A; Naluyima, Prossy; Cham, Fatim; Quinn, Thomas C; Serwadda, David; Sewankambo, Nelson K; Gray, Ronald H; Sandberg, Johan K; Michael, Nelson L; Wabwire-Mangen, Fred; Robb, Merlin L; Eller, Michael A

    2016-05-01

    The significance of HIV-associated immune activation and microbial translocation in Sub-Saharan African population remains poorly defined. We assessed biomarkers of inflammation, microbial translocation, and cellular activation and found most factors elevated in Ugandan HIV-1 seroconverters compared with community-matched controls. In contrast to previous findings in Western cohorts, C-reactive protein, neopterin, and intestinal fatty acid binding protein were not elevated. Higher T-cell activation and IL-6 were associated with faster disease progression. Surprisingly, intestinal fatty acid binding protein, indicative of enterocyte turnover, was higher in slow than in fast progressors. These data suggest differential relationships among biomarkers of intestinal barrier integrity and innate immune activation between developed countries and Sub-Saharan Africa. PMID:26630672

  13. NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

    PubMed

    D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

    2009-01-01

    The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed. PMID:18977333

  14. Association of L-FABP T94A and MTP I128T polymorphisms with hyperlipidemia in Chinese subjects.

    PubMed

    Tian, Yingying; Li, Hui; Wang, Shanbo; Yan, Jin; Chen, Zhiheng; Li, Zhenyu; Feng, Han; Zhou, Honghao; Ouyang, Dongsheng

    2015-03-01

    The purpose of this study was to evaluate the relation between the L-FABP T94A and MTP I128T polymorphisms and hyperlipidemia in Chinese subjects. We recruited 390 volunteers: 201 hyperlipidemic and 189 healthy volunteers. The L-FABP T94A and MTP I128T polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Anthropometry, lipid profile, and liver function of the subjects were determined. We observed that male carriers of the L-FABP A94 allele had significantly higher body weight (P = 0.012), higher body mass index (BMI) (P = 0.014), and higher plasma triacylglycerol levels (TAG) (P = 0.033) and lower ratios of high-density lipoprotein cholesterol (HDL-C) to total cholesterol (TC) (P = 0.008) than T94 homozygotes. The MTP T128 allele was associated with significantly lower serum TC (P < 0.001) and low-density lipoprotein cholesterol (LDL-C) (P < 0.001) levels in males. There was a direct correlation between the MTP T128 allele and a decreased risk of hyperlipidemia after adjusting for body mass index (OR = 0.327, 95 % CI: 0.178-0.600, P < 0.001). In conclusion, both the MTP I128T and the L-FABP T94A polymorphisms can affect serum lipid levels in the Chinese population. The MTP T128 allele offers protection against hyperlipidemia in the Chinese population. PMID:25663234

  15. Serum Fatty Acid-Binding Protein 4 Is a Predictor of Cardiovascular Events in End-Stage Renal Disease

    PubMed Central

    Furuhashi, Masato; Ishimura, Shutaro; Ota, Hideki; Hayashi, Manabu; Nishitani, Takahiro; Tanaka, Marenao; Yoshida, Hideaki; Shimamoto, Kazuaki; Hotamisligil, Gökhan S.; Miura, Tetsuji

    2011-01-01

    Background Fatty acid-binding protein 4 (FABP4/A-FABP/aP2), a lipid chaperone, is expressed in both adipocytes and macrophages. Recent studies have shown that FABP4 is secreted from adipocytes and that FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the impact of FABP4 concentrations on prognosis. We tested the hypothesis that FABP4 level predicts prognosis of patients with end-stage renal disease (ESRD), a group at high risk for atherosclerosis-associated morbidity and mortality. Methods and Results Biochemical markers including FABP4 were determined in 61 ESRD patients on chronic hemodialysis (HD). Serum FABP4 level in females (404.2±30.5 ng/ml) was significantly higher than that in males (315.8±30.0 ng/ml), and the levels in ESRD patients were about 20-times higher than those in age-, gender- and body mass index (BMI)-matched control subjects with normal renal function. FABP4 level was decreased by 57.2% after HD and was positively correlated with blood pressure, BMI, and levels of lipids and insulin. Multiple regression analysis indicated that HD duration, BMI, and triglycerides level were independent determinants for FABP4 level. ESRD patients with high FABP4 levels had higher cardiovascular mortality during the 7-year follow-up period. Cox proportional hazard regression analysis showed that logarithmically transformed FABP4 level was an independent predictor of cardiovascular death adjusted for age, gender, HD duration, BMI, and triglycerides level (hazard ratio, 7.75; 95% CI, 1.05–25.31). Conclusion These findings suggest that FABP4 level, being related to adiposity and metabolic disorders, is a novel predictor of cardiovascular mortality in ESRD. PMID:22102888

  16. Fatty Acid Binding Protein 4 Regulates VEGF-Induced Airway Angiogenesis and Inflammation in a Transgenic Mouse Model

    PubMed Central

    Ghelfi, Elisa; Yu, Chen-Wei; Elmasri, Harun; Terwelp, Matthew; Lee, Chun G.; Bhandari, Vineet; Comhair, Suzy A.; Erzurum, Serpil C.; Hotamisligil, Gökhan S.; Elias, Jack A.; Cataltepe, Sule

    2014-01-01

    Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function in vitro, but whether it plays a role in modulation of angiogenesis in vivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4−/− mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4−/− mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma. PMID:23391391

  17. Increased expression of fatty acid binding protein 4 and leptin in resident macrophages characterises atherosclerotic plaque rupture

    PubMed Central

    Lee, K.; Santibanez-Koref, M.; Polvikoski, T.; Birchall, D.; Mendelow, A.D.; Keavney, B.

    2013-01-01

    Objective Resident macrophages play an important role in atheromatous plaque rupture. The macrophage gene expression signature associated with plaque rupture is incompletely defined due to the complex cellular heterogeneity in the plaque. We aimed to characterise differential gene expression in resident plaque macrophages from ruptured and stable human atheromatous lesions. Methods and results We performed genome-wide expression analyses of isolated macrophage-rich regions of stable and ruptured human atherosclerotic plaques. Plaques present in carotid endarterectomy specimens were designated as stable or ruptured using clinical, radiological and histopathological criteria. Macrophage-rich regions were excised from 5 ruptured and 6 stable plaques by laser micro-dissection. Transcriptional profiling was performed using Affymetrix microarrays. The profiles were characteristic of activated macrophages. At a false discovery rate of 10%, 914 genes were differentially expressed between stable and ruptured plaques. The findings were confirmed in fourteen further stable and ruptured samples for a subset of eleven genes with the highest expression differences (p < 0.05). Pathway analysis revealed that components of the PPAR/Adipocytokine signaling pathway were the most significantly upregulated in ruptured compared to stable plaques (p = 5.4 × 10−7). Two key components of the pathway, fatty-acid binding-protein 4 (FABP4) and leptin, showed nine-fold (p = 0.0086) and five-fold (p = 0.0012) greater expression respectively in macrophages from ruptured plaques. Conclusions We found differences in gene expression signatures between macrophages isolated from stable and ruptured human atheromatous plaques. Our findings indicate the involvement of FABP4 and leptin in the progression of atherosclerosis and plaque rupture, and suggest that down-regulation of PPAR/adipocytokine signaling within plaques may have therapeutic potential. PMID:23122912

  18. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    SciTech Connect

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  19. Heart‐type fatty acid binding protein is a novel prognostic marker in patients with non‐ischaemic dilated cardiomyopathy

    PubMed Central

    Komamura, K; Sasaki, T; Hanatani, A; Kim, J; Hashimura, K; Ishida, Y; Ohkaru, Y; Asayama, K; Tanaka, T; Ogai, A; Nakatani, T; Kitamura, S; Kangawa, K; Miyatake, K; Kitakaze, M

    2006-01-01

    Objective To determine whether concentrations of heart‐type fatty acid binding protein (H‐FABP) measured before hospital discharge predict critical cardiac events in patients with idiopathic dilated cardiomyopathy (DCM). Patients 92 consecutive patients with DCM were enrolled and followed up for four years. Main outcome measures Serum concentrations of H‐FABP, brain natriuretic peptide (BNP), cardiac troponin T before hospital discharge and survival rate. Results 23 patients died of cardiac causes, received a left ventricular assist device or underwent heart transplantation during the four‐year follow up. Univariate analyses showed that New York Heart Association functional class, heart rate, ejection fraction, serum H‐FABP and plasma BNP were significant variables. According to multivariate analysis, serum H‐FABP and plasma BNP concentrations were independent predictors of critical cardiac events. Cardiac troponin T before hospital discharge was not a predictor. The area under the receiver operating characteristic curve for death from critical cardiac events was similar between H‐FABP and BNP. Patients with an H‐FABP concentration at or above the median (⩾ 5.4 ng/ml) had a significantly lower survival rate than those below the median, according to analysis by log rank test (p < 0.0001). When combined with BNP concentration at or above the median (⩾ 138 pg/ml), H‐FABP below the median predicted the worst prognosis among the combinations. Conclusions The concentration of serum H‐FABP before discharge from hospital may be an independent predictor for critical cardiac events in DCM. PMID:16387818

  20. Angiotensin II receptor blockers decrease serum concentration of fatty acid-binding protein 4 in patients with hypertension.

    PubMed

    Furuhashi, Masato; Mita, Tomohiro; Moniwa, Norihito; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Yoshida, Hideaki; Shimamoto, Kazuaki; Miura, Tetsuji

    2015-04-01

    Elevated circulating fatty acid-binding protein 4 (FABP4/A-FABP/aP2), an adipokine, is associated with obesity, insulin resistance, hypertension and cardiovascular events. However, how circulating FABP4 level is modified by pharmacological agents remains unclear. We here examined the effects of angiotensin II receptor blockers (ARBs) on serum FABP4 level. First, essential hypertensives were treated with ARBs: candesartan (8 mg day(-1); n=7) for 2 weeks, olmesartan (20 mg day(-1); n=9) for 12 weeks, and valsartan (80 mg day(-1); n=94) or telmisartan (40 mg day(-1); n=91) for 8 weeks added to amlodipine (5 mg day(-1)). Treatment with ARBs significantly decreased blood pressure and serum FABP4 concentrations by 8-20% without significant changes in adiposity or lipid variables, though the M value determined by hyperinsulinemic-euglycemic glucose clamp, a sensitive index of insulin sensitivity, was significantly increased by candesartan. Next, alterations in FABP4 secretion from 3T3-L1 adipocytes were examined under several agents. Lipolytic stimulation of the β-adrenoceptor in 3T3-L1 adipocytes by isoproterenol increased FABP4 secretion, and conversely, insulin suppressed FABP4 secretion. However, treatment of 3T3-L1 adipocytes with angiotensin II or ARBs for 2 h had no effect on gene expression or secretion of FABP4 regardless of β-adrenoceptor stimulation. In conclusion, treatment with structurally different ARBs similarly decreases circulating FABP4 concentrations in hypertensive patients as a class effect of ARBs, which is not attributable to blockade of the angiotensin II receptor in adipocytes. Reduction of FABP4 levels by ARBs might be involved in suppression of cardiovascular events. PMID:25672659

  1. Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPARalpha in rat liver.

    PubMed

    Carlsson, L; Lindén, D; Jalouli, M; Oscarsson, J

    2001-10-01

    The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products. PMID:11551854

  2. S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding

    PubMed Central

    Henkels, Karen M.; Mallets, Elizabeth R.; Dennis, Patrick B.; Gomez-Cambronero, Julian

    2015-01-01

    ., Gomez-Cambronero, J. S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding. PMID:25512366

  3. Sequence Comparison and Phylogeny of Nucleotide Sequence of Coat Protein and Nucleic Acid Binding Protein of a Distinct Isolate of Shallot virus X from India.

    PubMed

    Majumder, S; Baranwal, V K

    2011-06-01

    Shallot virus X (ShVX), a type species in the genus Allexivirus of the family Alfaflexiviridae has been associated with shallot plants in India and other shallot growing countries like Russia, Germany, Netherland, and New Zealand. Coat protein (CP) and nucleic acid binding protein (NB) region of the virus was obtained by reverse transcriptase polymerase chain reaction from scales leaves of shallot bulbs. The partial cDNA contained two open reading frames encoding proteins of molecular weights of 28.66 and 14.18 kDa belonging to Flexi_CP super-family and viral NB super-family, respectively. The percent identity and phylogenetic analysis of amino acid sequences of CP and NB region of the virus associated with shallot indicated that it was a distinct isolate of ShVX. PMID:23637504

  4. Identification of the messenger RNA for human cutaneous fatty acid-binding protein as a metastasis inducer.

    PubMed

    Jing, C; Beesley, C; Foster, C S; Rudland, P S; Fujii, H; Ono, T; Chen, H; Smith, P H; Ke, Y

    2000-05-01

    Using our recently developed systematic differential display and complete comparison of gene expression approaches combined with other methods, we have identified a large number of mRNAs that are expressed differentially between benign and malignant human cells. One such mRNA that is common to prostate and breast carcinoma cell lines encodes the human cutaneous fatty acid-binding protein (C-FABP). Northern and slot blot analyses confirm that the expression levels of C-FABP mRNA in the malignant prostate and breast carcinoma cell lines are 4.9+/-0.9- to 16.9+/-2.1-fold higher than those expressed in the benign cell lines. A similar difference between the benign and malignant cell lines was also detected at the protein level. In situ hybridization experiments have detected overexpression of the mRNA for C-FABP in human prostate carcinoma tissues. Transfection of a C-FABP expression construct into the benign, nonmetastatic rat mammary epithelial cell line Rama 37 and inoculation of the C-FABP expression transfectants into syngeneic Wistar-Furth rats produce a significant number (P < 0.05) of animals with metastases (6 of 26 animals), whereas the control transfectants generated by the vector alone yield no such metastases. Measurements of mRNA and protein levels with Northern and Western blotting show that C-FABP is not expressed in the control transfectant cells produced by the vector alone but is highly expressed in the pool of C-FABP transfectants and-the sublines established from their metastases. Immunocytochemical staining with antibodies to C-FABP shows that C-FABP is not expressed in the primary tumors developed from the control transfectants that have failed to metastasize, but it is expressed in both the primary tumors developed from the C-FABP transfectants and their metastases. Reinoculation of the sublines established from metastases in syngeneic rats has produced a higher proportion (50%) of animals (7 of 14 animals) with metastases than that obtained in

  5. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  6. Fatty Acid Binding Protein 5 Modulates Docosahexaenoic Acid-Induced Recovery in Rats Undergoing Spinal Cord Injury.

    PubMed

    Figueroa, Johnny D; Serrano-Illan, Miguel; Licero, Jenniffer; Cordero, Kathia; Miranda, Jorge D; De Leon, Marino

    2016-08-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) promote functional recovery in rats undergoing spinal cord injury (SCI). However, the precise molecular mechanism coupling n-3 PUFAs to neurorestorative responses is not well understood. The aim of the present study was to determine the spatiotemporal expression of fatty acid binding protein 5 (FABP5) after contusive SCI and to investigate whether this protein plays a role in n-3 PUFA-mediated functional recovery post-SCI. We found that SCI resulted in a robust spinal cord up-regulation in FABP5 mRNA levels (556 ± 187%) and protein expression (518 ± 195%), when compared to sham-operated rats, at 7 days post-injury (dpi). This upregulation coincided with significant alterations in the metabolism of fatty acids in the injured spinal cord, as revealed by metabolomics-based lipid analyses. In particular, we found increased levels of the n-3 series PUFAs, particularly docosahexaenoic acid (DHA; 22:6 n-3) and eicosapentaenoic acid (EPA; 20:5 n-3) at 7 dpi. Animals consuming a diet rich in DHA and EPA exhibited a significant upregulation in FABP5 mRNA levels at 7 dpi. Immunofluorescence showed low basal FABP5 immunoreactivity in spinal cord ventral gray matter NeuN(+) neurons of sham-operated rats. SCI resulted in a robust induction of FABP5 in glial (GFAP(+), APC(+), and NG2(+)) and precursor cells (DCX(+), nestin(+)). We found that continuous intrathecal administration of FABP5 silencing with small interfering RNA (2 μg) impaired spontaneous open-field locomotion post-SCI. Further, FABP5 siRNA administration hindered the beneficial effects of DHA to ameliorate functional recovery at 7 dpi. Altogether, our findings suggest that FABP5 may be an important player in the promotion of cellular uptake, transport, and/or metabolism of DHA post-SCI. Given the beneficial roles of n-3 PUFAs in ameliorating functional recovery, we propose that FABP5 is an important contributor to basic repair mechanisms in the

  7. Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin.

    PubMed

    Carlsson, L; Nilsson, I; Oscarsson, J

    1998-06-01

    Liver fatty acid-binding protein (LFABP) is an abundant protein in hepatocytes that binds most of the long chain fatty acids present in the cytosol. It is suggested to be of importance for fatty acid uptake and utilization in the hepatocyte. In the present study, the effects of bovine GH (bGH) and other hormones on the expression of LFABP and its messenger RNA (mRNA) were studied in hypophysectomized rats and in vitro using primary cultures of rat hepatocytes. One injection of bGH increased LFABP mRNA levels about 5-fold after 6 h, but there was no effect of this treatment on LFABP levels. However, 7 days of bGH treatment increased both LFABP mRNA and LFABP protein levels 2- to 5-fold. Female rats had higher levels of LFABP than male rats. Hypophysectomy of female rats, but not that of male rats, decreased LFABP levels markedly. Treatment of hypophysectomized rats with bGH for 7 days as two daily injections or as a continuous infusion increased LFABP levels to a similar degree. This finding indicates that the sex difference in the expression of LFABP is not regulated by the sexually dimorphic secretory pattern of GH. Neither insulin nor insulin-like growth factor I treatment of hypophysectomized rats for 6-7 days had any effect on LFABP mRNA or LFABP levels. In vitro, bGH dose-dependently increased the expression of LFABP mRNA, but only in the presence of insulin. Insulin alone had a marked dose-dependent effect on LFABP mRNA levels and was of importance for maintaining the expression of LFABP mRNA during the culture. Incubation with bGH increased LFABP mRNA levels within 3 h. GH had no effect on LFABP mRNA levels in the presence of actinomycin D, indicating a transcriptional effect of GH. Incubation with glucagon in vitro decreased LFABP mRNA levels markedly, indicating that glucagon, in contrast to GH, has an effect opposite that of insulin on LFABP mRNA expression. It is concluded that GH is an important regulator of LFABP in vivo and in vitro. In contrast to

  8. Elevated Cellular Retinoic Acid Binding Protein-I in Cerebrospinal Fluid of Patients with Hemorrhagic Cerebrovascular Diseases : Preliminary Study

    PubMed Central

    Jeon, Jin Pyeong; Cho, Won-Sang; Kang, Hyun-Seung; Kim, Seung-Ki; Oh, Chang Wan

    2015-01-01

    Objective Elevated cellular retinoic acid binding protein-I (CRABP-I) is thought to be related to the abnormal proliferation and migration of smooth muscle cells (SMCs). Accordingly, a higher CRABP-I level could cause disorganized vessel walls by causing immature SMC phenotypes and altering extracellular matrix proteins which could result in vulnerable arterial walls with inadequate responses to hemodynamic stress. We hypothesized that elevated CRABP-I level in the cerebrospinal fluid (CSF) could be related to subarachnoid hemorrhage (SAH). Moreover, we also extended this hypothesis in patients with vascular malformation according to the presence of hemorrhage. Methods We investigated the CSF of 26 patients : SAH, n=7; unruptured intracranial aneurysm (UIA), n=7; arteriovenous malformation (AVM), n=4; cavernous malformation (CM), n=3; control group, n=5. The optical density of CRABP-I was confirmed by Western blotting and presented as mean±standard error of the measurement. Results CRABP-I in SAH (0.33±0.09) was significantly higher than that in the UIA (0.12±0.01, p=0.033) or control group (0.10±0.01, p=0.012). Hemorrhage presenting AVM (mean 0.45, ranged 0.30-0.59) had a higher CRABP-I level than that in AVM without hemorrhage presentation (mean 0.16, ranged 0.14-0.17). The CRABP-I intensity in CM with hemorrhage was 0.21 and 0.31, and for CM without hemorrhage 0.14. Overall, the hemorrhage presenting group (n=11, 0.34±0.06) showed a significantly higher CRABP-I intensity than that of the non-hemorrhage presenting group (n=10, 0.13±0.01, p=0.001). Conclusion The results suggest that elevated CRABP-I in the CSF could be related with aneurysm rupture. Additionally, a higher CRABP-I level seems to be associated with hemorrhage development in vascular malformation. PMID:25733988

  9. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.

    PubMed

    Herr, F M; Aronson, J; Storch, J

    1996-01-30

    The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K

  10. Analysis of the regulation of fatty acid binding protein 7 expression in human renal carcinoma cell lines

    PubMed Central

    2011-01-01

    Background Improving the treatment of renal cell carcinoma (RCC) will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7), also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system. Results We determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293) were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI) bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2) and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines. Conclusions Three putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key factors regulating the

  11. A Study on the Role of Heart Type Fatty Acid Binding Protein in the Diagnosis of Acute Myocardial Infarction

    PubMed Central

    Kabekkodu, Shama Prakash; Mananje, Sudhindra Rao

    2016-01-01

    Introduction Heart type Fatty Acid Binding Protein (H-FABP) has been proposed as an early cardiac biomarker for the diagnosis of acute myocardial Infarction (AMI) using animal models and clinical samples. Aim The study aimed to evaluate the role of H-FABP in early detection of AMI by comparing its sensitivity, specificity and predictive value with Creatinine Kinase-MB (CK-MB) and Cardiac Troponin I (cTnI). Materials and Methods This is a cross-sectional descriptive study of 50 patients admitted with the diagnosis of AMI at a tertiary care hospital in South India. The study group was categorised in to those coming to the hospital within four hours of symptom onset and those coming in between 4 to 12 hours. H-FABP was compared with those of troponin T and myoglobin tests. Results Among patients presenting within four hours of symptom onset, the sensitivity of H-FABP was 60% and was significantly higher than that of cardiac Troponin I (cTnI, 18.8%) and Creatinine Kinase (CK)-MB (12.5%). But specificity was only 23.53% and was less than that of cTnI (66.67%) and CK-MB (100%). In patients presenting during 4 to 12 hours of symptom onset, the sensitivity of H-FABP was 86.96% which was comparable to that of cTnI (90.9%) and CK-MB (77.3%). The specificity was 60% in the 4-12 hours group which was comparable to that of cTnI (50%) and CK-MB (50%). Conclusion The H-FABP is a sensitive biomarker for the diagnosis of AMI in the initial hours after symptom onset when the standard biomarkers may not be elevated, but it is less specific. During 4-12 hours of symptom onset it is as sensitive and specific as standard cardiac biomarkers troponin and CK-MB. Due to these factors H-FABP can be considered as a promising cardiac biomarker which can be used along with troponins and CK-MB at present. PMID:26894106

  12. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein.

    PubMed Central

    Kirk, W R; Kurian, E; Prendergast, F G

    1996-01-01

    1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity. PMID:8770188

  13. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  14. /sup 113/Cd NMR studies of a 1:1 Cd adduct with an 18-residue finger peptide from HIV-1 nucleic acid binding protein, p7

    SciTech Connect

    South, T.L.; Kim, B.; Summers, M.F.

    1989-01-04

    The Zn/sup 2+/ and Cd/sup 2+/ adducts with the 18-residue peptide comprising the amino acid sequence of the first finger (residues 13 through 30) of retroviral nucleic acid binding proteins p7 from HIV-1 (the causative agent of AIDS) have been prepared. /sup 1/H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The /sup 113/Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures.

  15. N-Benzyl-indolo carboxylic acids: Design and synthesis of potent and selective adipocyte fatty-acid binding protein (A-FABP) inhibitors.

    PubMed

    Barf, Tjeerd; Lehmann, Fredrik; Hammer, Kristin; Haile, Saba; Axen, Eva; Medina, Carmen; Uppenberg, Jonas; Svensson, Stefan; Rondahl, Lena; Lundbäck, Thomas

    2009-03-15

    Small molecule inhibitors of adipocyte fatty-acid binding protein (A-FABP) have gained renewed interest following the recent publication of pharmacologically beneficial effects of such inhibitors. Despite the potential utility of selective A-FABP inhibitors within the fields of metabolic disease, inflammation and atherosclerosis, there are few examples of useful A-FABP inhibitors in the public domain. Herein, we describe the optimization of N-benzyl-tetrahydrocarbazole derivatives through the use of co-crystal structure guided medicinal chemistry efforts. This led to the identification of a potent and selective class of A-FABP inhibitors as illustrated by N-benzyl-hexahydrocyclohepta[b]indole 30. PMID:19217286

  16. Development of the Brazilian Anti Schistosomiasis Vaccine Based on the Recombinant Fatty Acid Binding Protein Sm14 Plus GLA-SE Adjuvant.

    PubMed

    Tendler, Miriam; Almeida, Marilia; Simpson, Andrew

    2015-01-01

    Data herein reported and discussed refer to vaccination with the recombinant fatty acid binding protein (FABP) family member of the schistosomes, called Sm14. This antigen was discovered and developed under a Brazilian platform led by the Oswaldo Cruz Foundation, from the Health Ministry in Brazil, and was assessed for safety and immunogenicity in healthy volunteers. This paper reviews past and recent outcomes of developmental phases of the Sm14-based anti schistosomiasis vaccine addressed to, ultimately, impact transmission of the second most prevalent parasitic endemic disease worldwide. PMID:26029206

  17. 1H, 15N and 13C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies.

    PubMed

    Tseng, Kai-Li; Lee, Yi-Zong; Chen, Yun-Ru; Lyu, Ping-Chiang

    2016-04-01

    Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C14-C18. However, the structural details are unknown, and the structure-function relationship has remained to be further investigated. In this study, we finished the (1)H, (15)N and (13)C chemical shift assignments of (15)N/(13)C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, Cα, Hα, C and side chain Cβ assignments. These results can provide the basis for further structural exploration of lcFABP. PMID:26373428

  18. Cellular Retinoic Acid Binding Protein 2 Is Strikingly Downregulated in Human Esophageal Squamous Cell Carcinoma and Functions as a Tumor Suppressor

    PubMed Central

    Xiao, Weifan; Sun, Fenyong; Yuan, Hong; Pan, Qiuhui

    2016-01-01

    Esophageal squamous cell carcinoma (ESCC) is the predominant pathotype of esophageal carcinoma (EC) in China, especially in Henan province, with poor prognosis and limited 5-year survival rate. Cellular retinoic acid binding protein 2 (CRABP2) is a member of the retinoic acid (RA) and lipocalin/cytosolic fatty-acid binding protein family and plays a completely contrary role in tumorigenesis through the retinoid signaling pathway, depending on the nuclear RA receptors (RAR) and PPARbeta/delta receptors. Presently, the biological role of CRABP2 in the development of ESCC has never been reported. Here, we firstly evaluated the expression of CRABP2 at both mRNA and protein levels and showed that it was remarkably downregulated in clinical ESCC tissues and closely correlated with the occurrence position, pathology, TNM stage, size, infiltration depth and cell differentiation of the tumor. Additionally, the biological function assays demonstrated that CRABP2 acted as a tumor suppressor in esophageal squamous carcinogenesis by significantly inhibiting cell growth, inducing cell apoptosis and blocking cell metastasis both in vitro and in vivo. All in all, our finding simplicate that CRABP2 is possibly an efficient molecular marker for diagnosing and predicting the development of ESCC. PMID:26839961

  19. A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication

    PubMed Central

    Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-01-01

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication. PMID:21589879

  20. Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids.

    PubMed

    Wang, Hsin; He, Yan; Kroenke, Christopher D; Kodukula, Sarala; Storch, Judith; Palmer, Arthur G; Stark, Ruth E

    2002-04-30

    Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed. PMID:11969406

  1. A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.

    PubMed

    He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E

    2011-03-01

    Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family. PMID:21226535

  2. Molecular cloning, tissue distribution, and expression of a 14-kDa bile acid-binding protein from rat ileal cytosol.

    PubMed Central

    Gong, Y Z; Everett, E T; Schwartz, D A; Norris, J S; Wilson, F A

    1994-01-01

    A cDNA clone encoding the major intestinal cytosolic 14-kDa bile acid-binding protein (14-kDa I-BABP) was isolated from a rat ileal lambda gt22A library following immunoscreening using a monospecific antiserum raised against a 14-kDa polypeptide found in the rat ileal cytosol. One clone of 516 bp encoded a 128-amino acid protein with a predicted molecular mass of 14,544 Da. The deduced amino acid sequence of 14-kDa I-BABP showed 100% homology to rat intestinal 15-kDa protein (I-15P) and 72% homology to porcine 15-kDa gastrotropin, whereas comparison of I-BABP to rat 14-kDa fatty acid-binding proteins of liver, intestine, and heart revealed homologies of 44%, 25%, and 28%, respectively. Northern blot analysis revealed a single transcript of approximately 0.5 kb in ileum and ovary; however, the abundance of I-BABP mRNA was much greater in ileum than in ovary. No transcript was seen in RNA extracted from stomach, jejunum, colon, liver, adrenal, brain, heart, kidney, or testis. Transfection of the I-BABP cDNA into COS-7 cells resulted in the expression of a 14-kDa protein that was identical to the ileal cytosolic I-BABP as determined by immunoblotting. Photoaffinity labeling of expressed 14-kDa protein was saturable with respect to increasing concentrations of 7,7-azo[3H]taurocholate (Km, 83.3 microM; Vmax, 6.7 pmol/mg per 5 min). Taurocholate inhibited 7,7-azotaurocholate labeling by > 96% with lesser inhibition by taurochenodeoxycholate (83.1%), chenodeoxycholate (74.6%), cholate (50.5%), and progesterone (38.5%), whereas oleic acid and estradiol did not inhibit binding. Images PMID:8197128

  3. A CCCH-Type Zinc Finger Nucleic Acid-Binding Protein Quantitatively Confers Resistance against Rice Bacterial Blight Disease1[W][OA

    PubMed Central

    Deng, Hanqing; Liu, Hongbo; Li, Xianghua; Xiao, Jinghua; Wang, Shiping

    2012-01-01

    Bacterial blight is a devastating disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv oryzae (Xoo). Zinc finger proteins harboring the motif with three conserved cysteine residues and one histidine residue (CCCH) belong to a large family. Although at least 67 CCCH-type zinc finger protein genes have been identified in the rice genome, their functions are poorly understood. Here, we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX8-CX5-CX3-H zinc finger motifs, is involved in the rice-Xoo interaction. Activation of C3H12 partially enhanced resistance to Xoo, accompanied by the accumulation of jasmonic acid (JA) and induced expression of JA signaling genes in rice. In contrast, knockout or suppression of C3H12 resulted in partially increased susceptibility to Xoo, accompanied by decreased levels of JA and expression of JA signaling genes in rice. C3H12 colocalized with a minor disease resistance quantitative trait locus to Xoo, and the enhanced resistance of randomly chosen plants in the quantitative trait locus mapping population correlated with an increased expression level of C3H12. The C3H12 protein localized in the nucleus and possessed nucleic acid-binding activity in vitro. These results suggest that C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and that its function is likely associated with the JA-dependent pathway. PMID:22158700

  4. NMR-based modeling and binding studies of a ternary complex between chicken liver bile acid binding protein and bile acids.

    PubMed

    Tomaselli, Simona; Ragona, Laura; Zetta, Lucia; Assfalg, Michael; Ferranti, Pasquale; Longhi, Renato; Bonvin, Alexandre M J J; Molinari, Henriette

    2007-10-01

    Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions. PMID:17607743

  5. Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain)

    PubMed Central

    Zeng, Xianglan; Ye, Haihui; Yang, Ya’nan; Wang, Guizhong; Huang, Huiyang

    2013-01-01

    Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab. PMID:23569421

  6. Uncoupling of Obesity from Insulin Resistance Through a Targeted Mutation in aP2, the Adipocyte Fatty Acid Binding Protein

    NASA Astrophysics Data System (ADS)

    Hotamisligil, Gokhan S.; Johnson, Randall S.; Distel, Robert J.; Ellis, Ramsey; Papaioannou, Virginia E.; Spiegelman, Bruce M.

    1996-11-01

    Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-α (TNF-α), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-α.

  7. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    PubMed

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. PMID:17661353

  8. Hepatocellular uptake of oleate is energy dependent, sodium linked, and inhibited by an antibody to a hepatocyte plasma membrane fatty acid binding protein.

    PubMed Central

    Stremmel, W; Strohmeyer, G; Berk, P D

    1986-01-01

    Several studies suggest that a portion of hepatocellular nonesterified fatty acid uptake may be carrier mediated. To further investigate this process, initial rates (Vo) of [14C]oleate uptake into rat hepatocytes, isolated by collagenase perfusion and incubated at 37 degrees C with oleate in the presence of bovine serum albumin, were studied as a function of the concentration of unbound [14C]oleate in the medium. Vo was saturable with increasing unbound oleate concentration (Km = 8.3 X 10(-8) M; Vmax = 197 pmol per min per 5 X 10(4) hepatocytes) and was not inhibited by up to 40 microM sulfobromophthalein, taurocholate, or cholic acid. Oleate uptake was sodium dependent. Vo was significantly diminished when Li+, K+, choline, or sucrose were substituted for Na+ in the incubation medium and was reduced 46% by 1 mM ouabain. Uptake was also markedly reduced after exposure of cells to metabolic inhibitors (e.g., 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, antimycin, KCN). To evaluate the physiologic significance of the previously isolated rat liver plasma membrane fatty acid-binding protein, the effect of an antibody directed against this protein on hepatocellular [14C]oleate uptake was examined. Preincubation of hepatocytes with the IgG fraction of this antiserum inhibited Vo of [14C]oleate by up to 65% in dose-related fashion, without altering Vo for [35S]sulfobromophthalein, [14C]taurocholate, or [3H]cholate. These data indicate that at least a portion of hepatocellular oleate uptake is energy dependent, sodium linked, and mediated by a specific liver plasma membrane-fatty acid-binding protein. PMID:3459144

  9. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  10. Role of Cardiac Myocytes Heart Fatty Acid Binding Protein Depletion (H-FABP) in Early Myocardial Infarction in Human Heart (Autopsy Study)

    PubMed Central

    Shabaiek, Amany; Ismael, Nour El-Hoda; Elsheikh, Samar; Amin, Hebat Allah

    2016-01-01

    BACKGROUND: Many immunohistochemical markers have been used in the postmortem detection of early myocardial infarction. AIM: In the present study we examined the role of Heart-type fatty acid binding protein (H-FABP), in the detection of early myocardial infarction. MATERIAL AND METHODS: We obtained samples from 40 human autopsy hearts with/without histopathological signs of ischemia. RESULTS: All cases of definite and probable myocardial infarction showed a well-defined area of H-FABP depletion. All of the control cases showed strong H-FABP expression, except two markedly autolysed myocardial samples that showed affected antigenicity. CONCLUSION: Thus, we suggest H-FABP as being one of the valuable tools facing the problem of postmortem detection of early myocardial infarction/ischemia, but not in autolysis.

  11. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination. PMID:26244568

  12. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGESBeta

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  13. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    SciTech Connect

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  14. The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.

    PubMed Central

    Michel, V; Lehoux, I; Depret, G; Anglade, P; Labadie, J; Hebraud, M

    1997-01-01

    The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes. PMID:9393697

  15. Quantitative Proteomics by SWATH-MS Reveals Altered Expression of Nucleic Acid Binding and Regulatory Proteins in HIV-1-Infected Macrophages

    PubMed Central

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems. PMID:24564501

  16. The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Córsico, Betina; Liou, Heng Ling; Storch, Judith

    2004-03-30

    Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP, transfer occurs by diffusion through the aqueous phase. Earlier, we had shown that the helical domain of IFABP is critical in determining its collisional FA transfer mechanism. In the study presented here, we have engineered a pair of chimeric proteins, one with the "body" (ligand binding domain) of IFABP and the alpha-helical region of LFABP (alphaLbetaIFABP) and the other with the ligand binding pocket of LFABP and the helical domain of IFABP (alphaIbetaLFABP). The objective of this work was to determine whether the change in the alpha-helical domain of each FABP would alter the rate and mechanism of transfer of FA from the chimeric proteins in comparison with those of the wild-type proteins. The fatty acid transfer properties of the FABP chimeras were examined using a fluorescence resonance transfer assay. The results showed a significant modification of the absolute rate of FA transfer from the chimeric proteins compared to that of the wild type, indicating that the slower rate of FA transfer observed for wild-type LFABP relative to that of wild-type IFABP is, in part, determined by the helical domain of the proteins. In addition to these quantitative changes, it was of great interest to observe that the apparent mechanism of FA transfer also changed when the alpha-helical domain was exchanged, with transfer from alphaLbetaIFABP occurring by aqueous diffusion and transfer from alphaIbetaLFABP occurring via protein-membrane collisional interactions. These results demonstrate that the alpha-helical region of LFABP is responsible for its diffusional mechanism of fatty acid transfer to membranes. PMID:15035630

  17. A novel polymorphism in the chicken adipocyte fatty acid-binding protein gene (FABP4) that alters ligand-binding and correlates with fatness.

    PubMed

    Wang, Qigui; Guan, Tianzhu; Li, Hui; Bernlohr, David A

    2009-11-01

    Similar to the mammalian FABP4 gene, the chicken (Gallus gallus) FABP4 gene consists of four exons separated by three introns and encodes a 132 amino acid protein termed the adipocyte fatty acid-binding protein (AFABP). In the current study, a novel G/A polymorphism in exon 3 of the chicken FABP4 gene was identified associated with different chicken breeds that leads to either Ser or Asn at amino acid 89 of the AFABP protein. The Baier chicken averages 0.89+/-0.12% abdominal fat and expresses the G allele (Ser 89 isoform) while the Broiler chicken typically has 3.74+/-0.23% abdominal fat and expresses the A allele (Asn 89 isoforms). cDNAs corresponding to the two AFABP isoforms were cloned and expressed in Escherichia coli as GST fusions, purified by using glutathione sepharose 4B chromatography and evaluated for lipid binding using the fluorescent surrogate ligand 1-anilinonaphthalene 8-sulphonic acid (1,8-ANS). The results showed that AFABP Ser89 exhibited a lower ligand-binding affinity with apparent dissociation constants (Kd) of 7.31+/-3.75 microM, while the AFABP Asn89 isoform bound 1,8-ANS with an apparent dissociation constant of 2.99+/-1.00 microM (P=0.02). These results suggest that the Ser89Asn polymorphism may influence chicken AFABP function and ultimately lipid deposition through changing the ligand-binding activity of AFABP. PMID:19595785

  18. Development of an ultra-rapid diagnostic method based on heart-type fatty acid binding protein levels in the CSF of CJD patients.

    PubMed

    Matsui, Yuki; Satoh, Katsuya; Mutsukura, Kazuo; Watanabe, Takuya; Nishida, Noriyuki; Matsuda, Hideo; Sugino, Masaichi; Shirabe, Susumu; Eguchi, Katsumi; Kataoka, Yasufumi

    2010-10-01

    Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck(®) H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck(®) H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck(®) H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck(®) H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein. PMID:20499272

  19. TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

    SciTech Connect

    Wang Z.; Xu C.; Benning, C.

    2012-05-01

    The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.

  20. Structure of ginseng major latex-like protein 151 and its proposed lysophosphatidic acid-binding mechanism.

    PubMed

    Choi, Sun Hye; Hong, Myoung Ki; Kim, Hyeon Joong; Ryoo, Nayeon; Rhim, Hyewhon; Nah, Seung Yeol; Kang, Lin Woo

    2015-05-01

    Lysophosphatidic acid (LPA) is a phospholipid growth factor with myriad effects on biological systems. LPA is usually present bound to animal plasma proteins such as albumin or gelsolin. When LPA complexes with plasma proteins, it binds to its cognate receptors with higher affinity than when it is free. Recently, gintonin from ginseng was found to bind to LPA and to activate mammalian LPA receptors. Gintonin contains two components: ginseng major latex-like protein 151 (GLP) and ginseng ribonuclease-like storage protein. Here, the crystal structure of GLP is reported, which belongs to the plant Bet v 1 superfamily, and a model is proposed for how GLP binds LPA. Amino-acid residues of GLP recognizing LPA were identified using site-directed mutagenesis and isothermal titration calorimetry. The resulting GLP mutants were used to study the activation of LPA receptor-dependent signalling pathways. In contrast to wild-type GLP, the H147A mutant did not bind LPA, elicit intracellular Ca(2+) transients in neuronal cells or activate Ca(2+)-dependent Cl(-) channels in Xenopus oocytes. Based on these results, a mechanism by which GLP recognizes LPA and its requirement to activate G protein-coupled LPA receptors to elicit diverse biological responses were proposed. PMID:25945569

  1. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes.

    PubMed

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  2. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes

    PubMed Central

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  3. The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

    PubMed Central

    Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

    2013-01-01

    Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

  4. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls.

    PubMed

    Butler, J H; Hu, S; Brady, S R; Dixon, M W; Muday, G K

    1998-02-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo. PMID:11536873

  5. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  6. Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1∙Nup98)

    SciTech Connect

    Quan, Beili; Seo, Hyuk-Soo; Blobel, Günter; Ren, Yi

    2014-07-01

    mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the β-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

  7. The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii.

    PubMed

    Nicosia, Aldo; Costa, Salvatore; Tagliavia, Marcello; Maggio, Teresa; Salamone, Monica; Adamo, Giorgia; Ragusa, Maria Antonietta; Bennici, Carmelo; Masullo, Tiziana; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

    2016-05-01

    Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5'-untranslated region (UTR) of 63 bp and a 3'-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection. PMID:26939892

  8. Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis)

    PubMed Central

    2010-01-01

    Background Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Results Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Conclusions Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes. PMID:20846381

  9. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    PubMed

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  10. Different functions of intestinal and liver-type fatty acid-binding proteins in intestine and in whole body energy homeostasis.

    PubMed

    Lagakos, William Stacy; Gajda, Angela Marie; Agellon, Luis; Binas, Bert; Choi, Victor; Mandap, Bernadette; Russnak, Timothy; Zhou, Yin Xiu; Storch, Judith

    2011-05-01

    It has long been known that mammalian enterocytes coexpress two members of the fatty acid-binding protein (FABP) family, the intestinal FABP (IFABP) and the liver FABP (LFABP). Both bind long-chain fatty acids and have similar though not identical distributions in the intestinal tract. While a number of in vitro properties suggest the potential for different functions, the underlying reasons for expression of both proteins in the same cells are not known. Utilizing mice genetically lacking either IFABP or LFABP, we directly demonstrate that each of the enterocyte FABPs participates in specific pathways of intestinal lipid metabolism. In particular, LFABP appears to target fatty acids toward oxidative pathways and dietary monoacylglycerols toward anabolic pathways, while IFABP targets dietary fatty acids toward triacylglycerol synthesis. The two FABP-null models also displayed differences in whole body response to fasting, with LFABP-null animals losing less fat-free mass and IFABP-null animals losing more fat mass relative to wild-type mice. The metabolic changes observed in both null models appear to occur by nontranscriptional mechanisms, supporting the hypothesis that the enterocyte FABPs are specifically trafficking their ligands to their respective metabolic fates. PMID:21350192

  11. Direct comparison of mice null for liver or intestinal fatty acid-binding proteins reveals highly divergent phenotypic responses to high fat feeding.

    PubMed

    Gajda, Angela M; Zhou, Yin Xiu; Agellon, Luis B; Fried, Susan K; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-10-18

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP(-/-) and LFABP(-/-) mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP(-/-) mice, whereas LFABP(-/-) mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP(-/-) mice fed high fat diets became obese relative to WT, whereas IFABP(-/-) mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP(-/-) mice preferentially utilizing lipids, and IFABP(-/-) mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP(-/-), perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP(-/-) mice, result in divergent downstream effects at the systemic level. PMID:23990461

  12. Fatty Acid Binding Protein 7 Is a Molecular Marker in Adenoid Cystic Carcinoma of the Salivary Glands: Implications for Clinical Significance12

    PubMed Central

    Phuchareon, Janyaporn; Overdevest, Jonathan B.; McCormick, Frank; Eisele, David W.; van Zante, Annemieke; Tetsu, Osamu

    2014-01-01

    Adenoid cystic carcinoma (ACC) is an aggressive malignant neoplasm of the salivary glands. Its diagnosis is difficult due to overlapping features with other salivary tumors. Gene expression analysis may complement traditional diagnostic methods. We searched gene expression patterns in the Gene Expression Omnibus (GEO) database and in our tumor and normal samples. The biologic and prognostic potential of the identified genes was analyzed. The GEO data set of primary xenografted ACCs revealed that expression of five genes, engrailed homeobox 1 (EN1), fatty acid binding protein 7 (FABP7), hemoglobin epsilon 1, MYB, and versican (VCAN), was dramatically increased. mRNA expression of EN1, FABP7, MYB, and VCAN distinguished our sporadic ACCs from normal tissues and benign tumors. FABP7 expression appeared to be regulated differently from EN1 and MYB and was crossly correlated with poor prognosis in our ACC cohort. Immunohistochemistry showed that FABP7 protein was predominantly expressed in the nucleus of myoepithelial cells of both tubular and cribriform subtypes. In contrast, in the solid subtype, which is often associated with a lower survival rate, FABP7 protein was uniformly expressed in cancerous cells. One case with cribriform architecture and the highest level of FABP7 mRNA showed strong FABP7 staining in both duct-type epithelial and myoepithelial cells, suggesting that diffuse expression of FABP7 protein might be related to aggressive tumor behavior and poor prognosis. We propose FABP7 as a novel biomarker in ACC. The molecule may be useful in diagnosis and for identifying more effective therapies targeting this protein or upstream molecules that regulate it. PMID:25500088

  13. Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3▿

    PubMed Central

    Serrano, Pedro; Johnson, Margaret A.; Chatterjee, Amarnath; Neuman, Benjamin W.; Joseph, Jeremiah S.; Buchmeier, Michael J.; Kuhn, Peter; Wüthrich, Kurt

    2009-01-01

    The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 310-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold. PMID:19828617

  14. Bombyx mori nucleopolyhedrovirus orf8 encodes a nucleic acid binding protein that colocalizes with IE1 during infection.

    PubMed

    Imai, N; Kurihara, M; Matsumoto, S; Kang, W-K

    2004-08-01

    This report describes the characterization of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf8 gene. Immunoblot analyses demonstrated that orf8 was expressed as an early gene. The ORF8 protein accumulated in the nucleus, and was maintained at relatively constant levels from 4 to 24 h postinfection. Immunoblot analysis failed to detect ORF8 protein associated with budded virus and occlusion derived virus. In addition, immunohistochemical analysis by confocal microscopy showed that ORF8 protein colocalized with IE1 to specific nuclear foci throughout infection. To further examine the function of ORF8, a reporter gene was inserted into the orf8 reading frame. One orf8 disruption mutant (BmD8), which expressed the N-terminal half of ORF8, was isolated. However, it was not possible to isolate a null mutant, suggesting that orf8 may have an important role during viral infection. Single-step growth curves showed that BV production was reduced in BmD8 infected cells. Biochemical analyses indicated that ORF8 bound to nucleic acids. Together, these results suggest that BmNPV ORF8 may be involved in viral DNA replication and/or transcription. PMID:15290382

  15. Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

    NASA Astrophysics Data System (ADS)

    Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

    2009-12-01

    In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

  16. Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

    PubMed

    Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

    2015-09-11

    Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. PMID:26206084

  17. Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner.

    PubMed

    Hughes, Maria L R; Liu, Bonan; Halls, Michelle L; Wagstaff, Kylie M; Patil, Rahul; Velkov, Tony; Jans, David A; Bunnett, Nigel W; Scanlon, Martin J; Porter, Christopher J H

    2015-05-29

    Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. PMID:25847235

  18. Fatty acid binding protein 10 in the orange-spotted grouper (Epinephelus coioides): characterization and regulation under pH and temperature stress.

    PubMed

    Qi, Zeng-hua; Liu, Yu-feng; Wang, Wei-Na; Xin, Yu; Xie, Fu-xing; Wang, An-Li

    2012-04-01

    We have isolated and characterized a fatty acid binding protein from the liver of the orange-spotted grouper (Epinephelus coioides). Amino acid sequence similarity of the Ec-FABP (E. coioides-FABP) was highest to FABP10s isolated from the livers of catfish, chicken, salamander and iguana. The open-reading frame of the Ec-FABP codes for a protein of 14.0 kDa with a calculated isoelectric point of 8.5. We first expressed a FABP10 protein from orange-spotted grouper (E. coioides) in Pichia pastoris, and then characterized the antioxidative potential of our recombinant Ec-FABP by DCF fluorescence assay. RT-PCR assays showed that endogenous Ec-FABP mRNA is most strongly expressed in liver with the most abundance and intestine. Change in the groupers' blood cells respiratory burst activity was examined during and after exposure to the pH and temperature stress using flow cytometry. Further analysis of Ec-FABP gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-FABP transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its mRNA expression level peaked differed under these stresses. Western blot analyses confirmed that the Ec-FABP protein was strongly expressed in the liver after exposure to the pH and temperature stress. These results suggest that Ec-FABP expression is stimulated by pH and temperature stress and that it may play important roles in general adaptive and antioxidant responses. PMID:22182678

  19. Long-Term Effect of Docosahexaenoic Acid Feeding on Lipid Composition and Brain Fatty Acid-Binding Protein Expression in Rats

    PubMed Central

    Elsherbiny, Marwa E.; Goruk, Susan; Monckton, Elizabeth A.; Richard, Caroline; Brun, Miranda; Emara, Marwan; Field, Catherine J.; Godbout, Roseline

    2015-01-01

    Arachidonic (AA) and docosahexaenoic acid (DHA) brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7), a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains. PMID:26506385

  20. The bovine fatty acid binding protein 4 gene is significantly associated with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses.

    PubMed

    Michal, J J; Zhang, Z W; Gaskins, C T; Jiang, Z

    2006-08-01

    Fatty acid binding protein 4 (FABP4), which is expressed in adipose tissue, interacts with peroxisome proliferator-activated receptors and binds to hormone-sensitive lipase and therefore, plays an important role in lipid metabolism and homeostasis in adipocytes. The objective of this study was to investigate associations of the bovine FABP4 gene with fat deposition. Both cDNA and genomic DNA sequences of the bovine gene were retrieved from the public databases and aligned to determine its genomic organization. Primers targeting two regions of the FABP4 gene were designed: from nucleotides 5433-6106 and from nucleotides 7417-7868 (AAFC01136716). Direct sequencing of polymerase chain reaction (PCR) products on two DNA pools from high- and low-marbling animals revealed two single nucleotide polymorphisms (SNPs): AAFC01136716.1:g.7516G>C and g.7713G>C. The former SNP, detected by PCR-restriction fragment length polymorphism using restriction enzyme MspA1I, was genotyped on 246 F2 animals in a Waygu x Limousin F2 reference population. Statistical analysis showed that the FABP4 genotype significantly affected marbling score (P = 0.0398) and subcutaneous fat depth (P = 0.0246). The FABP4 gene falls into a suggestive/significant quantitative trait loci interval for beef marbling that was previously reported on bovine chromosome 14 in three other populations. PMID:16879357

  1. Fatty acid binding protein 4 regulates VEGF-induced airway angiogenesis and inflammation in a transgenic mouse model: implications for asthma.

    PubMed

    Ghelfi, Elisa; Yu, Chen-Wei; Elmasri, Harun; Terwelp, Matthew; Lee, Chun G; Bhandari, Vineet; Comhair, Suzy A; Erzurum, Serpil C; Hotamisligil, Gökhan S; Elias, Jack A; Cataltepe, Sule

    2013-04-01

    Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function in vitro, but whether it plays a role in modulation of angiogenesis in vivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4(-/-) mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4(-/-) mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma. PMID:23391391

  2. Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis).

    PubMed

    Dubey, Praveen Kumar; Goyal, Shubham; Mishra, Shailendra Kumar; Arora, Reena; Mukesh, Manishi; Niranjan, Saket Kumar; Kathiravan, Periasamy; Kataria, Ranjit Singh

    2016-04-01

    The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes. PMID:26894500

  3. Lack of association between the fatty acid binding protein 2 (FABP2) polymorphism with obesity and insulin resistance in two aboriginal populations from Chile.

    PubMed

    Pérez-Bravo, F; Fuentes, M; Angel, B; Sanchez, H; Carrasco, E; Santos, J L; Lera, L; Albala, C

    2006-12-01

    The aim of this study was to assess the frequency of fatty acid binding protein 2 (FABP2) Ala54Thr genetic polymorphism and to evaluate its association with obesity and insulin resistance in Chilean aboriginal populations. A sample of 96 urban Aymara and 111 urban Mapuche subjects aged 20-80 years were recruited for this cross-sectional study. Glucose, insulin and lipid profile were measured in fasting plasma samples. Insulin resistance was estimated through the HOMA-IR model. FABP2 Ala54Thr genotypes were determined by PCR followed by RFLP analysis. The allele frequency of Thr54 variant was estimated as 18.2% in Aymara subjects, which is one of the lowest reported to date. The corresponding frequency in Mapuche subjects was 31.9% (p<0.002). Regarding genotype-phenotype associations, no significant differences were found in any of the anthropometric or metabolic variables according to Ala54Thr genotypes. After adjustment by BMI and metabolic variables through a logistic regression analysis, the association of the FABP2 polymorphism with ethnic group persisted (Mapuche group: OR=2.37, 95% CI 1.319-4.277, p=0.004) It is unlikely that Ala54Thr polymorphism of the FABP2 gene plays a relevant role in obesity and insulin resistance in Chilean ethnic groups. PMID:17211557

  4. Heart-type fatty acid-binding protein and its relation with morphological changes in rat myocardial damage model induced by isoproterenol.

    PubMed

    Hasić, Sabaheta; Jadrić, Radivoj; Cosović, Esad; Kiseljaković, Emina; Mornjaković, Zakira; Winterhalter-Jadrić, Mira

    2011-11-01

    We have investigated heart type fatty acid binding protein (H-FABP) rat serum values at different time point following subcutaneous (s.c) isoproterenol (ISO) administration and their correlation with severity of myocardial lesion. Thirty adult, male, Wistar rats were used for this study. Six rats per group were treated with a single dose of either ISO (ISO groups, dose 100 mg/kg, s.c.) at different time point (30', 60', 120', 240') or with saline (control group). Serum H-FABP was determined by enzyme-linked immunosorbent assay (ELISA) and histological analysis was performed by hematoxylin-eosin (HE) method of staining. The first serum H-FABP increase was obtained 30' following ISO administration, but maximal value was reached after 240'. Myocardial histological changes were time-dependent and correlated with serum H-FABP values (p<0.001). The results of the study suggest that H-FABP is sensitive marker for acute rat myocardial injury and its possible inclusion in myocardial injury screening studies in rats. PMID:22117831

  5. The Non-native Helical Intermediate State May Accumulate at Low pH in the Folding and Aggregation Landscape of the Intestinal Fatty Acid Binding Protein.

    PubMed

    Sarkar-Banerjee, Suparna; Chowdhury, Sourav; Paul, Simanta Sarani; Dutta, Debashis; Ghosh, Anisa; Chattopadhyay, Krishnananda

    2016-08-16

    There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly β-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely β-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein

  6. Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein.

    PubMed

    Chaurasiya, Kathy R; McCauley, Micah J; Wang, Wei; Qualley, Dominic F; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S B; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C

    2014-01-01

    The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription. PMID:24345943

  7. Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid binding protein

    PubMed Central

    Chaurasiya, Kathy R.; McCauley, Micah J.; Wang, Wei; Qualley, Dominic F.; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S.B.; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G.; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C.

    2014-01-01

    The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response and have broad activity against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalyzed DNA elongation. Here we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription. PMID:24345943

  8. The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix

    PubMed Central

    Barneda, David; Planas-Iglesias, Joan; Gaspar, Maria L; Mohammadyani, Dariush; Prasannan, Sunil; Dormann, Dirk; Han, Gil-Soo; Jesch, Stephen A; Carman, George M; Kagan, Valerian; Parker, Malcolm G; Ktistakis, Nicholas T; Klein-Seetharaman, Judith; Dixon, Ann M; Henry, Susan A; Christian, Mark

    2015-01-01

    Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat. DOI: http://dx.doi.org/10.7554/eLife.07485.001 PMID:26609809

  9. Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy R.; McCauley, Micah J.; Wang, Wei; Qualley, Dominic F.; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S. B.; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G.; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C.

    2014-01-01

    The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

  10. Ligand-specific and non-specific in vivo modulation of human epidermal cellular retinoic acid binding protein (CRABP).

    PubMed

    Hirschel-Scholz, S; Siegenthaler, G; Saurat, J H

    1989-04-01

    Retinoic acid (RA) is bound intracellularly by a specific, low molecular weight protein (CRABP), that is unrelated to its nuclear receptor and whose function and regulation are still unknown. In the present study we were able to obtain an in vivo modulation of CRABP by different stimuli in one of the major target organs of RA: the human skin. We found increased CRABP after daily application during 4 days of natural or synthetic retinoids (RA, acitretin, isotretinoin, Ro137410, retinol), that have either a high affinity to CRABP or can be transformed into RA. Only Ro150778 with no affinity and no reported transformation had no effect. No macro- or microscopical changes could be observed with any of the tested compounds. Induction of inflammatory and hyperproliferative changes in the skin by topical dithranol treatment, UVB irradiation or scotch tape stripping also induced a significant increase of CRABP 3 days after exposure. Topical diflucortolone showed not only a tendancy to decrease intrinsic CRABP levels, but significantly reduced the retinoid stimulated rise of CRABP. Thus we conclude that the increase of CRABP in a fully differentiated adult tissue seems to be a biological phenomenon following processes of inflammation and proliferation with a lag of several days, while retinoids seem to be able to induce such a rise independently of, or before, the appearance of such processes. Corticosteroids seem to be inhibitors of this reaction. We discuss the hypothesis that CRABP might function as an intracellular 'buffer' in the case of RA overload. PMID:2543582

  11. Fatty acid binding proteins 4 and 5 in overweight prepubertal boys: effect of nutritional counselling and supplementation with an encapsulated fruit and vegetable juice concentrate.

    PubMed

    Canas, Jose A; Damaso, L; Hossain, J; Balagopal, P Babu

    2015-01-01

    Elevated fatty acid binding proteins (FABP) may play a role in obesity and co-morbidities. The role of nutritional interventions in modulating these levels remains unclear. The aim of this post hoc study was to determine the effect of overweight (OW) on FABP4 and FABP5 in boys in relation to indices of adiposity, insulin resistance and inflammation, and to investigate the effects of a 6-month supplementation with an encapsulated fruit and vegetable juice concentrate (FVJC) plus nutritional counselling (NC) on FABP levels. A post hoc analysis of a double-blind, randomised, placebo-controlled study of children recruited from the general paediatric population was performed. A total of thirty age-matched prepubertal boys (nine lean and twenty-one OW; aged 6-10 years) were studied. Patients received NC by a registered dietitian and were randomised to FVJC or placebo capsules for 6 months. FABP4, FABP5, glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), glucose-induced acute insulin response (AIR), lipid-corrected β-carotene (LCβC), adiponectin, leptin, high-sensitivity C-reactive protein (hs-CRP), IL-6 and body composition by dual-energy X-ray absorptiometry were determined before and after the intervention. FABP were higher (P < 0·01) in the OW v. lean boys and correlated directly with HOMA-IR, abdominal fat mass (AFM), hs-CRP, IL-6, and LCβC (P < 0·05 for all). FABP4 was associated with adiponectin and AIR (P < 0·05). FVJC plus NC reduced FABP4, HOMA-IR and AFM (P < 0·05 for all) but not FABP5. OW boys showed elevated FABP4 and FABP5, but only FABP4 was lowered by the FVJC supplement. PMID:26688725

  12. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

  13. Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding*

    PubMed Central

    Gajda, Angela M.; Zhou, Yin Xiu; Agellon, Luis B.; Fried, Susan K.; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-01-01

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP−/− and LFABP−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP−/− mice, whereas LFABP−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP−/− mice fed high fat diets became obese relative to WT, whereas IFABP−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP−/− mice preferentially utilizing lipids, and IFABP−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP−/− mice, result in divergent downstream effects at the systemic level. PMID:23990461

  14. Effect of liver fatty acid binding protein (FABP) T94A missense mutation on plasma lipoprotein responsiveness to treatment with fenofibrate.

    PubMed

    Brouillette, Charles; Bossé, Yohan; Pérusse, Louis; Gaudet, Daniel; Vohl, Marie-Claude

    2004-01-01

    Fenofibrate, a peroxisome proliferated activated receptor alpha (PPARalpha) agonist, has been shown to decrease plasma triglyceride (TG) and increase plasma high-density lipoprotein (HDL) cholesterol levels despite a large interindividual variation in the response. Fenofibrate-activated PPARalpha binds to a DNA sequence element termed PPAR response element (PPRE) present in regulatory regions of target genes. A PPRE has been identified in the proximal 5' flanking region of the gene encoding the liver fatty acid binding protein (LFABP). LFABP is a small cytosolic protein of 14 kDa present in the liver and the intestine and is a member of the superfamily of the fatty acid binding proteins (FABPs). FABPs play a role in the solubilization of long-chain fatty acids (LCFAs) and their CoA-ester to various intracellular organelles. FABPs serves as intracellular acceptors of LCFAs, and they may also have an impact in ligand-dependent transactivation of PPARs in trafficking LCFAs to the nucleus. Since PPARs are known to regulate the transcription of many genes involved in lipid metabolism, the importance of LFABP in fatty acid uptake has to be considered. The aim of this study was to verify whether genetic variations in the LFABP gene may impact on plasma lipoprotein/lipid levels in the fasting state as well as on the response to a lipid-lowering therapy with fenofibrate on plasma lipids and obesity variables. We also wanted to verify whether the presence of the PPARalpha L162V mutation interacts with genetic variants in LFABP gene. To achieve this goal, we first determined the genomic structure of the human LFABP gene and then designed intronic primers to sequence the coding regions, all exon-intron splicing boundaries, and the promoter region of the gene in 24 patients showing divergent plasma lipoprotein/lipid response to fenofibrate. Sequence analysis revealed the presence of a T94A missense mutation in exon 3. Interspecies comparison revealed that threonine 94 is

  15. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    PubMed

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p

  16. Serum Level of Heart-Type Fatty Acid Binding Protein (H-FABP) Before and After Treatment of Congestive Heart Failure in Children.

    PubMed

    Zoair, Amr; Mawlana, Wegdan; Abo-Elenin, Amany; Korrat, Mostafa

    2015-12-01

    Remodeling of the heart following injury affects the morbidity and mortality in children presented with heart failure (HF). Heart-type fatty acid binding protein (H-FABP) is a novel biomarker that could be of help to predict the prognosis and risk stratification in those children. We aimed to evaluate the diagnostic and prognostic value of H-FABP in children with heart failure before and after treatment. The study was conducted as a prospective cohort study. It included 30 children with HF as a patient group and 20 healthy children matched for age and sex as a control group. Echocardiographic assessment of the heart was done using conventional Doppler echocardiography. Serum levels of (H-FABP) were measured using enzyme-linked immunosorbent assay before and after treatment of HF. All patients were observed during follow-up period of 3 months. There was a significant difference in the serum level of H-FABP in our patients before treatment (5.278 ± 3.253 ng/ml) compared with after treatment (2.089 ± 0.160 ng/ml) with significant difference compared with the control group. There was a significant increase in the serum level of H-FABP with increase in the severity of heart failure according to Ross classification. Significant increase in the H-FABP was associated with adverse outcome. Serum levels of H-FABP strongly correlated with clinical and echocardiographic assessment of LV performance of children with HF, and its levels significantly increased in children with adverse outcome suggesting its value as a useful diagnostic and prognostic predictor (with high sensitivity and specificity). PMID:26123812

  17. Effect of endurance training and/or fish oil supplemented diet on cytoplasmic fatty acid binding protein in rat skeletal muscles and heart.

    PubMed

    Clavel, Stéphan; Farout, L; Briand, M; Briand, Y; Jouanel, P

    2002-07-01

    Endurance training and/or a fish oil supplemented diet affect cytoplasmic fatty acid binding protein (FABP(c)) content in rat skeletal muscles and heart. After 8 weeks of swimming, trained rats exhibited higher FABP(c) content in the extensor digitorum longus (EDL) and in the gastrocnemius than did control rats (30%). The FABP(c) increase was associated with an increase of citrate synthase activity (85% and 93%, respectively, in the two muscles), whereas lactate dehydrogenase activity decreased significantly. In contrast, in the soleus and in the heart we did not observe any effect of exercise either on FABP(c) or on the metabolic profile. Therefore, increasing oxidative capacities of muscle by exercise resulted in a concomitant increase of the FABP(c) content. Giving a polyunsaturated fatty acid (omega-3) supplemented diet for eight weeks induced a large rise of the FABP(c) in EDL (300%), gastrocnemius (250%), soleus (50%) and heart (15%) without a concurrent accumulation of intramuscular triglycerides or modification of the citrate synthase activity, suggesting that polyunsaturated fatty acids may increase FABP(c) content by up-regulating fatty acid metabolism genes via peroxisome proliferator-activated receptor alpha activation. Endurance trained rats fed with an omega-3 diet had similar FABP(c) content in the gastrocnemius muscle when compared to sedentary omega-3 fed rats, whereas an additive effect of exercise and diet was observed in the EDL. The FABP(c) in the soleus and in the heart of rats fed with omega-3 supplements remained constant whether rats performed exercise or not. As a result, both exercise and omega-3-enriched diet influenced FABP(c) content in muscle. These two physiological treatments presumably acted on FABP(c) content by increasing fatty acid flux within the cell. PMID:12111278

  18. Fatty acid-binding protein 5 regulates diet-induced obesity via GIP secretion from enteroendocrine K cells in response to fat ingestion.

    PubMed

    Shibue, Kimitaka; Yamane, Shunsuke; Harada, Norio; Hamasaki, Akihiro; Suzuki, Kazuyo; Joo, Erina; Iwasaki, Kanako; Nasteska, Daniela; Harada, Takanari; Hayashi, Yoshitaka; Adachi, Yasuhiro; Owada, Yuji; Takayanagi, Ryoichi; Inagaki, Nobuya

    2015-04-01

    Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K cells in response to nutrient intake, especially fat. GIP is one of the contributing factors inducing fat accumulation that results in obesity. A recent study shows that fatty acid-binding protein 5 (FABP5) is expressed in murine K cells and is involved in fat-induced GIP secretion. We investigated the mechanism of fat-induced GIP secretion and the impact of FABP5-related GIP response on diet-induced obesity (DIO). Single oral administration of glucose and fat resulted in a 40% reduction of GIP response to fat but not to glucose in whole body FABP5-knockout (FABP5(-/-)) mice, with no change in K cell count or GIP content in K cells. In an ex vivo experiment using isolated upper small intestine, oleic acid induced only a slight increase in GIP release, which was markedly enhanced by coadministration of bile and oleic acid together with attenuated GIP response in the FABP5(-/-) sample. FABP5(-/-) mice exhibited a 24% reduction in body weight gain and body fat mass under a high-fat diet compared with wild-type (FABP5(+/+)) mice; the difference was not observed between GIP-GFP homozygous knock-in (GIP(gfp/gfp))-FABP5(+/+) mice and GIP(gfp/gfp)-FABP5(-/-) mice, in which GIP is genetically deleted. These results demonstrate that bile efficiently amplifies fat-induced GIP secretion and that FABP5 contributes to the development of DIO in a GIP-dependent manner. PMID:25628425

  19. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken.

    PubMed

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-10-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  20. Heart-type Fatty acid-binding protein in Acute Myocardial infarction Evaluation (FAME): Background and design of a diagnostic study in primary care

    PubMed Central

    Bruins Slot, Madeleine HE; van der Heijden, Geert JMG; Rutten, Frans H; van der Spoel, Onno P; Mast, E Gijs; Bredero, Ad C; Doevendans, Pieter A; Glatz, Jan FC; Hoes, Arno W

    2008-01-01

    Background Currently used biomarkers for cardiac ischemia are elevated in blood plasma after a delay of several hours and therefore unable to detect acute coronary syndrome (ACS) in a very early stage. General practitioners (GPs), however, are often confronted with patients suspected of ACS within hours after onset of complaints. This ongoing study aims to evaluate the added diagnostic value beyond clinical assessment for a rapid bedside test for heart-type fatty-acid binding protein (H-FABP), a biomarker that is detectable as soon as one hour after onset of ischemia. Methods Participating GPs perform a blinded H-FABP rapid bedside test (Cardiodetect®) in patients with symptoms suggestive of ACS such as chest pain or discomfort at rest. All patients, whether referred to hospital or not, undergo electrocardiography (ECG) and venapunction for a plasma troponin test within 12–36 hours after onset of complaints. A final diagnosis will be established by an expert panel consisting of two cardiologists and one general practitioner (blinded to the H-FABP test result), using all available patient information, also including signs and symptoms. The added diagnostic value of the H-FABP test beyond history taking and physical examination will be determined with receiver operating characteristic curves derived from multivariate regression analysis. Conclusion Reasons for presenting the design of our study include the prevention of publication bias and unacknowledged alterations in the study aim, design or data-analysis. To our knowledge this study is the first to assess the diagnostic value of H-FABP outside a hospital-setting. Several previous hospital-based studies showed the potential value of H-FABP in diagnosing ACS. Up to now however it is unclear whether these results are equally promising when the test is used in primary care. The first results are expected in the end of 2008. PMID:18412949

  1. Fasciola hepatica fatty acid binding protein inhibits TLR4 activation and suppresses the inflammatory cytokines induced by LPS in vitro and in vivo

    PubMed Central

    Martin, Ivelisse; Cabán-Hernández, Kimberly; Figueroa-Santiago, Olgary; Espino, Ana M.

    2015-01-01

    Toll-like receptor 4 (TLR4), the innate immunity receptor for bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. There is a need to develop molecules that block either activation through TLR4 or the downstream signaling pathways to inhibit the storm of inflammation typically elicited by bacterial lipopolysaccharide (LPS), which is a major cause of the high mortality associated with bacterial sepsis. We report here that a single intraperitoneal injection of 15μg Fasciola hepatica fatty acid binding protein (Fh12) 1 hour before exposure to LPS suppressed significantly the expression of serum inflammatory cytokines in a model of septic shock using C57BL/6 mice. Because macrophages are good source of IL12p70 and TNFα, and critical in driving adaptive immunity, we investigated the effect of Fh12 on the function of mouse bone marrow derived macrophages (bmMΦs). Whereas Fh12 alone did not induce cytokine expression, it significantly suppressed the expression of IL12, TNFα, IL6 and IL1β cytokines as well as iNOS2 in bmMΦs, and also impaired the phagocytic capacity of bmMΦs. Fh12 had a limited effect on the expression of inflammatory cytokines induced in response to other TLR-ligands. One mechanism used by Fh12 to exert its anti-inflammatory effect is binding to the CD14 co-receptor. Moreover, it suppresses phosphorylation of ERK, p38 and JNK. The potent anti-inflammatory properties of Fh12 demonstrated here open doors to further studies directed at exploring the potential of this molecule as a new class of drug against septic shock or other inflammatory diseases. PMID:25780044

  2. Heart-Type Fatty Acid-Binding Protein, in Early Detection of Acute Myocardial Infarction: Comparison with CK-MB, Troponin I and Myoglobin.

    PubMed

    Pyati, Anand K; Devaranavadagi, Basavaraj B; Sajjannar, Sanjeev L; Nikam, Shashikant V; Shannawaz, Mohd; Patil, Satish

    2016-10-01

    The study aimed to investigate whether heart-type fatty acid binding protein (H-FABP) measurement provides additional diagnostic value to that of conventional cardiac markers in acute myocardial infarction (AMI) within first 6 h after the onset of symptoms. The study included 120 subjects: 60 AMI cases and 60 age and sex matched controls. The cases and controls were further divided into 2 subgroups depending on the time since onset of chest pain as (1) subjects within 3 h and (2) between 3 and 6 h of onset of chest pain. In all the cases and controls, serum H-FABP concentration was measured by Immunoturbidimetric method, serum Troponin I and myoglobin concentrations by Chemiluminescence immunoassay and serum CK-MB concentration by Immuno-inhibition method. The sensitivity, specificity, positive and negative predictive values of H-FABP were significantly greater than CK-MB and myoglobin but were lesser than Troponin I in patients with suspected AMI in both within 3 h and 3-6 h groups. Receiver operating characteristic curves demonstrated greatest diagnostic ability for Troponin I (AUC = 0.99, p < 0.001) followed by H-FABP (AUC = 0.906, p < 0.001) within 3 h and 3-6 h after the onset of chest pain. In conclusion, the diagnostic value of H-FABP is greater than CK-MB and myoglobin but slightly lesser than troponin I for the early diagnosis of AMI within first 6 h of chest pain. H-FABP can be used as an additional diagnostic tool for the early diagnosis of AMI along with troponin I. PMID:27605741

  3. Association of Heart-Type Fatty Acid-Binding Protein with Cardiovascular Risk Factors and All-Cause Mortality in the General Population: The Takahata Study

    PubMed Central

    Otaki, Yoichiro; Watanabe, Tetsu; Takahashi, Hiroki; Hirayama, Atushi; Narumi, Taro; Kadowaki, Shinpei; Honda, Yuki; Arimoto, Takanori; Shishido, Tetsuro; Miyamoto, Takuya; Konta, Tsuneo; Shibata, Yoko; Fukao, Akira; Daimon, Makoto; Ueno, Yoshiyuki; Kato, Takeo; Kayama, Takamasa; Kubota, Isao

    2014-01-01

    Background Despite many recent advances in medicine, preventing the development of cardiovascular diseases remains a challenge. Heart-type fatty acid-binding protein (H-FABP) is a marker of ongoing myocardial damage and has been reported to be a useful indicator for future cardiovascular events. However, it remains to be determined whether H-FABP can predict all-cause and cardiovascular deaths in the general population. Methods and Results This longitudinal cohort study included 3,503 subjects who participated in a community-based health checkup with a 7-year follow-up. Serum H-FABP was measured in registered subjects. The results demonstrated that higher H-FABP levels were associated with increasing numbers of cardiovascular risk factors, including hypertension, diabetes mellitus, obesity, and metabolic syndrome. There were 158 deaths during the follow-up period, including 50 cardiovascular deaths. Deceased subjects had higher H-FABP levels compared to surviving subjects. Multivariate Cox proportional hazard regression analysis revealed that H-FABP is an independent predictor of all-cause and cardiovascular deaths after adjustments for confounding factors. Subjects were divided into four quartiles according to H-FABP level, and Kaplan-Meier analysis demonstrated that the highest H-FABP quartile was associated with the greatest risks for all-cause and cardiovascular deaths. Net reclassification index and integrated discrimination index were significantly increased by addition of H-FABP to cardiovascular risk factors. Conclusions H-FABP level was increased in association with greater numbers of cardiovascular risk factors and was an independent risk factor for all-cause and cardiovascular deaths. H-FABP could be a useful indicator for the early identification of high-risk subjects in the general population. PMID:24847804

  4. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

    PubMed Central

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-01-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  5. Heart-Type Fatty Acid Binding Protein: A Better Cardiac Biomarker than CK-MB and Myoglobin in the Early Diagnosis of Acute Myocardial Infarction

    PubMed Central

    Devaranavadagi, Basavaraj B; Sajjannar, Sanjeev L; Nikam, Shashikant V; Shannawaz, Mohd; Sudharani

    2015-01-01

    Background Early diagnosis and therapeutic intervention can improve the outcome of acute myocardial infarction (AMI). However, there are no satisfactory cardiac biomarkers for the diagnosis of AMI within 6 hours of onset of symptoms. Among novel biochemical markers of AMI, heart-type fatty acid binding protein (H-FABP) is of particular interest. Aim To compare the diagnostic value of H-FABP with that of CK-MB and myoglobin in suspected AMI patients within first 6 hours after the onset of symptoms. Settings and Design The study includes 40 AMI cases and 40 non-cardiac chest pain otherwise healthy controls. The cases and controls were further divided into 2 groups depending on the time since chest pain as those subjects within 3 hours and those between 3-6 hours of onset of chest pain. Materials and Methods In all the cases and controls, serum H-FABP, CK-MB and myoglobin concentrations were measured by Immunoturbidimetric method, immuno-inhibition method and Chemiluminescence immunoassay respectively. Statistical Analysis Data is presented as mean ± SD values. Differences between means of two groups were assessed by Student t-test. Sensitivity, Specificity, Positive predictive value, Negative predictive values were calculated and ROC curve analysis was done to assess the diagnostic validity of each study parameter. Results The sensitivity, specificity, PPV, NPV of H-FABP were greater than CK-MB and myoglobin and ROC curve analysis demonstrated highest area under curve for H-FABP followed by myoglobin and CK-MB in patients with suspected AMI both within 3 hours and 3-6 hours after the onset of chest pain. Conclusion The diagnostic efficiency of H-FABP is greater than CK-MB and myoglobin for the early diagnosis of AMI within first 6 hours of chest pain. H-FABP can be used as an additional diagnostic tool for the early diagnosis of AMI. PMID:26557510

  6. The Ala54Thr Polymorphism of the Fatty Acid Binding Protein 2 Gene Modulates HDL Cholesterol in Mexican-Americans with Type 2 Diabetes

    PubMed Central

    Salto, Lorena M.; Bu, Liming; Beeson, W. Lawrence; Firek, Anthony; Cordero-MacIntyre, Zaida; De Leon, Marino

    2015-01-01

    The alanine to threonine amino acid substitution at codon 54 (Ala54Thr) of the intestinal fatty acid binding protein (FABP2) has been associated with elevated levels of insulin and blood glucose as well as with dyslipidemia. The aim of this study was to characterize the effect of this FABP2 polymorphism in Mexican-Americans with type 2 diabetes (T2D) in the context of a three-month intervention to determine if the polymorphism differentially modulates selected clinical outcomes. For this study, we genotyped 43 participant samples and performed post-hoc outcome analysis of the profile changes in fasting blood glucose, HbA1c, insulin, lipid panel and body composition, stratified by the Ala54Thr polymorphism. Our results show that the Thr54 allele carriers (those who were heterozygous or homozygous for the threonine-encoding allele) had lower HDL cholesterol and higher triglyceride levels at baseline compared to the Ala54 homozygotes (those who were homozygous for the alanine-encoding allele). Both groups made clinically important improvements in lipid profiles and glycemic control as a response to the intervention. Whereas the Ala54 homozygotes decreased HDL cholesterol in the context of an overall total cholesterol decrease, Thr54 allele carriers increased HDL cholesterol as part of an overall total cholesterol decrease. We conclude that the Ala54Thr polymorphism of FABP2 modulates HDL cholesterol in Mexican-Americans with T2D and that Thr54 allele carriers may be responsive in interventions that include dietary changes. PMID:26703680

  7. Proteomic analysis of human papillomavirus-related oral squamous cell carcinoma: identification of thioredoxin and epidermal-fatty acid binding protein as upregulated protein markers in microdissected tumor tissue.

    PubMed

    Melle, Christian; Ernst, Günther; Winkler, Robert; Schimmel, Bettina; Klussmann, Jens Peter; Wittekindt, Claus; Guntinas-Lichius, Orlando; von Eggeling, Ferdinand

    2009-04-01

    Human papillomavirus (HPV) infection has been identified as an etiologic agent for a subset of oral squamous cell carcinoma (OSCC) with increasing incidence. HPV DNA-positivity may confer better prognosis but the related oncogenic mechanisms are unknown. For the identification of HPV relevant proteins, we analyzed microdissected cells from HPV DNA-positive (n = 17) and HPV DNA-negative (n = 7) OSCC tissue samples. We identified 18 proteins from tumor tissues by peptide fingerprint mapping and SELDI MS that were separated using 2-DE. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified thioredoxin (TRX) and epidermal-fatty acid binding protein as upregulated in HPV related tumor tissue. This study, investigating for the first time proteomic changes in microdissected HPV infected tumor tissue, provides an indication on the oncogenic potential of viruses. PMID:19337991

  8. Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.

    PubMed

    Jing, C; Beesley, C; Foster, C S; Chen, H; Rudland, P S; West, D C; Fujii, H; Smith, P H; Ke, Y

    2001-06-01

    Human cutaneous fatty acid-binding protein (C-FABP) gene is capable of inducing the metastatic phenotype when overexpressed in nonmetastatic rat Rama 37 cells. However, the mechanism of how it induces metastasis is not clear. Northern and slot blot analyses revealed that expression of the endogenous vascular endothelial growth factor (VEGF) gene was increased by 3.8-5.2-fold in the C-FABP-transfected cells (pSV-CFABP-R37) and in their metastatic sublines (e.g., Met-1) when compared with that in the nonmetastatic control transfectant pSV-R37 cells generated by transfection of only plasmid DNA. Higher levels of VEGF immunoreactive protein were also secreted from the malignant C-FABP-expressing cells. Reverse transcription-PCR detected two VEGF transcript isoforms, VEGF(164) and VEGF(188), in both the nonmetastatic control transfectant pSV-R37 cells and the malignant metastatic Met-1 cells. Chick chorioallantoic membrane assays showed that the conditioned medium of the control pSV-R37 cells possessed only very weak angiogenic activity, whereas conditioned media from the metastatic C-FABP transfectants and their sublines were strongly angiogenic and could be inhibited by antibodies to VEGF. Transfection of VEGF(164) cDNA in an expression vector into nonmetastatic Rama 37 cells produced a cell clone (R37-VEGF-2) that expressed high levels of VEGF. Inoculation of R37-VEGF-2 cells into syngeneic Wistar Furth rats produced metastases in a significant number (Fisher's exact test, P < 0.01) of animals (18 of 31 animals), whereas the control, vector alone-transfected R37-PSV cells produced no metastases (0 of 30 animals). Immunocytochemical methods demonstrated a strong positive staining for VEGF and an increased microvessel density in the primary tumors produced from PSV-VEGF-2 cells in comparison with tumors produced from control transfectants. Immunocytochemical staining for factor VIII detected a 3.5-fold increase in microvessel density of the primary tumors produced by

  9. Fatty acid binding protein 3 (fabp3) is associated with insulin, lipids and cardiovascular phenotypes of the metabolic syndrome through epigenetic modifications in a northern european family population

    PubMed Central

    2013-01-01

    Background Fatty acid-binding proteins (FABPs) play regulatory roles at the nexus of lipid metabolism and signaling. Dyslipidemia in clinical manifestation frequently co-occurs with obesity, insulin resistance and hypertension in the Metabolic Syndrome (MetS). Animal studies have suggested FABPs play regulatory roles in expressing MetS phenotypes. In our family cohort of Northern European descent, transcript levels in peripheral white blood cells (PWBCs) of a key FABPs, FABP3, is correlated with the MetS leading components. However, evidence supporting the functions of FABPs in humans using genetic approaches has been scarce, suggesting FABPs may be under epigenetic regulation. The objective of this study was to test the hypothesis that CpG methylation status of a key regulator of lipid homeostasis, FABP3, is a quantitative trait associated with status of MetS phenotypes in humans. Methods We used a mass-spec based quantitative method, EpiTYPER®, to profile a CpG island that extends from the promoter to the first exon of the FABP3 gene in our family-based cohort of Northern European descent (n=517). We then conducted statistical analysis of the quantitative relationship of CpG methylation and MetS measures following the variance-component association model. Heritability of each methylation and the effect of age and sex on CpG methylation were also assessed in our families. Results We find that methylation levels of individual CpG units and the regional average are heritable and significantly influenced by age and sex. Regional methylation was strongly associated with plasma total cholesterol (p=0.00028) and suggestively associated with LDL-cholesterol (p=0.00495). Methylation at individual units was significantly associated with insulin sensitivity, lipid particle sizing and diastolic blood pressure (p<0.0028, corrected for multiple testing for each trait). Peripheral white blood cell (PWBC) expression of FABP3 in a separate group of subjects (n=128) negatively

  10. The effect of charge reversal mutations in the alpha-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs, lysophospholipids and bile acids.

    PubMed

    Hagan, Robert M; Davies, Joanna K; Wilton, David C

    2002-10-01

    Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of alpha-helix II. The alpha-helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on alpha-helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the alpha-helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group. PMID:12479568

  11. Inhibition of gene expression of carnitine palmitoyltransferase I and heart fatty acid binding protein in cyclophosphamide and ifosfamide-induced acute cardiotoxic rat models.

    PubMed

    Sayed-Ahmed, Mohamed M; Aldelemy, Meshan L; Al-Shabanah, Othman A; Hafez, Mohamed M; Al-Hosaini, Khaled A; Al-Harbi, Naif O; Al-Sharary, Shakir D; Al-Harbi, Mohamed M

    2014-09-01

    This study investigated whether cyclophosphamide (CP) and ifosfamide (IFO) therapy alters the expression of the key genes engaged in long-chain fatty acid (LCFA) oxidation outside rat heart mitochondria, and if so, whether these alterations should be viewed as a mechanism during CP- and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of the six treatment groups: Rats in group 1 (control) and group 2 (L-carnitine) were injected intraperitoneal (i.p.) with normal saline and L-carnitine (200 mg/kg/day), respectively, for 10 successive days. Animals in group 3 (CP group) were injected i.p. with normal saline for 5 days before and 5 days after a single dose of CP (200 mg/kg, i.p.). Rats in group 4 (IFO group) received normal saline for 5 successive days followed by IFO (50 mg/kg/day, i.p.) for 5 successive days. Rats in group 5 (CP-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5 days before and 5 days after a single dose of CP as group 3. Rats in group 6 (IFO-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5 days before and 5 days concomitant with IFO as group 4. Immediately, after the last dose of the treatment protocol, blood samples were withdrawn and animals were killed for biochemical, histopathological and gene expression studies. Treatment with CP and IFO significantly decreased expression of heart fatty acid binding protein (H-FABP) and carnitine palmitoyltransferase I (CPT I) genes in cardiac tissues. Moreover, CP but not IFO significantly increased acetyl-CoA carboxylase2 mRNA expression. Conversely, IFO but not CP significantly decreased mRNA expression of malonyl-CoA decarboxylase. Both CP and IFO significantly increased serum lactate dehydrogenase, creatine kinase isoenzyme MB and malonyl-CoA content and histopathological lesions in cardiac tissues. Interestingly, carnitine supplementation completely reversed all the biochemical, histopathological and

  12. Uses of Phage Display in Agriculture: Sequence Analysis and Comparative Modeling of Late Embryogenesis Abundant Client Proteins Suggest Protein-Nucleic Acid Binding Functionality

    PubMed Central

    Kushwaha, Rekha; Downie, A. Bruce; Payne, Christina M.

    2013-01-01

    A group of intrinsically disordered, hydrophilic proteins—Late Embryogenesis Abundant (LEA) proteins—has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From this previous study, we identified 21 client proteins for which clones were recovered, sometimes repeatedly. Here, we use sequence analysis and homology modeling of the client proteins to ascertain common sequence and structural properties that may contribute to binding affinity with the protective LEA protein. Our methods uncover what appears to be a predilection for protein-nucleic acid interactions among LEA client proteins, which is suggestive of subcellular residence. The results from this initial computational study will guide future efforts to uncover the protein protective mechanisms during heat stress, potentially leading to phage-display-directed evolution of synthetic LEA molecules. PMID:23956788

  13. Sequence-specific {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments for intestinal fatty-acid-binding protein complexed with palmitate (15.4 kDA)

    SciTech Connect

    Hodsdon, M.E.; Toner, J.J.; Cistola, D.P.

    1994-12-01

    Intestinal fatty-acid-binding protein (I-FABP) belongs to a family of soluble, cytoplasmic proteins that are thought to function in the intracellular transport and trafficking of polar lipids. Individual members of this protein family have distinct specificities and affinities for fatty acids, cholesterol, bile salts, and retinoids. We are comparing several retinol- and fatty-acid-binding proteins from intestine in order to define the factors that control molecular recognition in this family of proteins. We have established sequential resonance assignments for uniformly {sup 13}C/{sup 15}N-enriched I-FABP complexed with perdeuterated palmitate at pH7.2 and 37{degrees}C. The assignment strategy was similar to that introduced for calmodulin. We employed seven three-dimensional NMR experiments to establish scalar couplings between backbone and sidechain atoms. Backbone atoms were correlated using triple-resonance HNCO, HNCA, TOCSY-HMQC, HCACO, and HCA(CO)N experiments. Sidechain atoms were correlated using CC-TOCSY, HCCH-TOCSY, and TOCSY-HMQC. The correlations of peaks between three-dimensional spectra were established in a computer-assisted manner using NMR COMPASS (Molecular Simulations, Inc.) Using this approach, {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments have been established for 120 of the 131 residues of I-FABP. For 18 residues, amide {sup 1}H and {sup 15}N resonances were unobservable, apparently because of the rapid exchange of amide protons with bulk water at pH 7.2. The missing amide protons correspond to distinct amino acid patterns in the protein sequence, which will be discussed. During the assignment process, several sources of ambiguity in spin correlations were observed. To overcome this ambiguity, the additional inter-residue correlations often observed in the HNCA experiment were used as cross-checks for the sequential backbone assignments.

  14. Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycans and of hyaluronic acid-binding proteins in successive cartilage extracts.

    PubMed Central

    Vilim, V; Krajickova, J

    1991-01-01

    Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn-HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1705114

  15. Crystal Structure of the Mp1p Ligand Binding Domain 2 Reveals Its Function as a Fatty Acid-binding Protein*

    PubMed Central

    Liao, Shuang; Tung, Edward T. K.; Zheng, Wei; Chong, Ken; Xu, Yuanyuan; Dai, Peng; Guo, Yingying; Bartlam, Mark; Yuen, Kwok-Yung; Rao, Zihe

    2010-01-01

    Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 Å. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (Kd) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen. PMID:20053994

  16. Rosuvastatin Decreases Intestinal Fatty Acid Binding Protein (I-FABP), but Does Not Alter Zonulin or Lipopolysaccharide Binding Protein (LBP) Levels, in HIV-Infected Subjects on Antiretroviral Therapy

    PubMed Central

    Funderburg, Nicholas T.; Boucher, Morgan; Sattar, Abdus; Kulkarni, Manjusha; Labbato, Danielle; Kinley, Bruce I.; McComsey, Grace A.

    2016-01-01

    Introduction Altered gastrointestinal (GI) barrier integrity and subsequent microbial translocation may contribute to immune activation in HIV infection. We have reported that rosuvastatin improved several markers of immune activation in HIV+ participants, but the effect of statin treatment on markers of GI barrier dysfunction is unknown. Methods SATURN-HIV is a randomized, double-blind, placebo-controlled trial assessing the effect of rosuvastatin (10mg/daily) on markers of cardiovascular disease, inflammation, and immune activation in ART-treated patients. Gut-barrier integrity was assessed by the surrogate markers intestinal fatty acid binding protein (I-FABP), a marker of enterocyte death, and zonulin-1, a marker of gut epithelial cell function. Levels of lipopolysaccharide binding protein (LBP) were measured as a marker of microbial translocation. Results Rosuvastatin significantly reduced levels of I-FABP during the treatment period compared to the placebo. There was no effect of rosuvastatin treatment on levels of zonulin or LBP. Baseline levels of LBP were directly related to several markers of immune activation in samples from all participants, including soluble CD163, IP-10, VCAM-1, TNFR-II, and the proportion of CD4+ and CD8+ T cells expressing CD38 and HLA-DR. Many of these relationships, however, were not seen in the statin arm alone at baseline or over time, as inflammatory markers often decreased and LBP levels were unchanged. Conclusions Forty-eight weeks of rosuvastatin treatment reduced levels of I-FABP, but did not affect levels of zonulin or LBP. The reduction in levels of inflammatory markers that we have reported with rosuvastatin treatment is likely mediated through other mechanisms not related to gut integrity or microbial translocation.

  17. Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

    PubMed

    Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

    2016-08-01

    Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein

  18. The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response.

    PubMed

    Slaymaker, David H; Navarre, Duroy A; Clark, Daniel; del Pozo, Olga; Martin, Gregory B; Klessig, Daniel F

    2002-09-01

    In plants, salicylic acid (SA) plays an important role in signaling both local and systemic defense responses. Previous efforts to identify SA effector proteins in tobacco have led to the isolation of two soluble cytoplasmic SA-binding proteins (SABPs): catalase, SABP, and an approximately 25-kDa protein, SABP2. Here we describe the identification of an SA-binding protein, SABP3, in the stroma of tobacco chloroplasts. SABP3 bound SA with an apparent dissociation constant (K(d)) of 3.7 microM and exhibited much greater affinity for biologically active than inactive analogs. Purification and partial sequencing of SABP3 indicated that it is the chloroplast carbonic anhydrase (CA). Confirming this finding, recombinant tobacco chloroplast CA exhibited both CA enzymatic and SA-binding activities. Expression of this protein in yeast also demonstrated that CA/SABP3 has antioxidant activity. A second gene encoding CA was also cloned, and its encoded protein was shown to behave similarly to that purified as SABP3. Finally, silencing of CA gene expression in leaves suppressed the Pto:avrPto-mediated hypersensitive response in disease resistance. These results demonstrate that SA may act through multiple effector proteins in plants and shed further light on the function of CA in chloroplasts. PMID:12185253

  19. The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Franchini, G R; Storch, J; Corsico, B

    2008-04-01

    Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes. PMID:18284926

  20. A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

    PubMed

    Fleming, Jonathan K; Glass, Thomas R; Lackie, Steve J; Wojciak, Jonathan M

    2016-09-01

    Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling. PMID:27444045

  1. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

    PubMed

    Pedersen, L B; Birkelund, S; Holm, A; Ostergaard, S; Christiansen, G

    1996-02-01

    The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1. PMID:8576073

  2. Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.

    PubMed

    Chen, G; Radominska-Pandya, A

    2000-10-17

    Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution. PMID:11027136

  3. Functional analysis of a dietary recombinant fatty acid binding protein 10 (FABP10) on the Epinephelus coioides in response to acute low temperature challenge.

    PubMed

    Luo, Sheng-Wei; Cai, Luo; Liu, Yuan; Wang, Wei-Na

    2014-02-01

    The effect of Ec-FABP10 (Epinephelus coiodes-FABP10) on growth performance, enzyme activity, respiratory burst, MDA level, ATP content, immune-related gene expression of juvenile orange-spotted grouper (E. coioides). The commercial diet supplemented with FABP10 protein was feed to orange-spotted grouper for six weeks. No significant difference was observed in the specific growth rates, while the survival rate in the FABP10 additive group was significantly higher. After the feeding trial, the groupers were exposed to acute low temperature challenge. The decreased level of respiratory burst activity was observed in the FABP10 additive group after the exposure to the acute low temperature stress, while the blood cell count increased significantly at 15 °C and a significant increase of ATP content was observed at 10 °C. Higher enzymatic activities of CAT and SOD were observed at 20 °C and 15 °C, respectively. Meanwhile, the lower level of MDA was observed after the exposure to acute low temperature challenge by comparing with the controls. Further transcript expression analyses of FABP10, SOD2, GPX4, HSPA4 and LIPC in liver by quantitative real-time PCR demonstrated that the up-regulated transcript expression of FABP10, SOD2, HSPA4 and LIPC was observed in FABP10 additive group at 15 °C, while the transcript expression of GPX4 increased significantly at 20 °C. Western blotting analysis confirmed that FABP10 protein expression strongly increased at 15 ± 0.5 °C in FABP10 additive group. These results showed that FABP10 additive diet could moderate the metabolic and immune abilities mainly via ROS pathway in the orange-spotted grouper. PMID:24412164

  4. Influence of liposomes rich in unsaturated or saturated fatty acids on the growth of human xenotransplanted mammary carcinomas and on the levels of heart type fatty acid binding protein.

    PubMed

    Naundorf, H; Zschiesche, W; Reszka, R; Fichtner, I

    1995-01-01

    A panel of 4 human mammary carcinomas passaged in nude mice were subjected to intraperitoneal application of cholesterol-free liposomes enriched with linoleic (unsaturated fatty acid) or stearic acid (saturated fatty acid). The liposomes were examined with regard to their influence on the tumor growth and level of heart type fatty acid binding protein (FABP). Liposomes with different fatty acid composition influenced the growth of mammary carcinomas 3366, BO, 4000 and 4151 in distinct ways. Liposomes with a high content of stearic acid significantly inhibited the growth of mammary carcinomas 3366 and BO, whereas mammary carcinomas 4000 and 4151 were not affected. The growth of mammary carcinoma 3366 was moderately increased after supplementation of liposomes rich in linoleic acid, the tumor BO was significantly inhibited and the growth of MaCa 4000 and 4151 was unchanged. Liposome treatment led to a significant increase in heart type FABP in mammary carcinomas 3366 and BO regardless of whether the animals were treated with liposomes rich in stearic or linoleic acid. Such significant changes of FABP level could not be observed in mammary carcinomas 4000 or 4151. We suggest that the lipid-mediated growth modulation seems to be dependent on an increase of heart type FABPs in these tumor models. PMID:8562891

  5. Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41

    PubMed Central

    Samuel, Buck S.; Shaito, Abdullah; Motoike, Toshiyuki; Rey, Federico E.; Backhed, Fredrik; Manchester, Jill K.; Hammer, Robert E.; Williams, S. Clay; Crowley, Jan; Yanagisawa, Masashi; Gordon, Jeffrey I.

    2008-01-01

    The distal human intestine harbors trillions of microbes that allow us to extract calories from otherwise indigestible dietary polysaccharides. The products of polysaccharide fermentation include short-chain fatty acids that are ligands for Gpr41, a G protein-coupled receptor expressed by a subset of enteroendocrine cells in the gut epithelium. To examine the contribution of Gpr41 to energy balance, we compared Gpr41−/− and Gpr41+/+ mice that were either conventionally-raised with a complete gut microbiota or were reared germ-free and then cocolonized as young adults with two prominent members of the human distal gut microbial community: the saccharolytic bacterium, Bacteroides thetaiotaomicron and the methanogenic archaeon, Methanobrevibacter smithii. Both conventionally-raised and gnotobiotic Gpr41−/− mice colonized with the model fermentative community are significantly leaner and weigh less than their WT (+/+) littermates, despite similar levels of chow consumption. These differences are not evident when germ-free WT and germ-free Gpr41 knockout animals are compared. Functional genomic, biochemical, and physiologic studies of germ-free and cocolonized Gpr41−/− and +/+ littermates disclosed that Gpr41-deficiency is associated with reduced expression of PYY, an enteroendocrine cell-derived hormone that normally inhibits gut motility, increased intestinal transit rate, and reduced harvest of energy (short-chain fatty acids) from the diet. These results reveal that Gpr41 is a regulator of host energy balance through effects that are dependent upon the gut microbiota. PMID:18931303

  6. Xanthurenic Acid Binds to Neuronal G-Protein-Coupled Receptors That Secondarily Activate Cationic Channels in the Cell Line NCB-20

    PubMed Central

    Taleb, Omar; Maammar, Mohammed; Brumaru, Daniel; Bourguignon, Jean-Jacques; Schmitt, Martine; Klein, Christian; Kemmel, Véronique; Maitre, Michel; Mensah-Nyagan, Ayikoe Guy

    2012-01-01

    Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca2+ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. PMID:23139790

  7. Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

    PubMed

    Taleb, Omar; Maammar, Mohammed; Brumaru, Daniel; Bourguignon, Jean-Jacques; Schmitt, Martine; Klein, Christian; Kemmel, Véronique; Maitre, Michel; Mensah-Nyagan, Ayikoe Guy

    2012-01-01

    Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+) concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. PMID:23139790

  8. Fatty acid binding protein 4 and 5 play a crucial role in thermogenesis under the conditions of fasting and cold stress.

    PubMed

    Syamsunarno, Mas Rizky A A; Iso, Tatsuya; Yamaguchi, Aiko; Hanaoka, Hirofumi; Putri, Mirasari; Obokata, Masaru; Sunaga, Hiroaki; Koitabashi, Norimichi; Matsui, Hiroki; Maeda, Kazuhisa; Endo, Keigo; Tsushima, Yoshito; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

    2014-01-01

    Hypothermia is rapidly induced during cold exposure when thermoregulatory mechanisms, including fatty acid (FA) utilization, are disturbed. FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipose tissues and macrophages, have been identified as key molecules in the pathogenesis of overnutrition-related diseases, such as insulin resistance and atherosclerosis. We have recently shown that FABP4/5 are prominently expressed in capillary endothelial cells in the heart and skeletal muscle and play a crucial role in FA utilization in these tissues. However, the role of FABP4/5 in thermogenesis remains to be determined. In this study, we showed that thermogenesis is severely impaired in mice lacking both FABP4 and FABP5 (DKO mice), as manifested shortly after cold exposure during fasting. In DKO mice, the storage of both triacylglycerol in brown adipose tissue (BAT) and glycogen in skeletal muscle (SkM) was nearly depleted after fasting, and a biodistribution analysis using 125I-BMIPP revealed that non-esterified FAs (NEFAs) are not efficiently taken up by BAT despite the robustly elevated levels of serum NEFAs. In addition to the severe hypoglycemia observed in DKO mice during fasting, cold exposure did not induce the uptake of glucose analogue 18F-FDG by BAT. These findings strongly suggest that DKO mice exhibit pronounced hypothermia after fasting due to the depletion of energy storage in BAT and SkM and the reduced supply of energy substrates to these tissues. In conclusion, FABP4/5 play an indispensable role in thermogenesis in BAT and SkM. Our study underscores the importance of FABP4/5 for overcoming life-threatening environments, such as cold and starvation. PMID:24603714

  9. Development of a therapeutic monoclonal antibody that targets secreted fatty acid-binding protein aP2 to treat type 2 diabetes.

    PubMed

    Burak, M Furkan; Inouye, Karen E; White, Ariel; Lee, Alexandra; Tuncman, Gurol; Calay, Ediz S; Sekiya, Motohiro; Tirosh, Amir; Eguchi, Kosei; Birrane, Gabriel; Lightwood, Daniel; Howells, Louise; Odede, Geofrey; Hailu, Hanna; West, Shauna; Garlish, Rachel; Neale, Helen; Doyle, Carl; Moore, Adrian; Hotamisligil, Gökhan S

    2015-12-23

    The lipid chaperone aP2/FABP4 has been implicated in the pathology of many immunometabolic diseases, including diabetes in humans, but aP2 has not yet been targeted for therapeutic applications. aP2 is not only an intracellular protein but also an active adipokine that contributes to hyperglycemia by promoting hepatic gluconeogenesis and interfering with peripheral insulin action. Serum aP2 levels are markedly elevated in mouse and human obesity and strongly correlate with metabolic complications. These observations raise the possibility of a new strategy to treat metabolic disease by targeting serum aP2 with a monoclonal antibody (mAb) to aP2. We evaluated mAbs to aP2 and identified one, CA33, that lowered fasting blood glucose, improved systemic glucose metabolism, increased systemic insulin sensitivity, and reduced fat mass and liver steatosis in obese mouse models. We examined the structure of the aP2-CA33 complex and resolved the target epitope by crystallographic studies in comparison to another mAb that lacked efficacy in vivo. In hyperinsulinemic-euglycemic clamp studies, we found that the antidiabetic effect of CA33 was predominantly linked to the regulation of hepatic glucose output and peripheral glucose utilization. The antibody had no effect in aP2-deficient mice, demonstrating its target specificity. We conclude that an aP2 mAb-mediated therapeutic constitutes a feasible approach for the treatment of diabetes. PMID:26702093

  10. [Clinical implication of urinary protein markers in diabetic nephropathy and interventional effects of Chinese herbal medicine].

    PubMed

    Shi, Xi-Miao; Meng, Xian-Jie; Wan, Yi-Gang; Shen, Shan-Mei; Luo, Xun-Yang; Gu, Liu-Bao; Yao, Jian

    2014-07-01

    In clinic, some urinary protein makers can dynamically and noninvasively reflect the degree of renal tubular injury in patients with diabetic nephropathy (DN). These urinary biomarkers of tubular damage are broadly divided into two categories. One is newfound, including kidney injury molecule-1 (Kim-1), neutrophil getatinase-associated lipocalin (NGAL), liver-type fatty acid-binding protein (L-FABP) and cystatin C (CysC); the other one is classical, including beta2 microglobulin (beta2-MG), retinal binding protein (RBP) and N-acetyl-beta-D-glucosaminidase (NAG). It is reported that, the increases in urinary protein markers are not only closely related to the damage of tubular epithelial cells in DN patients, but also can be ameliorated by the treatment with Chinese herbal compound preparations or Chinese herbal medicine. Recently, although urinary proteomics are used in the protein separation and identification, the traditional associated detection of urinary protein markers is more practical in clinic. At present, it is possible that the associated detection of urinary biomarkers of glomerular and tubular damages may be a feasible measure to reveal the clinical significance of urinary protein markers in DN patients and the interventional effects of Chinese herbal medicine. PMID:25272479

  11. A single-nucleotide polymorphism in the 3′-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple-negative breast cancer

    PubMed Central

    Wang, Wenmiao; Yuan, Peng; Yu, Dianke; Du, Feng; Zhu, Anjie; Li, Qing; Zhang, Pin; Lin, Dongxin; Xu, Binghe

    2016-01-01

    Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and high heterogeneity. The aim of this study was to screen patients for single-nucleotide polymorphisms (SNPs) associated with the prognosis of TNBC. Database-derived SNPs (NextBio, Ensembl, NCBI and MirSNP) located in the 3′-untranslated regions (3′-UTRs) of genes that are differentially expressed in breast cancer were selected. The possible associations between 111 SNPs and progression risk among 323 TNBC patients were investigated using a two-step case-control study with a discovery cohort (n=162) and a validation cohort (n=161). We identified the rs1054135 SNP in the adipocyte fatty acid binding protein 4 (FABP4) gene as a predictor of TNBC recurrence. The G allele of rs1054135 was associated with a reduced risk of disease progression as well as a prolonged disease-free survival time (DFS), with a hazard ratio (HR) for recurrence in the combined sample of 0.269 [95%CI: 0.098−0.735;P=0.001]. Notably, for individuals having the rs1054135 SNP with the AA/AG genotype, the magnitude of increased tumour recurrence risk for overweight patients (BMI≥25kg/m2) was significantly elevated (HR2.53; 95%CI: 1.06–6.03). Immunohistochemical staining of adipocytes adjacent to TNBC tissues showed that the expression level of FABP4 was statistically significantly lower in patients with the rs1054135-GG genotype and those in the disease-free group (P=0.0004 and P=0.0091, respectively). These results suggested that the expression of a lipid metabolism-related gene and an important SNP in the 3′-UTR of FABP4 are associated with TNBC prognosis, which may aid in the screening of high-risk patients with TNBC recurrence and the development of novel chemotherapeutic agents. PMID:26959740

  12. Heart-type fatty acid binding protein and high-sensitivity troponin T are myocardial damage markers that could predict adverse clinical outcomes in patients with peripheral artery disease

    PubMed Central

    Otaki, Yoichiro; Takahashi, Hiroki; Watanabe, Tetsu; Yamaura, Gensai; Funayama, Akira; Arimoto, Takanori; Shishido, Tetsuro; Miyamoto, Takuya; Kubota, Isao

    2015-01-01

    Background Despite many recent advances in endovascular therapy (EVT), peripheral artery disease (PAD) is an increasing health problem with high mortality. Heart-type fatty acid-binding protein (H-FABP) and high-sensitivity troponin T (hsTnT) are markers of ongoing myocardial damage and have been reported to be useful indicators of future cardiovascular events. However, it remains to be determined whether H-FABP and hsTnT can predict adverse clinical outcomes in patients with PAD. Methods and results We enrolled 208 de novo PAD patients who underwent EVT. Serum H-FABP and hsTnT were measured in all patients before EVT. During the median follow-up period of 694 days, there were 40 major adverse cardiovascular and cerebrovascular events (MACCEs) including all-cause deaths, and re-hospitalizations due to cardiovascular and cerebrovascular diseases and amputations. H-FABP and hsTnT were found to be higher in patients with critical limb ischemia (CLI) compared to those without this condition. Multivariate Cox proportional hazard regression analysis revealed that both H-FABP and hsTnT were independent predictors of MACCEs after adjustment for confounding factors. Kaplan–Meier analysis demonstrated that patients in the highest tertile according to H-FABP levels, as well as those in the highest hsTnT tertile, were at greatest risk for MACCEs. The net reclassification index was significantly improved by the addition of H-FABP as well as the addition of hsTnT to traditional risk factors. Conclusion The myocardial damage markers H-FABP and hsTnT were increased in PAD patients with CLI and could predict MACCEs in PAD patients. PMID:26673681

  13. Comparison of a qualitative measurement of heart-type fatty acid-binding protein with other cardiac markers as an early diagnostic marker in the diagnosis of non-ST - segment elevation myocardial infarction

    PubMed Central

    Gerede, Demet Menekşe; Güleç, Sadi; Kılıçkap, Mustafa; Kaya, Cansın Tulunay; Vurgun, Veysel Kutay; Özcan, Özgür Ulaş; Göksülük, Hüseyin; Erol, Çetin

    2015-01-01

    Summary Objective: Heart-type fatty acid-binding protein (H-FABP) is a novel cardiac marker used in the early diagnosis of acute myocardial infarction (AMI), which shows myocyte injury. Our study aimed to compare bedside H-FABP measurements with routine creatine kinase-MB (CK-MB) and troponin I (TnI) tests for the early diagnosis of non-ST-elevation MI (NSTEMI), as well as for determining its exclusion capacity. Methods A total of 48 patients admitted to the emergency room within the first 12 hours of onset of ischaemic-type chest pain lasting more than 30 minutes and who did not have ST-segment elevation on electrocardiography (ECG) were included in the study. Definite diagnoses of NSTEMI were made in 24 patients as a result of 24-hour follow up, and the remaining 24 patients did not develop MI. Results When various subgroups were analysed according to admission times, H-FABP was found to be a better diagnostic marker compared to CK-MB and TnI (accuracy index 85%), with a high sensitivity (79%) and specificity (93%) for early diagnosis (≤ six hours). The respective sensitivities of bedside H-FABP and TnI tests were 89 vs 33% (p < 0.05) for patients presenting within three hours of onset of symptoms. Conclusion Bedside H-FABP measurements may contribute to correct early diagnoses, as its levels are elevated soon following MI, and measurement is easy, with a rapid result. PMID:26212703

  14. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

  15. Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein

    PubMed Central

    Alqahtani, Mashael F.; Smith, Craig M.; Weiss, Scott L.; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S.

    2016-01-01

    Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004–0.174, 13), day 2 (0.020, 0.002–0.109, 10), and day 3 (0.018, 0.003–0.058, 23) compared with febrile (0.705, 0.187–1.778, 22) and healthy (0.7, 0.4–1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2–54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3–20.6, 11). MMP-9/TIMP-1 ratios

  16. DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy.

    PubMed

    Cortés, A; Huertas, D; Fanti, L; Pimpinelli, S; Marsellach, F X; Piña, B; Azorín, F

    1999-07-01

    The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Deltascp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin. PMID:10393197

  17. Escherichia coli DnaB Helicase–DnaC Protein Complex: Allosteric Effects of the Nucleotides on the Nucleic Acid Binding and the Kinetic Mechanism of NTP Hydrolysis. 3†

    PubMed Central

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2011-01-01

    Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB–DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB–DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB–DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB–DnaC complex. In the tertiary DnaB–DnaC–ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB–DnaC complex is discussed. PMID:19432487

  18. Crystal Structures of the Staphylococcal Toxin SSL5 in Complex With Sialyl-Lewis X Reveal a Conserved Binding Site That Shares Common Features With Viral And Bacterial Sialic Acid-Binding Proteins

    SciTech Connect

    Baker, H.M.; Basu, I.; Chung, M.C.; Caradoc-Davies, T.; Fraser, J.D.; Baker, E.N.

    2009-06-02

    Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.

  19. Induction of the hyaluronic acid-binding protein, tumor necrosis factor-stimulated gene-6, in cervical smooth muscle cells by tumor necrosis factor-alpha and prostaglandin E(2).

    PubMed

    Fujimoto, Toshio; Savani, Rashmin C; Watari, Michiko; Day, Anthony J; Strauss, Jerome F

    2002-04-01

    Immediately before parturition the cervix undergoes striking changes in structure (ripening) that facilitate dilatation and effacement. Cervical ripening shares many features in common with inflammation-associated tissue remodeling, making it a valuable process to explore with respect to the biochemical events in extracellular matrix restructuring. Cervical ripening can be pharmacologically induced with prostaglandin E(2) (PGE(2)). Among the biochemical changes in the cervix at parturition is a marked increase in the hyaluronic acid (HA) content. HA and HA-binding proteins have been implicated in tissue hydration, release of collagenase, and leukocyte migration, but their roles in cervical ripening have not been explored. In the present study we examined the ability of PGE(2) to induce expression of the HA-binding protein, tumor necrosis factor-stimulated gene (TSG)-6, in human cervical smooth muscle cells (hCSMCs) and compared the PGE(2) response to that of tumor necrosis factor-alpha (TNF-alpha), an established inducer of TSG-6. TNF-alpha stimulated TSG-6 mRNA accumulation in a dose- and time-dependent manner, with the maximal response observed at 10 ng/ml after 6 hours of incubation. PGE(2) stimulated TSG-6 mRNA expression, but the magnitude of response was substantially less than that produced by TNF-alpha, and it was maximal only after 24 hours of incubation. Quantitative real-time polymerase chain reaction was performed to assess the induction of TSG-6 mRNA and nascent transcripts at 24 hours of treatment. Induction of TSG-6 mRNA and nascent transcripts in response to 10 micromol/L of PGE(2) was 5.7-fold and 6.3-fold greater than control values, respectively, whereas TNF-alpha (10 ng/ml) induced TSG-6 mRNA and nascent transcripts by 80-fold and 134-fold, respectively. TNF-alpha and PGE(2) stimulated secretion of TSG-6 into the culture medium as detected by Western blotting. The effects of PGE(2) on secretion of TSG-6 were delayed compared to TNF-alpha. A 1

  20. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  1. Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

    PubMed

    Chintapalli, Sree V; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L; van Rossum, Damian B; Anishkin, Andriy; Adams, Sean H

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  2. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-12-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy…) are applied. We found that i) the tools have been greatly improved over the years; ii) some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii) RNA binding and DNA binding appear to follow similar driving forces and iv) dataset bias may exist in some methods. PMID:26681179

  3. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy…) are applied. We found that i) the tools have been greatly improved over the years; ii) some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii) RNA binding and DNA binding appear to follow similar driving forces and iv) dataset bias may exist in some methods. PMID:26681179

  4. Folic acid binds DNA and RNA at different locations.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2015-03-01

    We located multiple binding sites for folic acid on DNA and tRNA at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural analysis revealed that folic acid binds DNA and tRNA at multiple sites via hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of Kfolic acid-DNA=1.1 (±0.3)×10(4) M(-1) and Kfolic acid-tRNA=6.4 (±0.5)×10(3) M(-1). Molecular modeling showed the participation of several nucleobases in folic acid complexes with DNA and tRNA, stabilized by H-bonding network. Two types of complexes were located for folic acid-tRNA adducts, one at the major groove and the other with TΨC loop, while acid binding occurs at major and minor grooves of DNA duplex. Folic acid complexation induced more alterations of DNA structure than tRNA. PMID:25555838

  5. Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

    PubMed

    Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

    2016-08-01

    The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis. PMID:27606346

  6. Characterization of phosphonic acid binding to zinc oxide

    SciTech Connect

    Hotchkiss, Peter J.; Malicki, Michał; Giordano, Anthony J.; Armstrong, Neal R.; Marder, Seth R.

    2011-01-24

    Radio Frequency (RF) sputter-deposited zinc oxide (ZnO) films have been modified with alkylphosphonic acids in order to study both the binding of the phosphonic acid (PA) group to the ZnO surface and the packing of the alkyl chain. The characterization of these PA-modified ZnO substrates by X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRRAS), atomic force microscopy (AFM) and contact angle measurements is presented herein. The surface modification procedure is straightforward and was adapted from earlier work. XPS analysis shows that oxygen plasma (OP) treatment creates reactive oxygen species on the surface of ZnO, allowing for a more robust binding of PAs to the ZnO surface. IRRAS analysis indicates that octadecylphosphonic acid binds to the ZnO surface in a predominantly tridentate fashion, forming dense, well-packed monolayers with alkyl chains in a fully anti-conformation. AFM and contact angle measurements indicate good surface coverage of the PAs with little to no multilayer formation.

  7. RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2016-07-01

    RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/. PMID:27084939

  8. Evaluating Healthful Properties of Cereals and Cereal Fractions by Their Bile-Acid-Binding Potential

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The healthful, cholesterol-lowering (atherosclerosis amelioration) or detoxification of harmful metabolites (cancer prevention) potential of cereals and cereal fractions could be predicted by evaluating their in vitro bile acid binding under physiological conditions. Using equal dry matter per incu...

  9. Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

    PubMed Central

    Mitra, Mithun; Hercík, Kamil; Byeon, In-Ja L.; Ahn, Jinwoo; Hill, Shawn; Hinchee-Rodriguez, Kathyrn; Singer, Dustin; Byeon, Chang-Hyeock; Charlton, Lisa M.; Nam, Gabriel; Heidecker, Gisela; Gronenborn, Angela M.; Levin, Judith G.

    2014-01-01

    Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A’s deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A’s potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. PMID:24163103

  10. Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II.

    PubMed Central

    Kontermann, R. E.; Kobor, M.; Bautz, E. K.

    1993-01-01

    The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases. PMID:8443600

  11. Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

    PubMed

    Matsumoto, Kenji; Yokoyama, Shin-ichiro; Gato, Nobuki

    2010-02-01

    The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice. PMID:19585467

  12. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs. PMID:26487699

  13. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  14. Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor.

    PubMed

    Van Zeebroeck, Griet; Bonini, Beatriz Monge; Versele, Matthias; Thevelein, Johan M

    2009-01-01

    Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle. PMID:19060912

  15. In Vitro bile acid binding of kale, mustard greens, broccoli, cabbage and green bell pepper improves with microwave cooking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding potential of foods and food fractions has been related to lowering the risk of heart disease and that of cancer. Sautéing or steam cooking has been observed to significantly improve bile acid binding of green/leafy vegetables. It was hypothesized that microwave cooking could impr...

  16. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  17. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif

    PubMed Central

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-01-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain 161PPLP164 regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the 161PPLP164 domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  18. Cost-effectiveness analysis of acute kidney injury biomarkers in pediatric cardiac surgery

    PubMed Central

    Petrovic, Stanislava; Lakic, Dragana; Peco-Antic, Amira; Vulicevic, Irena; Ivanisevic, Ivana; Kotur-Stevuljevic, Jelena; Jelic-Ivanovic, Zorana

    2015-01-01

    Introduction Acute kidney injury (AKI) is significant problem in children with congenital heart disease (CHD) who undergo cardiac surgery. The economic impact of a biomarker-based diagnostic strategy for AKI in pediatric populations undergoing CHD surgery is unknown. The aim of this study was to perform the cost effectiveness analysis of using serum cystatin C (sCysC), urine neutrophil gelatinase-associated lipocalin (uNGAL) and urine liver fatty acid-binding protein (uL-FABP) for the diagnosis of AKI in children after cardiac surgery compared with current diagnostic method (monitoring of serum creatinine (sCr) level). Materials and methods We developed a decision analytical model to estimate incremental cost-effectiveness of different biomarker-based diagnostic strategies compared to current diagnostic strategy. The Markov model was created to compare the lifetime cost associated with using of sCysC, uNGAL, uL-FABP with monitoring of sCr level for the diagnosis of AKI. The utility measurement included in the analysis was quality-adjusted life years (QALY). The results of the analysis are presented as the incremental cost-effectiveness ratio (ICER). Results Analysed biomarker-based diagnostic strategies for AKI were cost-effective compared to current diagnostic method. However, uNGAL and sCys C strategies yielded higher costs and lower effectiveness compared to uL-FABP strategy. uL-FABP added 1.43 QALY compared to current diagnostic method at an additional cost of $8521.87 per patient. Therefore, ICER for uL-FABP compared to sCr was $5959.35/QALY. Conclusions Our results suggest that the use of uL-FABP would represent cost effective strategy for early diagnosis of AKI in children after cardiac surgery. PMID:26110039

  19. Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

    PubMed

    Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

    2012-01-01

    Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

  20. Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

    PubMed Central

    Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

    2012-01-01

    Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

  1. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors

    PubMed Central

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  2. Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

    PubMed

    Urano-Tashiro, Yumiko; Takahashi, Yukihiro; Oguchi, Riyo; Konishi, Kiyoshi

    2016-01-01

    Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins. PMID:27101147

  3. Urinary Kidney Injury Molecules in Children with Iron-Deficiency Anemia

    PubMed Central

    Güneş, Ali; Ece, Aydın; Aktar, Fesih; Tan, İlhan; Söker, Murat; Karabel, Duran; Balık, Hasan; Uluca, Ünal; Şen, Velat; Yolbaş, İlyas

    2015-01-01

    Background The aim of this study was to investigate the urine levels of human kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-β-D-glucosaminidase (NAG), and liver-type fatty acid-binding protein (L-FABP) in children with iron-deficiency anemia (IDA). Material/Methods Thirty-five children with IDA and 32 matched healthy controls were recruited. We assessed complete blood count, serum iron, iron-binding capacity, ferritin, serum levels of urea, creatinine (Cr), sodium (Na), potassium (K), calcium (Ca), and glucose levels. Estimated glomerular filtration rate (eGFR) was calculated. Urinary NAG, NGAL, KIM-1, and L-FABP were measured and divided by urine creatinine for comparisons. Results There were no significant differences in serum urea, Cr, or eGFR between the IDA group and the control group (p>0.05, for all). IDA patients had significantly higher urine NGAL/Cr, L-FABP/Cr, KIM-1/Cr, and NAG/Cr compared with the control group (p<0.05). There were significant negative correlations between hemoglobin, hematocrit, red blood cell count, and urine NGAL/Cr, NAG/Cr, L-FABP/Cr, KIM-1/Cr levels (p<0.05). Conclusions Higher urinary kidney injury molecule levels in IDA patients suggest a possible subclinical renal injury in pediatric IDA patients whose renal functions and serum electrolytes were normal. PMID:26697893

  4. Measurements of the acid-binding capacity of ingredients used in pig diets

    PubMed Central

    2005-01-01

    Some feed ingredients bind more acid in the stomach than others and for this reason may be best omitted from pig starter foods if gastric acidity is to be promoted. The objective of this study was to measure the acid-binding capacity (ABC) of ingredients commonly used in pig starter foods. Ingredients were categorised as follows: (i) milk products (n = 6), (ii) cereals (n = 10), (iii) root and pulp products (n = 5), (iv) vegetable proteins (n = 11), (v) meat and fish meal (n = 2), (vi) medication (n = 3), (vii) amino acids (n = 4), (viii) minerals (n = 16), (ix) acid salts (n = 4), (x) acids (n = 10). A 0.5 g sample of food was suspended in 50 ml distilled de-ionised water with continuous stirring. This suspension was titrated with 0.1 mol/L HCl or 0.1 mol/L NaOH so that approximately 10 additions of titrant was required to reach pH 3.0. The pH readings after each addition were recorded following equilibration for three minutes. ABC was calculated as the amount of acid in milliequivalents (meq) required to lower the pH of 1 kg food to (a) pH 4.0 (ABC-4) and (b) pH 3.0 (ABC-3). Categories of food had significantly different (P < 0.01) ABC values. Mean ABC-4 and ABC-3 values of the ten categories were: (i) 623 (s.d. 367.0) and 936 (s.d. 460.2), (ii) 142 (s.d. 79.2) and 324 (s.d. 146.4), (iii) 368 (s.d. 65.3) and 804 (s.d. 126.7), (iv) 381 (s.d. 186.1) and 746 (s.d. 227.0), (v) 749 (s.d. 211.6) and 1508 (s.d. 360.8), (vi) 120 (s.d. 95.6) and 261 (s.d. 163.2), (vii) 177 (s.d. 60.7) and 1078 (s.d. 359.0), (viii) 5064 (s.d. 5525.1) and 7051 (s.d. 5911.6), (ix) 5057 (s.d. 1336.6) and 8945 (s.d. 2654.1) and (x) -5883 (s.d. 4220.5) and -2591 (s.d. 2245.4) meq HCl per kg, respectively. Within category, ABC-3 and ABC- 4 values were highly correlated: R2 values of 0.80 and greater for food categories i, iv, v, vi, vii and viii. The correlation between predicted and observed ABC values of 34 mixed diets was 0.83 for ABC-4 and 0.71 for ABC-3. It was concluded that complete diets

  5. Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex

    PubMed Central

    Ellisdon, Andrew M.; Dimitrova, Lyudmila; Hurt, Ed; Stewart, Murray

    2012-01-01

    The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively-transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3–Thp1–Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3–Thp1–Sem1 forms a novel PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Structure-guided mutations underline the essential requirement of the Thp1–Sac3 interaction for mRNA binding and for the coupling of transcription and processing with mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor and, identify how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly. PMID:22343721

  6. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    SciTech Connect

    Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Szyczew, Alex J.; Kiefel, Milton J.; Itzstein, Mark von; Blanchard, Helen

    2005-06-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.

  7. Steam Cooking Significantly Improves in Vitro Bile Acid Binding of Beets, Eggplant, Asparagus, Carrots, Green Beans and Cauliflower

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The relative healthful potential of cooked beets, okra, eggplant, asparagus, carrots, green beans, cauliflower and turnips was evaluated by determining their in vitro bile acid binding using a mixture of bile acids secreted in human bile at a duodenal physiological pH of 6.3. Six treatments and two...

  8. Health promoting potential of cereals, grain fractions and beans as determined by their in vitro bile acid binding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Health promoting potential (Cholesterol lowering and cancer risk reduction) of foods have been determined by in-vitro bile acid binding under physiological conditions. Lowered bile acids result in reduced fat absorption, conversion of cholesterol to bile acids and reduced cancer causing secondary b...

  9. Pre-transplant Evaluation of Donor Urinary Biomarkers can Predict Reduced Graft Function After Deceased Donor Kidney Transplantation

    PubMed Central

    Koo, Tai Yeon; Jeong, Jong Cheol; Lee, Yonggu; Ko, Kwang-Pil; Lee, Kyoung-Bun; Lee, Sik; Park, Suk Joo; Park, Jae Berm; Han, Miyeon; Lim, Hye Jin; Ahn, Curie; Yang, Jaeseok

    2016-01-01

    Abstract Several recipient biomarkers are reported to predict graft dysfunction, but these are not useful in decision making for the acceptance or allocation of deceased donor kidneys; thus, it is necessary to develop donor biomarkers predictive of graft dysfunction. To address this issue, we prospectively enrolled 94 deceased donors and their 109 recipients who underwent transplantation between 2010 and 2013 at 4 Korean transplantation centers. We investigated the predictive values of donor urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and L-type fatty acid binding protein (L-FABP) for reduced graft function (RGF). We also developed a prediction model of RGF using these donor biomarkers. RGF was defined as delayed or slow graft function. Multiple logistic regression analysis was used to generate a prediction model, which was internally validated using a bootstrapping method. Multiple linear regression analysis was used to assess the association of biomarkers with 1-year graft function. Notably, donor urinary NGAL levels were associated with donor AKI (P = 0.014), and donor urinary NGAL and L-FABP were predictive for RGF, with area under the receiver-operating characteristic curves (AUROC) of 0.758 and 0.704 for NGAL and L-FABP, respectively. The best-fit model including donor urinary NGAL, L-FABP, and serum creatinine conveyed a better predictive value for RGF than donor serum creatinine alone (P = 0.02). In addition, we generated a scoring method to predict RGF based on donor urinary NGAL, L-FABP, and serum creatinine levels. Diagnostic performance of the RGF prediction score (AUROC 0.808) was significantly better than that of the DGF calculator (AUROC 0.627) and the kidney donor profile index (AUROC 0.606). Donor urinary L-FABP levels were also predictive of 1-year graft function (P = 0.005). Collectively, these findings suggest donor urinary NGAL and L-FABP to be useful biomarkers for RGF, and support

  10. Pre-transplant Evaluation of Donor Urinary Biomarkers can Predict Reduced Graft Function After Deceased Donor Kidney Transplantation.

    PubMed

    Koo, Tai Yeon; Jeong, Jong Cheol; Lee, Yonggu; Ko, Kwang-Pil; Lee, Kyoung-Bun; Lee, Sik; Park, Suk Joo; Park, Jae Berm; Han, Miyeon; Lim, Hye Jin; Ahn, Curie; Yang, Jaeseok

    2016-03-01

    Several recipient biomarkers are reported to predict graft dysfunction, but these are not useful in decision making for the acceptance or allocation of deceased donor kidneys; thus, it is necessary to develop donor biomarkers predictive of graft dysfunction. To address this issue, we prospectively enrolled 94 deceased donors and their 109 recipients who underwent transplantation between 2010 and 2013 at 4 Korean transplantation centers. We investigated the predictive values of donor urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and L-type fatty acid binding protein (L-FABP) for reduced graft function (RGF). We also developed a prediction model of RGF using these donor biomarkers. RGF was defined as delayed or slow graft function. Multiple logistic regression analysis was used to generate a prediction model, which was internally validated using a bootstrapping method. Multiple linear regression analysis was used to assess the association of biomarkers with 1-year graft function. Notably, donor urinary NGAL levels were associated with donor AKI (P = 0.014), and donor urinary NGAL and L-FABP were predictive for RGF, with area under the receiver-operating characteristic curves (AUROC) of 0.758 and 0.704 for NGAL and L-FABP, respectively. The best-fit model including donor urinary NGAL, L-FABP, and serum creatinine conveyed a better predictive value for RGF than donor serum creatinine alone (P = 0.02). In addition, we generated a scoring method to predict RGF based on donor urinary NGAL, L-FABP, and serum creatinine levels. Diagnostic performance of the RGF prediction score (AUROC 0.808) was significantly better than that of the DGF calculator (AUROC 0.627) and the kidney donor profile index (AUROC 0.606). Donor urinary L-FABP levels were also predictive of 1-year graft function (P = 0.005). Collectively, these findings suggest donor urinary NGAL and L-FABP to be useful biomarkers for RGF, and support the use of

  11. In vitro bile acid binding and short-chain fatty acid profile of flax fiber and ethanol co-products.

    PubMed

    Fodje, Adele M L; Chang, Peter R; Leterme, Pascal

    2009-10-01

    Fibers from flaxseed and co-products from ethanol production could be potential sources of dietary fiber in human diet. In vitro fermentation and bile acid binding models were used to investigate the metabolic effects of lignaMax (Bioriginal Food and Science Corp., Saskatoon, SK, Canada) flax meal, spent flax meal, soluble flax gum, wheat insoluble fiber (WIF), and rye insoluble fiber (RIF). Wheat and rye bran were used as reference samples. Bile acid binding of substrates was analysed at taurocholate ([(14)C]taurocholate) concentration of 12.5 mM. Soluble flax gum showed the highest bile acid binding (0.57 micromol/mg of fiber) (P acid binding between wheat bran (0.2 micromol/mg of fiber) and WIF (0.26 micromol/mg of fiber). RIF had higher (P acid binding (0.20 micromol/mg of fiber) than rye bran (0.13 micromol/mg of fiber). Substrates were hydrolyzed and incubated with pig fecal samples. Short-chain fatty acid (SCFA) profile and gas accumulation (G(f)) were compared. Soluble flax gum generated the highest amount of acetic and propionic acids. SCFA profiles of wheat/rye brans and WIF/RIF were similar (except for butyric acid). G(f) for soluble flax gum was greater (P < .001) than that of spent flax meal. G(f) values of the wheat samples were similar, whereas the G(f) of the rye bran was higher (P < .001) than that of RIF. Fractional degradation rate (micro(t = T/2)) (P < .001) was also recorded. The highest mu(t = T/2) was observed for the soluble flax gum. Oil-depleted flaxseed fractions and WIF/RIF (co-products from ethanol production) could be potential sources of dietary fiber in human nutrition. PMID:19857071

  12. Susceptibility of L-FABP−/− mice to oxidative stress in early-stage alcoholic liver[S

    PubMed Central

    Smathers, Rebecca L.; Galligan, James J.; Shearn, Colin T.; Fritz, Kristofer S.; Mercer, Kelly; Ronis, Martin; Orlicky, David J.; Davidson, Nicholas O.; Petersen, Dennis R.

    2013-01-01

    Chronic ethanol consumption is a prominent cause of liver disease worldwide. Dysregulation of an important lipid uptake and trafficking gene, liver-fatty acid binding protein (L-FABP), may contribute to alterations in lipid homeostasis during early-stage alcoholic liver. We have reported the detrimental effects of ethanol on the expression of L-FABP and hypothesize this may deleteriously impact metabolic networks regulating fatty acids. Male wild-type (WT) and L-FABP−/− mice were fed a modified Lieber-DeCarli liquid diet for six weeks. To assess the response to chronic ethanol ingestion, standard biochemical indicators for alcoholic liver disease (ALD) and oxidative stress were measured. Ethanol ingestion resulted in attenuation of hepatic triglyceride accumulation and elevation of cholesterol in L-FABP−/− mice. Lipidomics analysis validated multiple alterations in hepatic lipids resulting from ethanol treatment. Increased immunohistochemical staining for the reactive aldehydes 4-hydroxynonenal and malondialdehyde were observed in WT mice ingesting ethanol; however, L-FABP−/− mice displayed prominent protein adducts in liver sections evaluated from pair-fed and ethanol-fed mice. Likewise, alterations in glutathione, thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyl content all indicated L-FABP−/− mice exhibit high sustained oxidative stress in the liver. These data establish that L-FABP is an indirect antioxidant protein essential for sequestering FFA and that its impairment could contribute to in the pathogenesis of ALD. PMID:23359610

  13. Pressurized water extraction of β-glucan enriched fractions with bile acids-binding capacities obtained from edible mushrooms.

    PubMed

    Palanisamy, Marimuthu; Aldars-García, Laila; Gil-Ramírez, Alicia; Ruiz-Rodríguez, Alejandro; Marín, Francisco R; Reglero, Guillermo; Soler-Rivas, Cristina

    2014-01-01

    A pressurized water extraction (PWE) method was developed in order to extract β-glucans with bile acids-binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly β-glucans (including chitooligosaccharides deriving from chitin hydrolysis), α-glucans, and other PSCs (hetero-/proteo-glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids-binding capacities of some extracts were similar to a β-glucan enriched fraction obtained from cereals. PMID:24399760

  14. Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life

    PubMed Central

    Austin, James A.; Wright, Gareth S. A.; Watanabe, Seiji; Grossmann, J. Günter; Antonyuk, Svetlana V.; Yamanaka, Koji; Hasnain, S. Samar

    2014-01-01

    Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype. PMID:24591609

  15. Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis.

    PubMed

    Beck-Fruchter, Ronit; Shalev, Eliezer; Weiss, Amir

    2016-03-01

    The human oocyte is surrounded by hyaluronic acid, which acts as a natural selector of spermatozoa. Human sperm that express hyaluronic acid receptors and bind to hyaluronic acid have normal shape, minimal DNA fragmentation and low frequency of chromosomal aneuploidies. Use of hyaluronic acid binding assays in intracytoplasmic sperm injection (ICSI) cycles to improve clinical outcomes has been studied, although none of these studies had sufficient statistical power. In this systematic review and meta-analysis, electronic databases were searched up to June 2015 to identify studies of ICSI cycles in which spermatozoa able to bind hyaluronic acid was selected. The main outcomes were fertilization rate and clinical pregnancy rate. Secondary outcomes included cleavage rate, embryo quality, implantation rate, spontaneous abortion and live birth rate. Seven studies and 1437 cycles were included. Use of hyaluronic acid binding sperm selection technique yielded no improvement in fertilization and pregnancy rates. A meta-analysis of all available studies showed an improvement in embryo quality and implantation rate; an analysis of prospective studies only showed an improvement in embryo quality. Evidence does not support routine use of hyaluronic acid binding assays in all ICSI cycles. Identification of patients that might benefit from this technique needs further study. PMID:26776822

  16. Steam cooking significantly improves in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage.

    PubMed

    Kahlon, Talwinder Singh; Chiu, Mei-Chen M; Chapman, Mary H

    2008-06-01

    Bile acid binding capacity has been related to the cholesterol-lowering potential of foods and food fractions. Lowered recirculation of bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of cancer. Bile acid binding potential has been related to lowering the risk of heart disease and that of cancer. Previously, we have reported bile acid binding by several uncooked vegetables. However, most vegetables are consumed after cooking. How cooking would influence in vitro bile acid binding of various vegetables was investigated using a mixture of bile acids secreted in human bile under physiological conditions. Eight replicate incubations were conducted for each treatment simulating gastric and intestinal digestion, which included a substrate only, a bile acid mixture only, and 6 with substrate and bile acid mixture. Cholestyramine (a cholesterol-lowering, bile acid binding drug) was the positive control treatment and cellulose was the negative control. Relative to cholestyramine, in vitro bile acid binding on dry matter basis was for the collard greens, kale, and mustard greens, 13%; broccoli, 10%; Brussels sprouts and spinach, 8%; green bell pepper, 7%; and cabbage, 5%. These results point to the significantly different (P < or = .05) health-promoting potential of collard greens = kale = mustard greens > broccoli > Brussels sprouts = spinach = green bell pepper > cabbage as indicated by their bile acid binding on dry matter basis. Steam cooking significantly improved the in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage compared with previously observed bile acid binding values for these vegetables raw (uncooked). Inclusion of steam-cooked collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage in our daily diet as health-promoting vegetables should be emphasized. These green

  17. Acetylcholinesterase (AChE) inhibition aggravates fasting-induced triglyceride accumulation in the mouse liver.

    PubMed

    Yokota, Shin-Ichi; Nakamura, Kaai; Ando, Midori; Kamei, Hiroyasu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Shibata, Shigenobu

    2014-01-01

    Although fasting induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. Because parasympathetic nervous system activity tends to attenuate the secretion of very-low-density-lipoprotein-triglyceride (VLDL-TG) and increase TG stores in the liver, and serum cholinesterase activity is elevated in fatty liver disease, the inhibition of the parasympathetic neurotransmitter acetylcholinesterase (AChE) may have some influence on hepatic lipid metabolism. To assess the influence of AChE inhibition on lipid metabolism, the effect of physostigmine, an AChE inhibitor, on fasting-induced increase in liver TG was investigated in mice. In comparison with ad libitum-fed mice, 30 h fasting increased liver TG accumulation accompanied by a downregulation of sterol regulatory element-binding protein 1 (SREBP-1) and liver-fatty acid binding-protein (L-FABP). Physostigmine promoted the 30 h fasting-induced increase in liver TG levels in a dose-dependent manner, accompanied by a significant fall in plasma insulin levels, without a fall in plasma TG. Furthermore, physostigmine significantly attenuated the fasting-induced decrease of both mRNA and protein levels of SREBP-1 and L-FABP, and increased IRS-2 protein levels in the liver. The muscarinic receptor antagonist atropine blocked these effects of physostigmine on liver TG, serum insulin, and hepatic protein levels of SREBP-1 and L-FABP. These results demonstrate that AChE inhibition facilitated fasting-induced TG accumulation with up regulation of the hepatic L-FABP and SREBP-1 in mice, at least in part via the activation of muscarinic acetylcholine receptors. Our studies highlight the crucial role of parasympathetic regulation in fasting-induced TG accumulation, and may be an important source of information on the mechanism of hepatic disorders of lipid metabolism. PMID:25383314

  18. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

    PubMed Central

    Wang, S; Kool, E T

    1994-01-01

    We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD >> RRR > RDR >> DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones

  19. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

    PubMed

    Wang, S; Kool, E T

    1994-06-25

    We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD > RRR > RDR > DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex

  20. Retinoic acid binding properties of the lipocalin member beta-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modeling methods.

    PubMed

    Zsila, Ferenc; Bikádi, Zsolt; Simonyi, Miklós

    2002-12-01

    Interaction between the Vitamin A derivative all-trans retinoic acid and the lipocalin member bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted pi-pi(*) absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD-pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the beta-barrel. PMID:12429354

  1. Acute kidney injury after using contrast during cardiac catheterization in children with heart disease.

    PubMed

    Hwang, Young Ju; Hyun, Myung Chul; Choi, Bong Seok; Chun, So Young; Cho, Min Hyun

    2014-08-01

    Acute kidney injury (AKI) is closely associated with the mortality of hospitalized patients and long-term development of chronic kidney disease, especially in children. The purpose of our study was to assess the evidence of contrast-induced AKI after cardiac catheterization in children with heart disease and evaluate the clinical usefulness of candidate biomarkers in AKI. A total of 26 children undergoing cardiac catheterization due to various heart diseases were selected and urine and blood samples were taken at 0 hr, 6 hr, 24 hr, and 48 hr after cardiac catheterization. Until 48 hr after cardiac catheterization, there was no significant increase in serum creatinine level in all patients. Unlike urine kidney injury molecule-1, IL-18 and neutrophil gelatinase-associated lipocalin, urine liver-type fatty acid-binding protein (L-FABP) level showed biphasic pattern and the significant difference in the levels of urine L-FABP between 24 and 48 hr. We suggest that urine L-FABP can be one of the useful biomarkers to detect subclinical AKI developed by the contrast before cardiac surgery. PMID:25120320

  2. Interstitial renal fibrosis due to multiple cisplatin treatments is ameliorated by semicarbazide-sensitive amine oxidase inhibition.

    PubMed

    Katagiri, Daisuke; Hamasaki, Yoshifumi; Doi, Kent; Negishi, Kousuke; Sugaya, Takeshi; Nangaku, Masaomi; Noiri, Eisei

    2016-02-01

    Elucidation of acute kidney diseases and disorders (AKD), including acute kidney injury (AKI), is important to prevent their progression to chronic kidney disease. Current animal AKI models are often too severe for use in evaluating human AKI. Therefore, new animal models of mild kidney injury are needed. Here a new clinically relevant animal model using multiple low doses of cisplatin (CP) was used to evaluate AKD. When 10 mg/kg CP was administered intraperitoneally once weekly for three times to L-type fatty acid-binding protein (L-FABP) transgenic mice, moderate renal interstitial fibrosis and tubule dilatation occurred, accompanied by brush-border loss. Urinary L-FABP, a promising biomarker of AKI, changed more drastically than blood urea nitrogen or creatinine. Preventing fibrosis in organs was also studied. Oral administration of a recently reported selective semicarbazide-sensitive amine oxidase inhibitor, PXS-4728A, for 1 week attenuated kidney injury and interstitial fibrosis compared with vehicle. Inhibition of renal lipid accumulation in semicarbazide-sensitive amine oxidase inhibitor-treated mice, together with reduced oxidative stress and L-FABP suppression in proximal tubules, suggested an antifibrotic effect of semicarbazide-sensitive amine oxidase inhibition in this CP-AKD model, a representative onco-nephrology. Thus, semicarbazide-sensitive amine oxidase inhibitors may be promising candidates for the prevention of chronic kidney disease in patients using CP to treat malignancy. PMID:26535996

  3. MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

    PubMed

    Pereira, Lloyd A; Hugo, Honor J; Malaterre, Jordane; Huiling, Xu; Sonza, Secondo; Cures, Alina; Purcell, Damian F J; Ramsland, Paul A; Gerondakis, Steven; Gonda, Thomas J; Ramsay, Robert G

    2015-01-01

    MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation. PMID:25853889

  4. Salivary and serum biomarkers for the study of side effects of aripiprazole coprescribed with mirtazapine in rats

    PubMed Central

    Bogdan, Maria; Silosi, Isabela; Surlin, Petra; Tica, Andrei Adrian; Tica, Oana Sorina; Balseanu, Tudor-Adrian; Rauten, Anne-Marie; Camen, Adrian

    2015-01-01

    The aim of this study was to investigate whether the co-administration of aripiprazole and mirtazapine could determine weight gain and lipid metabolism disorders in Wistar rats, compared to the same side effects produced by mirtazapine alone, and the risk of hepatotoxicity due to the combination of the two substances. Tumor necrosis factor alpha (TNF-α), liver fatty acid binding protein (L-FABP/FABP1) and repulsive guidance molecule C/hemojuvelin (RGM-C/HJV) levels were determined in serum and in saliva. Also, serum levels for total cholesterol (TC), low and high-density lipoprotein (LDL, HDL), triglycerides (TG), aspartate aminotransferase (ASAT) and alanine amino transferase (ALAT) were assessed. We found positive and statistically significant correlations between serum and salivary levels of TNF-α, L-FABP/FABP1 and RGM-C/HJV. Mirtazapine determined significantly differences of TNF-α and L-FABP serum levels; final body weight; TC and LDL levels, leading to higher concentrations than its association with aripiprazole. Although not statistically significant, mirtazapine group experienced higher values for salivary levels of TNF-α, TG and ASAT, and lower values for HDL, compared to aripiprazole + mirtazapine group. The results suggest that aripiprazole might improve some of the disturbances caused by mirtazapine, and that the two drugs combination cause no additional alterations in liver function. Also, the findings indicate that TNF-α, L-FABP/FABP1 and RGM-C/HJV levels can be helpful as biomarkers for metabolic disturbances and impaired function of hepatocytes, and that their salivary determination can replace serum determination. PMID:26221370

  5. Early prediction of acute kidney injury biomarkers after endovascular stent graft repair of aortic aneurysm: a prospective observational study

    PubMed Central

    2014-01-01

    Background Acute kidney injury (AKI) is a common and serious condition usually detected some time after onset by changes in serum creatinine (sCr). Although stent grafting to repair aortic aneurysms is associated with AKI caused by surgical procedures or the use of contrast agents, early biomarkers for AKI have not been adequately examined in stent graft recipients. We studied biomarkers including urinary neutrophil gelatinase-associated lipocalin (NGAL), blood NGAL, N-acetyl-β-d-glucosaminidase (NAG), microalbumin (Alb), and liver fatty acid-binding protein (L-FABP) as prospective early biomarkers for AKI in patients who had received stent graft repairs of aortic aneurysms. Methods In addition to pre-surgical sampling, at 2 to 6 h and at 1, 3 to 4, and 5 days or later (until stable) after surgery, urine and serum biomarkers were sampled from 47 patients who underwent stent graft repair of aortic aneurysms. Results Using Acute Kidney Injury Network criteria, 6 (14%) of 42 retained patients developed AKI. NGAL corrected with urine Cr (NGAL/Cr) values demonstrated the best predictive value for AKI (97% specificity, 83% sensitivity at a 65.1 μg/gCr cutoff). The area under the receiver-operator characteristic curve of NGAL/Cr value 2 h after surgery was 0.9. Although NGAL/Cr, L-FABP corrected with urine Cr (L-FABP/Cr), L-FABP, NAG, and Alb corrected by urine Cr (Alb/Cr) all reached peak values before AKI detection by sCr in AKI patients, all biomarkers reached the cutoff value before AKI detection after adaption of cutoff value. Conclusions After stent graft repair of aortic aneurysm, NGAL/Cr is a potentially useful early biomarker for AKI. PMID:25960881

  6. Salivary and serum biomarkers for the study of side effects of aripiprazole coprescribed with mirtazapine in rats.

    PubMed

    Bogdan, Maria; Silosi, Isabela; Surlin, Petra; Tica, Andrei Adrian; Tica, Oana Sorina; Balseanu, Tudor-Adrian; Rauten, Anne-Marie; Camen, Adrian

    2015-01-01

    The aim of this study was to investigate whether the co-administration of aripiprazole and mirtazapine could determine weight gain and lipid metabolism disorders in Wistar rats, compared to the same side effects produced by mirtazapine alone, and the risk of hepatotoxicity due to the combination of the two substances. Tumor necrosis factor alpha (TNF-α), liver fatty acid binding protein (L-FABP/FABP1) and repulsive guidance molecule C/hemojuvelin (RGM-C/HJV) levels were determined in serum and in saliva. Also, serum levels for total cholesterol (TC), low and high-density lipoprotein (LDL, HDL), triglycerides (TG), aspartate aminotransferase (ASAT) and alanine amino transferase (ALAT) were assessed. We found positive and statistically significant correlations between serum and salivary levels of TNF-α, L-FABP/FABP1 and RGM-C/HJV. Mirtazapine determined significantly differences of TNF-α and L-FABP serum levels; final body weight; TC and LDL levels, leading to higher concentrations than its association with aripiprazole. Although not statistically significant, mirtazapine group experienced higher values for salivary levels of TNF-α, TG and ASAT, and lower values for HDL, compared to aripiprazole + mirtazapine group. The results suggest that aripiprazole might improve some of the disturbances caused by mirtazapine, and that the two drugs combination cause no additional alterations in liver function. Also, the findings indicate that TNF-α, L-FABP/FABP1 and RGM-C/HJV levels can be helpful as biomarkers for metabolic disturbances and impaired function of hepatocytes, and that their salivary determination can replace serum determination. PMID:26221370

  7. Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

    PubMed Central

    Burbaum, J. J.; Schimmel, P.

    1992-01-01

    Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304356

  8. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) mediates periarticular bone loss, but not joint destruction, in murine antigen-induced arthritis.

    PubMed

    Shimizu, Tomohiro; Takahata, Masahiko; Kameda, Yusuke; Endo, Tsutomu; Hamano, Hiroki; Hiratsuka, Shigeto; Ota, Masahiro; Iwasaki, Norimasa

    2015-10-01

    Osteoclastogenesis requires immunoreceptor tyrosine-based activation motif signaling. Multiple immunoreceptors associated with immunoreceptor tyrosine-based activation motif adaptor proteins, including DNAX-activating protein 12 kDa (DAP12) and Fc receptor common γ (FcRγ), have been identified in osteoclast lineage cells, and some are involved in arthritis-induced bone destruction. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is an immunoreceptor that regulates osteoclast development and bone resorption in association with DAP12. Whether Siglec-15 is involved in arthritis-induced bone lesions, however, remains unknown. Here we used a murine antigen-induced arthritis model to examine the role of Siglec-15 in the development of bone lesions induced by joint inflammation. Arthritis was unilaterally induced in the knee joints of 8-week-old female wild-type (WT) and Siglec-15(-/-) mice, and the contralateral knees were used as a control. The degree of joint inflammation, and cartilage and subchondral bone destruction in Siglec-15(-/-) mice was comparable to that in WT mice, indicating that Siglec-15 is not involved in the development of arthritis and concomitant cartilage and subchondral bone destruction. On the other hand, the degree of periarticular bone loss in the proximal tibia of the arthritic knee was significantly lower in Siglec-15(-/-) mice compared to WT mice. Although osteoclast formation in the metaphysis was enhanced in both WT and Siglec-15(-/-) mice after arthritis induction, mature multinucleated osteoclast formation was impaired in Siglec-15(-/-) mice, indicating impaired osteoclast bone resorptive function in the periarticular regions of the arthritic joint in Siglec-15(-/-) mice. Confirming this result, Siglec-15(-/-) primary unfractionated bone marrow cells harvested from arthritic femurs and tibiae failed to develop into mature multinuclear osteoclasts. Our findings suggest that Siglec-15 is a therapeutic target for periarticular

  9. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  10. Molecular dynamic simulations reveal the structural determinants of fatty acid binding to oxy-myoglobin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolite...

  11. Bile acid-binding ability of kaki-tannin from young fruits of persimmon (Diospyros kaki) in vitro and in vivo.

    PubMed

    Matsumoto, Kenji; Kadowaki, Akio; Ozaki, Natsumi; Takenaka, Makiko; Ono, Hiroshi; Yokoyama, Shin-ichiro; Gato, Nobuki

    2011-04-01

    The bile acid-binding ability of a highly polymerized tannin (kaki-tannin) extracted from dried-young fruits of persimmon (Diospyros kaki) was examined. The kaki-tannin was composed mainly of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Bile acid-binding ability of kaki-tannin was examined against cholic acid, glycocholic acid, taurocholic acid and deoxycholic acid in vitro, and its effect on fecal bile acid excretion in mice was also examined. Although the bile acid-binding ability of kaki-tannin was weaker than that of cholestyramine, kaki-tannin adsorbed all the bile acids tested and significantly promoted fecal bile acid excretion in mice when supplied at 1% (w/w) in the diet. PMID:20922818

  12. Identification and Characterization of Linoleic Acid as an Endogenous Modulator of in Vitro N-1-Naphthylphthalamic Acid Binding.

    PubMed Central

    Suttle, J. C.

    1997-01-01

    An endogenous inhibitor of the in vitro binding of the phytotropin N-1-naphthylphthalamic acid to microsomal membranes was detected in extracts prepared from etiolated pea (Pisum sativum L.) epicotyls. Following extensive purification, the inhibitor was identified as linoleic acid. Authentic linoleic acid inhibited N-1-naphthylphthalamic acid binding noncompetitively in a dose-dependent manner, exhibiting a 50% inhibitory concentration of approximately 24 ([mu]M. Using a variety of fatty acids and their derivatives, this inhibition was found to exhibit strict structural requirements, with both linoleic and linolenic acids being the most inhibitory. A variety of membrane-solubilizing detergents elicited no such inhibitory activity when tested at equivalent concentrations. The possible physiological significance of this interaction is discussed and it is proposed that linoleic acid serves as an intracellular modulator of phytotropin binding and therefore polar auxin transport. PMID:12223622

  13. The Sialic Acid Binding Protein, Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species

    PubMed Central

    Zhou, Peng; Liu, Jinman; Li, Xiaoli; Takahashi, Yukihiro; Qi, Fengxia

    2015-01-01

    Dental biofilm development involves initial colonization of the tooth’s surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S. gordonii also is a pathogen in infective endocarditis in susceptible humans. A surface adhesin, Hsa, has been shown to play a critical role in colonization of S. gordonii on the heart tissue; however, its role in oral biofilm development has not been reported. In this study we demonstrate that Hsa is essential for coaggregation between S. gordonii and Veillonella sp., which are bridging species connecting the pioneer colonizers to the late colonizers. Interestingly, the same domains shown to be required for Hsa binding to sialic acid on the human cell surface are also required for coaggregation with Veillonella sp. However, sialic acid appeared not to be required for this intergeneric coaggregation. This result suggests that although the same domains of Hsa are involved in binding to eukaryotic as well as Veillonella cells, the binding mechanism is different. The gene expression pattern of hsa was also studied and shown not to be induced by coaggregation with Veillonella sp. PMID:26606595

  14. Saturated fatty-acids regulate retinoic acid signaling and suppress tumorigenesis by targeting fatty-acid-binding protein 5

    PubMed Central

    Levi, Liraz; Wang, Zeneng; Doud, Mary Kathryn; Hazen, Stanley L.; Noy, Noa

    2015-01-01

    Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes, and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPARβ/δ. The data indicate that these activities of LCFA are mediated by FABP5 which delivers ligands from the cytosol to nuclear PPARβ/δ. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPARβ/δ pathway. We show further that, by concomitantly promoting activation of RAR and inhibiting the activation of PPARβ/δ, SLCFA suppress the oncogenic properties of FABP5-expressing carcinoma cells in cultured cells and in vivo. The observations suggest that compounds that inhibit FABP5 may constitute a new class of drugs for therapy of certain types of cancer. PMID:26592976

  15. Saturated fatty acids regulate retinoic acid signalling and suppress tumorigenesis by targeting fatty acid-binding protein 5.

    PubMed

    Levi, Liraz; Wang, Zeneng; Doud, Mary Kathryn; Hazen, Stanley L; Noy, Noa

    2015-01-01

    Long chain fatty acids (LCFA) serve as energy sources, components of cell membranes and precursors for signalling molecules. Here we show that these biological compounds also regulate gene expression and that they do so by controlling the transcriptional activities of the retinoic acid (RA)-activated nuclear receptors RAR and PPARβ/δ. The data indicate that these activities of LCFA are mediated by FABP5, which delivers ligands from the cytosol to nuclear PPARβ/δ. Both saturated and unsaturated LCFA (SLCFA, ULCFA) bind to FABP5, thereby displacing RA and diverting it to RAR. However, while SLCFA inhibit, ULCFA activate the FABP5/PPARβ/δ pathway. We show further that, by concomitantly promoting the activation of RAR and inhibiting the activation of PPARβ/δ, SLCFA suppress the oncogenic properties of FABP5-expressing carcinoma cells in cultured cells and in vivo. The observations suggest that compounds that inhibit FABP5 may constitute a new class of drugs for therapy of certain types of cancer. PMID:26592976

  16. Bovine adipose triglyceride lipase is not altered and adipocyte fatty acid binding protein is increased by dietary flaxseed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this paper, we report the full length coding sequence of bovine ATGL cDNA are reported and analyze its expression in bovine tissues. Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic function in mice and humans. Thi...

  17. Chemical composition and bile acid binding activity of products obtained from amaranth (Amaranthus cruentus) seeds.

    PubMed

    Tiengo, Andréa; Motta, Eliana Maria Pettirossi; Netto, Flavia Maria

    2011-11-01

    Cardiovascular diseases are currently the greatest cause of mortality in the world, and dislipidemia is appearing as one of the most important risk factors. The binding of bile acids (BAs) has been hypothesized as a possible mechanism by which dietary fibers lower blood cholesterol levels. Besides the fibers, other components in the amaranth seeds may be related to this hypocholesterolemic effect. The objective of the present study was to evaluate the BA binding capacity of some products obtained from defatted amaranth flour (DAF) and from the amaranth protein concentrate (APC). The alkaline residue, rich in fibers (8.6%), presented the lowest binding activity for the BAs tested, with the exception of glycocholic acid. The DAF showed intermediary binding activity for all the BAs tested, although similar to that of the APC for deoxycholic acid, and to that of the amaranth protein hydrolysate (APH) for taurocholic acid. The DAF and APC showed binding activity for secondary bile acids toxic to the intestinal mucus. From the results, amaranth products were shown to have the ability to bind BAs, but it was not possible to affirm whether the main component responsible for this activity was the proteins, fibers or eventually some other non-evaluated component. PMID:21901402

  18. In vitro bile-acid binding and fermentation of high, medium, and low molecular weight beta-glucan.

    PubMed

    Kim, Hyun Jung; White, Pamela J

    2010-01-13

    The impact of beta-glucan molecular weight (MW) on in vitro bile-acid binding and in vitro fermentation with human fecal flora was evaluated. beta-Glucan extracted from oat line 'N979-5-4' was treated with lichenase (1,3-1,4-beta-D-glucanase) to yield high (6.87x10(5) g/mol), medium (3.71x10(5) g/mol), and low (1.56x10(5) g/mol) MW fractions. The low MW beta-glucan bound more bile acid than did the high MW beta-glucan (p<0.05). If the positive control, cholestyramine, was considered to bind bile acid at 100%, the relative bile-acid binding of the original oat flour and the extracted beta-glucan with high, medium, and low MW was 15, 27, 24, and 21%, respectively. Significant effects of high, medium, and low MW beta-glucans on total SCFA were observed compared to the blank without substrate (p<0.05). There were no differences in pH changes and total gas production among high, medium, and low MW beta-glucans, and lactulose. The low MW beta-glucan produced greater amounts of SCFA than the high MW after 24 h of fermentation. Among the major SCFA, more propionate was produced from all MW fractions of extracted beta-glucans than from lactulose. In vitro fermentation of extracted beta-glucan fractions with different MW lowered pH and produced SCFA, providing potential biological function. PMID:20020684

  19. A high-fat diet and the threonine-encoding allele (Thr54) polymorphism of fatty acid–binding protein 2 reduce plasma triglyceride–rich lipoproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Thr54 allele of the fatty acid binding protein 2 (FABP2) DNA polymorphism is associated with increased triglyceride-rich lipoproteins and insulin resistance. We investigated whether the triglyceride-rich lipoprotein response to diets of varied fat content is affected by the fatty acid binding pr...

  20. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  1. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    SciTech Connect

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

  2. Stacking interaction and its role in kynurenic acid binding to glutamate ionotropic receptors.

    PubMed

    Zhuravlev, Alexander V; Zakharov, Gennady A; Shchegolev, Boris F; Savvateeva-Popova, Elena V

    2012-05-01

    Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation. PMID:21833825

  3. Steam Cooking Significantly Improves In Vitro Bile Acid Binding of Collard Greens, Kale, Mustard Greens, Broccoli, Green Bell Pepper and Cabbage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding capacity has been related to the cholesterol-lowering potential of foods and food fractions. Lowering recirculating bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increasing the r...

  4. Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers

    SciTech Connect

    Hamilton, J.A. )

    1989-04-01

    Temperature-dependent (5-42{degree}C) {sup 13}C NMR spectra of albumin complexes with 90% isotopically substituted (1-{sup 13}C)octanoic or (1-{sup 13}C)decanoic acids showed a single peak at >30{degree}C but three peaks at lower temperatures. The chemical-shift differences result from different ionic and/or hydrogen-bonding interactions between amino acid side chains and the fatty acid carboxyl carbon. Rapid exchange of fatty acid among binding sites obscures these sites at temperatures >30{degree}C. Rate constants for exchange at 33{degree}C were 350 sec{sup {minus}1} for octanoate and 20 sec {sup {minus}1} for decanoate. Temperature-dependent data for octanoate showed an activation energy of 2 kcal/mol for exchange. Spectra of albumin complexes with the 12-carbon saturated fatty acid, lauric acid, had several narrow laurate carboxyl peaks at 35{degree}C, indicating longer lifetimes in the different binding sites. Fatty acid exchange between albumin and model membranes (phosphatidylcholine bilayers) occurred on a time scale comparable to that for exchange among albumin binding sites, following the order octanoate > decanoate > laurate. The equilibrium distribution of fatty acid between lipid bilayers and protein was measured directly from NMR spectra. Decreasing pH increased the relative affinity of fatty acid for the lipid bilayer. The results predict that the relative affinity of octanoic acid for albumin and membranes will be similar to that of long-chain fatty acids, but the rate of equilibration will be {approx} 10{sup 4} faster for octanoic acid.

  5. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

    PubMed Central

    Mukherjee, Sourav; Hanson, Alicia M.; Shadrick, William R.; Ndjomou, Jean; Sweeney, Noreena L.; Hernandez, John J.; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J.; Heck, Julie A.; Arnold, Leggy A.; Schoenen, Frank J.; Frick, David N.

    2012-01-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors. PMID:22740655

  6. CD36 Binds Oxidized Low Density Lipoprotein (LDL) in a Mechanism Dependent upon Fatty Acid Binding*

    PubMed Central

    Jay, Anthony G.; Chen, Alexander N.; Paz, Miguel A.; Hung, Justin P.; Hamilton, James A.

    2015-01-01

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes. PMID:25555908

  7. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

    PubMed Central

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

    2016-01-01

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  8. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

    PubMed

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

    2016-01-19

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  9. Techno-functional properties and in vitro bile acid-binding capacities of tamarillo (Solanum betaceum Cav.) hydrocolloids.

    PubMed

    Gannasin, Sri Puvanesvari; Adzahan, Noranizan Mohd; Mustafa, Shuhaimi; Muhammad, Kharidah

    2016-04-01

    Hydrocolloids were extracted from seed mucilage and the pulp fractions from red tamarillo (Solanum betaceum Cav.) mesocarp, and characterisation of their techno-functional properties and in vitro bile acid-binding capacities was performed. The seed mucilage hydrocolloids that were extracted, using either 1% citric acid (THC) or water (THW), had a good foaming capacity (32-36%), whereas the pulp hydrocolloids that were extracted, using 72% ethanol (THE) or 20mM HEPES buffer (THH), had no foaming capacity. The pulp hydrocolloid, however, possessed high oil-holding and water-holding capacities in the range of 3.3-3.6 g oil/g dry sample and 25-27 g water/g dry sample, respectively. This enabled the pulp hydrocolloid to entrap more bile acids (35-38% at a hydrocolloid concentration of 2%) in its gelatinous network in comparison to commercial oat fibre and other hydrocolloids studied. The exceptional emulsifying properties (80-96%) of both hydrocolloids suggest their potential applications as food emulsifiers and bile acid binders. PMID:26593571

  10. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    SciTech Connect

    Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

    2011-06-10

    Highlights: {yields} Siglec-15 was identified as a gene overexpressed in giant cell tumor. {yields} Siglec-15 mRNA expression increased in association with osteoclast differentiation. {yields} Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor {kappa}B ligand (RANKL) or tumor necrosis factor (TNF)-{alpha}. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D{sub 3} and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  11. A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    PubMed Central

    Vallström, Anna; Olofsson, Annelie; Öhman, Carina; Rakhimova, Lena; Borén, Thomas; Engstrand, Lars; Brännström, Kristoffer; Arnqvist, Anna

    2014-01-01

    During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors. PMID:24991812

  12. Regulation of B Cell Functions by the Sialic Acid-Binding Receptors Siglec-G and CD22

    PubMed Central

    Jellusova, Julia; Nitschke, Lars

    2011-01-01

    B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells. PMID:22566885

  13. Non-extractable procyanidins and lignin are important factors in the bile acid binding and radical scavenging properties of cell wall material in some fruits.

    PubMed

    Hamauzu, Yasunori; Mizuno, Yukari

    2011-03-01

    The cell wall components and the food functions of alcohol-insoluble solids (AIS) of Chinese quince, quince, hawthorn, apple, pear and blueberry fruits were analyzed. Chinese quince contained characteristically high contents of cellulose, lignin, and non-extractable procyanidins (NEPCs). On the other hand, the quince AIS contained the highest proportion of NEPCs, the highest mean degree of polymerization (mDP), the strongest radical scavenging activity, and strong bile acid binding activity. In fruit AIS, the lignin and NEPC contents both showed positive correlations with the bile acid binding and radical scavenging activities. The value for mDP × NEPC content was a good index for the radical scavenging activity. The results suggest that highly polymerized NEPCs and lignin are important factors of cell wall components of fruits to having a high functionality, and Chinese quince and quince are interesting fruits from this view point. PMID:21243435

  14. Identification of a Novel Hypocholesterolemic Protein, Major Royal Jelly Protein 1, Derived from Royal Jelly

    PubMed Central

    Asai, Saori; Kusada, Mio; Watanabe, Suzuyo; Kawashima, Takuji; Nakamura, Tadashi; Shimada, Masaya; Goto, Tsuyoshi; Nagaoka, Satoshi

    2014-01-01

    Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats. PMID:25144734

  15. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model. PMID:27029427

  16. The unique ligand binding features of subfamily-II iLBPs with respect to bile salts and related drugs.

    PubMed

    Favretto, Filippo; Ceccon, Alberto; Zanzoni, Serena; D'Onofrio, Mariapina; Ragona, Laura; Molinari, Henriette; Assfalg, Michael

    2015-04-01

    Intracellular lipid binding proteins (iLBPs) are a family of evolutionarily related small cytoplasmic proteins implicated in the transcellular transport of lipophilic ligands. Subfamily-II iLBPs include the liver fatty acid binding protein (L-FABP), and the ileal and the liver and ileal bile acid binding proteins (L-BABP and I-BABP). Atomic-level investigations during the past 15-20 years have delivered relevant information on bile acid binding by this protein group, revealing unique features including binding cooperativity, promiscuity, and site selectivity. Using NMR spectroscopy and other biophysical techniques, our laboratories have contributed to an understanding of the molecular determinants of some of these properties and their generality among proteins from different animal species. We focused especially on formation of heterotypic complexes, considering the mixed compositions of physiological bile acid pools. Experiments performed with synthetic bile acid derivatives showed that iLBPs could act as targets for cell-specific contrast agents and, more generally, as effective carriers of amphiphilic drugs. This review collects the major findings related to bile salt interactions with iLBPs aiming to provide keys for a deeper understanding of protein-mediated intracellular bile salt trafficking. PMID:25468388

  17. Urinary levels of early kidney injury molecules in children with vitamin B12 deficiency.

    PubMed

    Güneş, Ali; Aktar, Fesih; Tan, İlhan; Söker, Murat; Uluca, Ünal; Balık, Hasan; Mete, Nuriye

    2016-10-01

    The aim of this study was to investigate urine early kidney injury molecules, including human kidney injury molecule-1 (KIM-1), liver-type fatty-acid binding protein (L-FABP), N-acetyl-b-D-glucosaminidase A (NAG), and neutrophil gelatinase-associated lipocalin (NGAL) in children with vitamin B12 (cobalamin) deficiency (CD). Twelve children with vitamin B12 deficiency and 20 healthy matched controls were included. Hematologic parameters, serum urea, creatinine (Cr), electrolytes, B12 and folate levels were recorded. Estimated glomerular filtration rate (eGFR) was calculated. Urine protein, electrolytes, andurinary early markers were measured. Patients with CD had significantly higher urine electrolyte/Cr ratios (p <0.05). Significantly higher urinary KIM-1/Cr, L-FABP/Cr, NAG/Cr and NGAL/Cr were found in CD group (p <0.05). Significant negative correlations were found between levels of serum B12 and urinary markers in the patients (p <0.05). Increased urinary kidney injury molecules and electrolytes in children with B12 deficiency suggest a possible subclinical renal dysfunction, which cannot be determined by conventional kidney function tests. PMID:27606644

  18. Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis1

    PubMed Central

    Murphy, Angus S.; Hoogner, Karen R.; Peer, Wendy Ann; Taiz, Lincoln

    2002-01-01

    Polar transport of the plant hormone auxin is regulated at the cellular level by inhibition of efflux from a plasma membrane (PM) carrier. Binding of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to a regulatory site associated with the carrier has been characterized, but the NPA-binding protein(s) have not been identified. Experimental disparities between levels of high-affinity NPA binding and auxin transport inhibition can be explained by the presence of a low-affinity binding site and in vivo hydrolysis of NPA. In Arabidopsis, colocalization of NPA amidase and aminopeptidase (AP) activities, inhibition of auxin transport by artificial β-naphthylamide substrates, and saturable displacement of NPA by the AP inhibitor bestatin suggest that PM APs may be involved in both low-affinity NPA binding and hydrolysis. We report the purification and molecular cloning of NPA-binding PM APs and associated proteins from Arabidopsis. This is the first report of PM APs in plants. PM proteins were purified by gel permeation, anion exchange, and NPA affinity chromatography monitored for tyrosine-AP activity. Lower affinity fractions contained two orthologs of mammalian APs involved in signal transduction and cell surface-extracellular matrix interactions. AtAPM1 and ATAPP1 have substrate specificities and inhibitor sensitivities similar to their mammalian orthologs, and have temporal and spatial expression patterns consistent with previous in planta histochemical data. Copurifying proteins suggest that the APs interact with secreted cell surface and cell wall proline-rich proteins. AtAPM1 and AtAPP1 are encoded by single genes. In vitro translation products of ATAPM1 and AtAPP1 have enzymatic activities similar to those of native proteins. PMID:11891249

  19. Associations of Perfusate Biomarkers and Pump Parameters With Delayed Graft Function and Deceased Donor Kidney Allograft Function.

    PubMed

    Parikh, C R; Hall, I E; Bhangoo, R S; Ficek, J; Abt, P L; Thiessen-Philbrook, H; Lin, H; Bimali, M; Murray, P T; Rao, V; Schröppel, B; Doshi, M D; Weng, F L; Reese, P P

    2016-05-01

    Hypothermic machine perfusion (HMP) is increasingly used in deceased donor kidney transplantation, but controversy exists regarding the value of perfusion biomarkers and pump parameters for assessing organ quality. We prospectively determined associations between perfusate biomarkers (neutrophil gelatinase-associated lipocalin [NGAL], kidney injury molecule 1, IL-18 and liver-type fatty acid-binding protein [L-FABP]) and pump parameters (resistance and flow) with outcomes of delayed graft function (DGF) and 6-mo estimated GFR (eGFR). DGF occurred in 230 of 671 (34%) recipients. Only 1-h flow was inversely associated with DGF. Higher NGAL or L-FABP concentrations and increased resistance were inversely associated with 6-mo eGFR, whereas higher flow was associated with higher adjusted 6-mo eGFR. Discarded kidneys had consistently higher median resistance and lower median flow than transplanted kidneys, but median perfusate biomarker concentrations were either lower or not significantly different in discarded compared with transplanted kidneys. Notably, most recipients of transplanted kidneys with isolated "undesirable" biomarker levels or HMP parameters experienced acceptable 6-mo allograft function, suggesting these characteristics should not be used in isolation for discard decisions. Additional studies must confirm the utility of combining HMP measurements with other characteristics to assess kidney quality. PMID:26695524

  20. Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy

    PubMed Central

    Simard, J. R.; Zunszain, P. A.; Ha, C.-E.; Yang, J. S.; Bhagavan, N. V.; Petitpas, I.; Curry, S.; Hamilton, J. A.

    2005-01-01

    Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [13C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA–HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [13C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [13C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites. PMID:16330771

  1. Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy.

    PubMed

    Simard, J R; Zunszain, P A; Ha, C-E; Yang, J S; Bhagavan, N V; Petitpas, I; Curry, S; Hamilton, J A

    2005-12-13

    Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [(13)C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [(13)C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [(13)C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites. PMID:16330771

  2. Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response

    PubMed Central

    Baek, Yu Mi; Yoon, Soojin; Hwang, Yeo Eun

    2016-01-01

    Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I. PMID:27574504

  3. Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response.

    PubMed

    Baek, Yu Mi; Yoon, Soojin; Hwang, Yeo Eun; Kim, Dong-Eun

    2016-08-01

    Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I. PMID:27574504

  4. The effect of acute alcohol intoxication on gut wall integrity in healthy male volunteers; a randomized controlled trial.

    PubMed

    de Jong, W J; Cleveringa, A M; Greijdanus, B; Meyer, P; Heineman, E; Hulscher, J B

    2015-02-01

    The aim of the study is to determine the effect of acute alcohol consumption on enterocytes. Chronic alcohol consumption has been known to induce a decrease in gut wall integrity in actively drinking alcoholics and patients with alcohol-induced liver disease. Data on the extent of the damage induced by acute alcohol consumption in healthy human beings is scarce. Studies show that heavy incidental alcohol consumption is a growing problem in modern society. Data on this matter may provide insights into the consequences of this behavior for healthy individuals. In a randomized clinical trial in crossover design, 15 healthy volunteers consumed water one day and alcohol the other. One blood sample was collected pre-consumption, five every hour post-consumption, and one after 24 h. Intestinal fatty acid binding protein (I-FABP) was used as a marker for enterocyte damage. Liver fatty acid binding protein (L-FABP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) were used as markers for hepatocyte damage. Lipopolysaccharide binding protein (LBP) and soluble CD14 (sCD14) were used as a measure of translocation. Interleukin-6 (IL-6) was used to assess the acute inflammatory response to endotoxemia. Alcohol consumption caused a significant increase in serum I- and L-FABP levels, compared to water consumption. Levels increased directly post-consumption and decreased to normal levels within 4 h. LBP, sCD14, and IL-6 levels were not significantly higher in the alcohol group. Moderate acute alcohol consumption immediately damages the enterocyte but does not seem to cause endotoxemia. PMID:25559494

  5. CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

    PubMed Central

    Kellett-Clarke, Helena; Stegmann, Monika; Barclay, A. Neil; Metcalfe, Clive

    2015-01-01

    CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies. PMID:26379032

  6. Bile acid derivatives as ligands of the farnesoid x receptor: molecular determinants for bile acid binding and receptor modulation.

    PubMed

    Gioiello, Antimo; Cerra, Bruno; Mostarda, Serena; Guercini, Chiara; Pellicciari, Roberto; Macchiarulo, Antonio

    2014-01-01

    Bile acids are a peculiar class of steroidal compounds that never cease to amaze. From being simple detergents with a primary role in aiding the absorption of fats and fat-soluble vitamins, bile acids are now widely considered as crucial hormones endowed with genomic and non-genomic functions that are mediated by their interaction with several proteins including the nuclear receptor Farnesoid X Receptor (FXR). Taking advantages of the peculiar properties of bile acids in interacting with the FXR receptor, several biliary derivatives have been synthesized and tested as FXR ligands. The availability of these compounds has contributed to characterize the receptor from a structural, patho-physiological and therapeutic standpoint. Among these, obeticholic acid is a first-in-class FXR agonist that is demonstrating hepatoprotective effects upon FXR activation in patients with liver diseases such as primary biliary cirrhosis and nonalcoholic steatohepatitis. This review provides an historical overview of the rationale behind the discovery of obeticholic acid and chemical tools generated to depict the molecular features and bio-pharmacological relevance of the FXR receptor, as well as to summarize structure-activity relationships of bile acid-based FXR ligands so far reported. PMID:25388535

  7. In vitro bile acid binding of mustard greens, kale, broccoli, cabbage and green bell pepper improves with sautéing compared with raw or other methods of preparation.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid binding capacity has been related to cholesterol-lowering potential of foods and food fractions. Lowered recirculating bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of can...

  8. Excessive reactive oxygen species induces apoptosis in fibroblasts: Role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

    SciTech Connect

    Chowdhury, Anindya Roy; Ghosh, Ilora Datta, Kasturi

    2008-02-01

    Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities alongwith initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca{sup 2+} influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.

  9. Thio-ketosides of sialic acid containing aryl azides: potential photo-affinity probes for analysis of neuraminidases and sialic acid binding proteins

    SciTech Connect

    Warner, T.G.; Lee, L.A.

    1986-05-01

    To date, only a single report describing the synthesis of thio-ketosides of sialic acid has appeared. In this procedure, the pseudo thiourea of acetoneuraminic acid methyl ester (NTU) was used to prepare the sodium thiolate salt. However, in their hands, the preparation of NTU was not straight-forward, and in subsequent reactions thio glycosides were not obtained. Therefore, they have developed an alternate route for introduction of the sulfhydryl group and have prepared novel thio-ketosides with aryl azides. The thio linkage is advantageous since it is not easily cleaved by neuraminidases and it allows incorporation of /sup 35/S as a convenient radioactive label. 2-deoxy-2-S-acetyl-4,7,8,9,- tetra-0-acetyl-N-acetyl neuraminic acid methyl ester was prepared (70% yield) from 2-chloro aceto- neuraminic acid methyl ester and potassium thioacetate in acetone at room temperature (RT) for 90 min. Selective hydrolysis of the thio acetate group was accomplished with equimolar sodium methoxide in DMF. After 10 min at RT, 4-fluoro-3-nitrophenyl azide was added and reaction continued for 60 min. Silicic acid purification, base hydrolysis, and gel filtration chromatography, gave 2'-deoxy-2'-(2-nitro-4-azido-thiophenyl)-..cap alpha..-D-N-acetyl neuraminic acid (35% yield). Other thio-arylazido ketosides were prepared similarly.

  10. Common Variants of the Liver Fatty Acid Binding Protein Gene Influence the Risk of Type 2 Diabetes and Insulin Resistance in Spanish Population

    PubMed Central

    Mansego, Maria Luisa; Martínez, Fernando; Martínez-Larrad, Maria Teresa; Zabena, Carina; Rojo, Gemma; Morcillo, Sonsoles; Soriguer, Federico; Martín-Escudero, Juan Carlos; Serrano-Ríos, Manuel; Redon, Josep; Chaves, Felipe Javier

    2012-01-01

    Summary The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. Methods 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. Results One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. Conclusions The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians. PMID:22396741

  11. Severity of soybean meal induced distal intestinal inflammation, enterocyte proliferation rate, and fatty acid binding protein (Fabp2) staining differ between strains of rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Complete replacement of fishmeal in feeds for carnivorous fishes often causes reduced growth and can negatively affect health. Salmonids fed diets containing full fat or defatted soybean meal develop dose dependent inflammation in the distal intestine (DI). Little is known about the sensitivity of d...

  12. Advancing the Development of Glycated Protein Biosensing Technology

    PubMed Central

    Kameya, Miho; Sakaguchi-Mikami, Akane; Ferri, Stefano; Tsugawa, Wakako; Sode, Koji

    2015-01-01

    Research advances in biochemical molecules have led to the development of convenient and reproducible biosensing molecules for glycated proteins, such as those based on the enzymes fructosyl amino acid oxidase (FAOX) or fructosyl peptide oxidase (FPOX). Recently, more attractive biosensing molecules with potential applications in next-generation biosensing of glycated proteins have been aggressively reported. We review 2 such molecules, fructosamine 6-kinase (FN6K) and fructosyl amino acid-binding protein, as well as their recent applications in the development of glycated protein biosensing systems. Research on FN6K and fructosyl amino acid-binding protein has been opening up new possibilities for the development of highly sensitive and proteolytic-digestion-free biosensing systems for glycated proteins. PMID:25627465

  13. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected. PMID:12417619

  14. Sialic Acid-Binding Immunoglobulin-like Lectin G Promotes Atherosclerosis and Liver Inflammation by Suppressing the Protective Functions of B-1 Cells

    PubMed Central

    Gruber, Sabrina; Hendrikx, Tim; Tsiantoulas, Dimitrios; Ozsvar-Kozma, Maria; Göderle, Laura; Mallat, Ziad; Witztum, Joseph L.; Shiri-Sverdlov, Ronit; Nitschke, Lars; Binder, Christoph J.

    2016-01-01

    Summary Atherosclerosis is initiated and sustained by hypercholesterolemia, which results in the generation of oxidized LDL (OxLDL) and other metabolic byproducts that trigger inflammation. Specific immune responses have been shown to modulate the inflammatory response during atherogenesis. The sialic acid-binding immunoglobulin-like lectin G (Siglec-G) is a negative regulator of the functions of several immune cells, including myeloid cells and B-1 cells. Here, we show that deficiency of Siglec-G in atherosclerosis-prone mice inhibits plaque formation and diet-induced hepatic inflammation. We further demonstrate that selective deficiency of Siglec-G in B cells alone is sufficient to mediate these effects. Levels of B-1 cell-derived natural IgM with specificity for OxLDL were significantly increased in the plasma and peritoneal cavity of Siglec-G-deficient mice. Consistent with the neutralizing functions of OxLDL-specific IgM, Siglec-G-deficient mice were protected from OxLDL-induced sterile inflammation. Thus, Siglec-G promotes atherosclerosis and hepatic inflammation by suppressing protective anti-inflammatory effector functions of B cells. PMID:26947073

  15. Impact of SCP-2/SCP-x gene ablation and dietary cholesterol on hepatic lipid accumulation

    PubMed Central

    Klipsic, Devon; Landrock, Danilo; Martin, Gregory G.; McIntosh, Avery L.; Landrock, Kerstin K.; Mackie, John T.; Schroeder, Friedhelm

    2015-01-01

    While a high-cholesterol diet induces hepatic steatosis, the role of intracellular sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) proteins is unknown. We hypothesized that ablating SCP-2/SCP-x [double knockout (DKO)] would impact hepatic lipids (cholesterol and cholesteryl ester), especially in high-cholesterol-fed mice. DKO did not alter food consumption, and body weight (BW) gain decreased especially in females, concomitant with hepatic steatosis in females and less so in males. DKO-induced steatosis in control-fed wild-type (WT) mice was associated with 1) loss of SCP-2; 2) upregulation of liver fatty acid binding protein (L-FABP); 3) increased mRNA and/or protein levels of sterol regulatory element binding proteins (SREBP1 and SREBP2) as well as increased expression of target genes of cholesterol synthesis (Hmgcs1 and Hmgcr) and fatty acid synthesis (Acc1 and Fas); and 4) cholesteryl ester accumulation was also associated with increased acyl-CoA cholesterol acyltransferase-2 (ACAT2) in males. DKO exacerbated the high-cholesterol diet-induced hepatic cholesterol and glyceride accumulation, without further increasing SREBP1, SREBP2, or target genes. This exacerbation was associated both with loss of SCP-2 and concomitant downregulation of Ceh/Hsl, apolipoprotein B (ApoB), MTP, and/or L-FABP protein expression. DKO diminished the ability to secrete excess cholesterol into bile and oxidize cholesterol to bile acid for biliary excretion, especially in females. This suggested that SCP-2/SCP-x affects cholesterol transport to particular intracellular compartments, with ablation resulting in less to the endoplasmic reticulum for SREBP regulation, making more available for cholesteryl ester synthesis, for cholesteryl-ester storage in lipid droplets, and for bile salt synthesis and/or secretion. These alterations are significant findings, since they affect key processes in regulation of sterol metabolism. PMID:26113298

  16. Impact of SCP-2/SCP-x gene ablation and dietary cholesterol on hepatic lipid accumulation.

    PubMed

    Klipsic, Devon; Landrock, Danilo; Martin, Gregory G; McIntosh, Avery L; Landrock, Kerstin K; Mackie, John T; Schroeder, Friedhelm; Kier, Ann B

    2015-09-01

    While a high-cholesterol diet induces hepatic steatosis, the role of intracellular sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) proteins is unknown. We hypothesized that ablating SCP-2/SCP-x [double knockout (DKO)] would impact hepatic lipids (cholesterol and cholesteryl ester), especially in high-cholesterol-fed mice. DKO did not alter food consumption, and body weight (BW) gain decreased especially in females, concomitant with hepatic steatosis in females and less so in males. DKO-induced steatosis in control-fed wild-type (WT) mice was associated with 1) loss of SCP-2; 2) upregulation of liver fatty acid binding protein (L-FABP); 3) increased mRNA and/or protein levels of sterol regulatory element binding proteins (SREBP1 and SREBP2) as well as increased expression of target genes of cholesterol synthesis (Hmgcs1 and Hmgcr) and fatty acid synthesis (Acc1 and Fas); and 4) cholesteryl ester accumulation was also associated with increased acyl-CoA cholesterol acyltransferase-2 (ACAT2) in males. DKO exacerbated the high-cholesterol diet-induced hepatic cholesterol and glyceride accumulation, without further increasing SREBP1, SREBP2, or target genes. This exacerbation was associated both with loss of SCP-2 and concomitant downregulation of Ceh/Hsl, apolipoprotein B (ApoB), MTP, and/or L-FABP protein expression. DKO diminished the ability to secrete excess cholesterol into bile and oxidize cholesterol to bile acid for biliary excretion, especially in females. This suggested that SCP-2/SCP-x affects cholesterol transport to particular intracellular compartments, with ablation resulting in less to the endoplasmic reticulum for SREBP regulation, making more available for cholesteryl ester synthesis, for cholesteryl-ester storage in lipid droplets, and for bile salt synthesis and/or secretion. These alterations are significant findings, since they affect key processes in regulation of sterol metabolism. PMID:26113298

  17. Assessment of Sialic Acid Diversity in Cancer- and Non-Cancer Related CA125 Antigen Using Sialic Acid-Binding Ig-Like Lectins (Siglecs)

    PubMed Central

    Mitic, N.; Milutinovic, B.; Jankovic, M.

    2012-01-01

    This study was aimed at obtaining insight into the diversity of sialic acids in cancer- and non-cancer-related CA125 antigen, tumour marker of serous ovarian cancer. Starting from available data suggesting the possible relevance of sialic acids for discriminating CA125 antigens of different origin, we have employed a new experimental approach based on the use of human sialic acid-binding Ig-like lectins, Siglecs, as tools for the investigation of sialylation. Siglec−2, belonging to the group of evolutionarily conserved Siglecs, and Siglec−3, −6, −7, −9 and −10, which are CD33-like Siglecs, were probed in solid-phase binding assays with cancer-related CA125 antigens from pleural fluid of patients with ovarian carcinoma (pfCA125), the OVCAR-3 ovarian carcinoma cell line (clCA125) and a non-cancer-related CA125 antigen, i.e. pregnancy-associated pCA125 antigen. All Siglecs used showed detectable binding to pCA125 antigen. Siglec−3, Siglec−7 and Siglec−2 exhibited moderately stronger binding to pCA125 antigen than the others. In contrast to this, Siglec−2 and Siglec−3 preferentially recognized pfCA125 with greater total binding than for pCA125, whereas Siglec−9 and Siglec−10 were highly selective for clCA125. Siglecs promise to be powerful tools for discriminating CA125 of different origin and could propagate further research on other molecular markers of biomedical and diagnostic importance. PMID:22377735

  18. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  19. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  20. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  1. Icaritin ameliorates carbon tetrachloride-induced acute liver injury mainly because of the antioxidative function through estrogen-like effects.

    PubMed

    Liu, Peng; Jin, Xiang; Lv, Hao; Li, Jing; Xu, Wen; Qian, Hai-hua; Yin, Zhengfeng

    2014-12-01

    To investigate the effects of icaritin, an active ingredient extracted from Epimedium Sagittatum (Sieb. et Zucc.), on CCl4-induced liver injury and its possible mechanisms. Hepatocytes isolated from Sprague-Dawley male rats were treated with 3 mmol/L CCl4 for 24 h to induce acute liver cell injury, then icaritin (0.1, 1, 10, 100 μmol/L, respectively) was administrated to the cells, and estrogen receptor antagonist ICI182,780 (1 μmol/L) was co-treated with 10 μmol/L icaritin. Biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD)) and cell apoptosis were detected to evaluate the injury degree. Protein expressions of Bax, Bcl-2, liver fatty acid-binding protein (L-FABP), and peroxisome proliferator-activated receptor-α (PPAR-α) as well as reactive oxygen species (ROS) generation were determined by western blot. Icaritin alleviated CCl4-induced liver cell injury in a concentration-dependent manner and 10 μmol/L was the optimal concentration. Icaritin (10 μmol/L) significantly reduced activities of ALT, AST in cell culture medium and MDA level of the impaired liver cells, but increased the intercellular SOD activity. The apoptotic rate of the impaired liver cells was also decreased by icaritin (10 μmol/L) treatment. Icaritin might exert antioxidative and anti-apoptotic functions via estrogen-like effect, as the ratio of Bcl-2/Bax was significantly increased, while protein expressions of L-FABP and PPAR-α were markedly increased, and this function was blocked by the estrogen receptor antagonist ICI182,780 efficiently. Icaritin may be a promising drug candidate for acute liver injury benefiting from the antioxidative and anti-apoptotic functions via estrogen-like effect. PMID:25148823

  2. Epoetin beta pegol alleviates oxidative stress and exacerbation of renal damage from iron deposition, thereby delaying CKD progression in progressive glomerulonephritis rats.

    PubMed

    Hirata, Michinori; Tashiro, Yoshihito; Aizawa, Ken; Kawasaki, Ryohei; Shimonaka, Yasushi; Endo, Koichi

    2015-12-01

    The increased deposition of iron in the kidneys that occurs with glomerulopathy hinders the functional and structural recovery of the tubules and promotes progression of chronic kidney disease (CKD). Here, we evaluated whether epoetin beta pegol (continuous erythropoietin receptor activator: CERA), which has a long half-life in blood and strongly suppresses hepcidin-25, exerts renoprotection in a rat model of chronic progressive glomerulonephritis (cGN). cGN rats showed elevated urinary total protein excretion (uTP) and plasma urea nitrogen (UN) from day 14 after the induction of kidney disease (day 0) and finally declined into end-stage kidney disease (ESKD), showing reduced creatinine clearance with glomerulosclerosis, tubular dilation, and tubulointerstitial fibrosis. A single dose of CERA given on day 1, but not on day 16, alleviated increasing uTP and UN, thereby delaying ESKD. In the initial disease phase, CERA significantly suppressed urinary 8-OHdG and liver-type fatty acid-binding protein (L-FABP), a tubular damage marker. CERA also inhibited elevated plasma hepcidin-25 levels and alleviated subsequent iron accumulation in kidneys in association with elevated urinary iron excretion and resulted in alleviation of growth of Ki67-positive tubular and glomerular cells. In addition, at day 28 when the exacerbation of uTP occurs, a significant correlation was observed between iron deposition in the kidney and urinary L-FABP. In our study, CERA mitigated increasing kidney damage, thereby delaying CKD progression in this glomerulonephritis rat model. Alleviation by CERA of the exacerbation of kidney damage could be attributable to mitigation of tubular damage that might occur with lowered iron deposition in tubules. PMID:26634903

  3. Associations between Deceased-Donor Urine Injury Biomarkers and Kidney Transplant Outcomes.

    PubMed

    Reese, Peter P; Hall, Isaac E; Weng, Francis L; Schröppel, Bernd; Doshi, Mona D; Hasz, Rick D; Thiessen-Philbrook, Heather; Ficek, Joseph; Rao, Veena; Murray, Patrick; Lin, Haiqun; Parikh, Chirag R

    2016-05-01

    Assessment of deceased-donor organ quality is integral to transplant allocation practices, but tools to more precisely measure donor kidney injury and better predict outcomes are needed. In this study, we assessed associations between injury biomarkers in deceased-donor urine and the following outcomes: donor AKI (stage 2 or greater), recipient delayed graft function (defined as dialysis in first week post-transplant), and recipient 6-month eGFR. We measured urinary concentrations of microalbumin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), IL-18, and liver-type fatty acid binding protein (L-FABP) from 1304 deceased donors at organ procurement, among whom 112 (9%) had AKI. Each biomarker strongly associated with AKI in adjusted analyses. Among 2441 kidney transplant recipients, 31% experienced delayed graft function, and mean±SD 6-month eGFR was 55.7±23.5 ml/min per 1.73 m(2) In analyses adjusted for donor and recipient characteristics, higher donor urinary NGAL concentrations associated with recipient delayed graft function (highest versus lowest NGAL tertile relative risk, 1.21; 95% confidence interval, 1.02 to 1.43). Linear regression analyses of 6-month recipient renal function demonstrated that higher urinary NGAL and L-FABP concentrations associated with slightly lower 6-month eGFR only among recipients without delayed graft function. In summary, donor urine injury biomarkers strongly associate with donor AKI but provide limited value in predicting delayed graft function or early allograft function after transplant. PMID:26374609

  4. Urine Injury Biomarkers and Risk of Adverse Outcomes in Recipients of Prevalent Kidney Transplants: The Folic Acid for Vascular Outcome Reduction in Transplantation Trial.

    PubMed

    Bansal, Nisha; Carpenter, Myra A; Weiner, Daniel E; Levey, Andrew S; Pfeffer, Marc; Kusek, John W; Cai, Jianwen; Hunsicker, Lawrence G; Park, Meyeon; Bennett, Michael; Liu, Kathleen D; Hsu, Chi-Yuan

    2016-07-01

    Recipients of kidney transplants (KTR) are at increased risk for cardiovascular events, graft failure, and death. It is unknown whether urine kidney injury biomarkers are associated with poor outcomes among KTRs. We conducted a post hoc analysis of the Folic Acid for Vascular Outcome Reduction in Transplantation (FAVORIT) Trial using a case-cohort study design, selecting participants with adjudicated cardiovascular events, graft failure, or death. Urine neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), IL-18, and liver-type fatty acid binding protein (L-FABP) were measured in spot urine samples and standardized to urine creatinine concentration. We adjusted for demographics, cardiovascular risk factors, eGFR, and urine albumin-to-creatinine ratio. Patients had 291 cardiovascular events, 257 graft failure events, and 359 deaths. Each log increase in urine NGAL/creatinine independently associated with a 24% greater risk of cardiovascular events (adjusted hazard ratio [aHR], 1.24; 95% confidence interval [95% CI], 1.06 to 1.45), a 40% greater risk of graft failure (aHR, 1.40; 95% CI, 1.16 to 1.68), and a 44% greater risk of death (aHR, 1.44; 95% CI, 1.26 to 1.65). Urine KIM-1/creatinine and IL-18/creatinine independently associated with greater risk of death (aHR, 1.29; 95% CI, 1.03 to 1.61 and aHR, 1.25; 95% CI, 1.04 to 1.49 per log increase, respectively) but not with risk of cardiovascular events or graft failure. Urine L-FABP did not associate with any study outcomes. In conclusion, among prevalent KTRs, higher urine NGAL, KIM-1, and IL-18 levels independently and differentially associated with greater risk of adverse outcomes. PMID:26538631

  5. Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

    PubMed

    Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

    2016-05-01

    Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation. PMID:27037624

  6. Analysis of protein-protein interactions in MCF-7 and MDA-MB-231 cell lines using phthalic acid chemical probes.

    PubMed

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein-protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  7. Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

    PubMed Central

    Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghu S.; Smith, Thomas J.; Shah, Dilip M.

    2013-01-01

    MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA. PMID:24324798

  8. The "Jekyll and Hyde" Actions of Nucleic Acids on the Prion-like Aggregation of Proteins.

    PubMed

    Silva, Jerson L; Cordeiro, Yraima

    2016-07-22

    Protein misfolding results in devastating degenerative diseases and cancer. Among the culprits involved in these illnesses are prions and prion-like proteins, which can propagate by converting normal proteins to the wrong conformation. For spongiform encephalopathies, a real prion can be transmitted among individuals. In other disorders, the bona fide prion characteristics are still under investigation. Besides inducing misfolding of native proteins, prions bind nucleic acids and other polyanions. Here, we discuss how nucleic acid binding might influence protein misfolding for both disease-related and benign, functional prions and why the line between bad and good amyloids might be more subtle than previously thought. PMID:27288413

  9. Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry

    SciTech Connect

    Sagaram, Uma S.; El-Mounadi, Kaoutar; Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghoottama; Smith, Thomas J.; Shah, Dilip

    2013-12-04

    A highly conserved plant defensin MtDef4 potently inhibits the growth of a filamentous fungus Fusarium graminearum. MtDef4 is internalized by cells of F. graminearum. To determine its mechanism of fungal cell entry and antifungal action, NMR solution structure of MtDef4 has been determined. The analysis of its structure has revealed a positively charged patch on the surface of the protein consisting of arginine residues in its γ-core signature, a major determinant of the antifungal activity of MtDef4. Here, we report functional analysis of the RGFRRR motif of the γ-core signature of MtDef4. The replacement of RGFRRR to AAAARR or to RGFRAA not only abolishes fungal cell entry but also results in loss of the antifungal activity of MtDef4. MtDef4 binds strongly to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Mutations of RGFRRR which abolish fungal cell entry of MtDef4 also impair its binding to PA. Our results suggest that RGFRRR motif is a translocation signal for entry of MtDef4 into fungal cells and that this positively charged motif likely mediates interaction of this defensin with PA as part of its antifungal action.

  10. The Three-dimensional Structure of the Extracellular Adhesion Domain of the Sialic Acid-binding Adhesin SabA from Helicobacter pylori

    PubMed Central

    Pang, Siew Siew; Nguyen, Stanley Thai Son; Perry, Andrew J.; Day, Christopher J.; Panjikar, Santosh; Tiralongo, Joe; Whisstock, James C.; Kwok, Terry

    2014-01-01

    The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties LewisB and sialyl-LewisX, respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-LewisX and LewisX but not to LewisA, LewisB, or LewisY. Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-LewisX and LewisX. Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding. PMID:24375407

  11. Characterization of the differences in the cyclopiazonic acid binding mode to mammalian and P. Falciparum Ca2+ pumps: A computational study

    PubMed Central

    Di Marino, Daniele; D'Annessa, Ilda; Coletta, Andrea; Via, Allegra; Tramontano, Anna

    2015-01-01

    Despite the investments in malaria research, an effective vaccine has not yet been developed and the causative parasites are becoming increasingly resistant to most of the available drugs. PfATP6, the sarco/endoplasmic reticulum Ca2+ pump (SERCA) of P. falciparum, has been recently genetically validated as a potential antimalarial target and cyclopiazonic acid (CPA) has been found to be a potent inhibitor of SERCAs in several organisms, including P. falciparum. In position 263, PfATP6 displays a leucine residue, whilst the corresponding position in the mammalian SERCA is occupied by a glutamic acid. The PfATP6 L263E mutation has been studied in relation to the artemisinin inhibitory effect on P. falciparum and recent studies have provided evidence that the parasite with this mutation is more susceptible to CPA. Here, we characterized, for the first time, the interaction of CPA with PfATP6 and its mammalian counterpart to understand similarities and differences in the mode of binding of the inhibitor to the two Ca2+ pumps. We found that, even though CPA does not directly interact with the residue in position 263, the presence of a hydrophobic residue in this position in PfATP6 rather than a negatively charged one, as in the mammalian SERCA, entails a conformational arrangement of the binding pocket which, in turn, determines a relaxation of CPA leading to a different binding mode of the compound. Our findings highlight differences between the plasmodial and human SERCA CPA-binding pockets that may be exploited to design CPA derivatives more selective toward PfATP6. Proteins 2015; 83:564–574. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25581715

  12. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects

    NASA Technical Reports Server (NTRS)

    Ruegger, M.; Dewey, E.; Hobbie, L.; Brown, D.; Bernasconi, P.; Turner, J.; Muday, G.; Estelle, M.

    1997-01-01

    Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

  13. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    SciTech Connect

    Zhang, Yang-De Li, Hao; Liu, Hui; Pan, Yi-Feng

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  14. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

    PubMed Central

    Ruegger, M; Dewey, E; Hobbie, L; Brown, D; Bernasconi, P; Turner, J; Muday, G; Estelle, M

    1997-01-01

    Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development. PMID:9165751

  15. The Crystalline Structure of Escherichia Coli Derived, - and Holo-Rat Cellular Retinol Binding Protein II

    NASA Astrophysics Data System (ADS)

    Winter, Nathan Shoup

    1993-01-01

    Crystal of apo- and holo-rat cellular retinol binding protein II from the recombinant protein isolated from E. coli were grown. X-ray data to about 2A resolution for both crystal forms were collected. The phases for both data sets were determined by the molecular replacement technique using cellular retinol binding protein. The structures were then refined. The electron density from bound retinol was observed in the holo-form. Other than the presence or absence of bound retinol, little difference was noted in the structures of the apo- and holo-protein. The retinol was bound in a interior cavity with the hydroxyl group in the center of the protein, and the ionone ring near the surface. The hydroxyl group of the retinol made a hydrogen bond to glutamine 108, and the amine group of lysine 40 came into Van der Waals contact with the isoprene chain. The structure of cellular retinol binding protein II was then compared with the structures of five other intracellular lipid binding proteins: adipocyte lipid binding protein, cellular retinol binding protein, intestinal fatty acid binding protein, p2 protein from myelin sheaths, and a midgut fatty acid binding protein.

  16. Female Mice are Resistant to Fabp1 Gene Ablation-Induced Alterations in Brain Endocannabinoid Levels.

    PubMed

    Martin, Gregory G; Chung, Sarah; Landrock, Danilo; Landrock, Kerstin K; Dangott, Lawrence J; Peng, Xiaoxue; Kaczocha, Martin; Murphy, Eric J; Kier, Ann B; Schroeder, Friedhelm

    2016-09-01

    Although liver fatty acid binding protein (FABP1, L-FABP) is not detectable in the brain, Fabp1 gene ablation (LKO) markedly increases endocannabinoids (EC) in brains of male mice. Since the brain EC system of females differs significantly from that of males, it was important to determine if LKO differently impacted the brain EC system. LKO did not alter brain levels of arachidonic acid (ARA)-containing EC, i.e. arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), but decreased non-ARA-containing N-acylethanolamides (OEA, PEA) and 2-oleoylglycerol (2-OG) that potentiate the actions of AEA and 2-AG. These changes in brain potentiating EC levels were not associated with: (1) a net decrease in levels of brain membrane proteins associated with fatty acid uptake and EC synthesis; (2) a net increase in brain protein levels of cytosolic EC chaperones and enzymes in EC degradation; or (3) increased brain protein levels of EC receptors (CB1, TRVP1). Instead, the reduced or opposite responsiveness of female brain EC levels to loss of FABP1 (LKO) correlated with intrinsically lower FABP1 level in livers of WT females than males. These data show that female mouse brain endocannabinoid levels were unchanged (AEA, 2-AG) or decreased (OEA, PEA, 2-OG) by complete loss of FABP1 (LKO). PMID:27450559

  17. Structure of YqgQ Protein from Bacillus subtilis, a Conserved Hypothetical Protein

    SciTech Connect

    Lakshminarasimhan, D.; Eswaramoorthy, S; Burley, S; Swaminathan, S

    2010-01-01

    The crystal structure of the hypothetical protein YqgQ from Bacillus subtilis has been determined to 2.1 {angstrom} resolution. The crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 51.85, b = 41.25, c = 55.18 {angstrom}, {beta} = 113.4{sup o}, and contained three protein molecules in the asymmetric unit. The structure was determined by the single-wavelength anomalous dispersion method using selenium-labeled protein and was refined to a final R factor of 24.7% (R{sub free} = 28.0%). The protein molecule mainly comprises a three-helical bundle. Its putative function is inferred to be single-stranded nucleic acid binding based on sequence and structural homology.

  18. The Alba protein family: Structure and function.

    PubMed

    Goyal, Manish; Banerjee, Chinmoy; Nag, Shiladitya; Bandyopadhyay, Uday

    2016-05-01

    Alba family proteins are small, basic, dimeric nucleic acid-binding proteins, which are widely distributed in archaea and a number of eukaryotes. This family of proteins bears the distinct features of regulation through acetylation/deacetylation, hence named as acetylation lowers binding affinity (Alba). Alba family proteins bind DNA cooperatively with no apparent sequence specificity. Besides DNA, Alba proteins also interact with diverse RNA species and associate with ribonucleo-protein complexes. Initially, Alba proteins were recognized as chromosomal proteins and supposed to be involved in the maintenance of chromatin architecture and transcription repression. However, recent studies have shown increasing evidence of functional plasticity among Alba family of proteins that widely range from genome packaging and organization, transcriptional and translational regulation, RNA metabolism, and development and differentiation processes. In recent years, Alba family proteins have attracted growing interest due to their widespread occurrence in large number of organisms. Presence in multiple copies, functional crosstalk, differential binding affinity, and posttranslational modifications are some of the key factors that might regulate the biological functions of Alba family proteins. In this review article, we present an overview of the Alba family proteins, their salient features and emphasize their functional role in different organisms reported so far. PMID:26900088

  19. Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone.

    PubMed

    Fonseca, Cristina T; Pacífico, Lucila G G; Barsante, Michele M; Rassi, Tatiana; Cassali, Geovanni D; Oliveira, Sérgio C

    2006-08-01

    Schistosomiasis is an endemic disease that affects 200 million people worldwide. DNA-based vaccine is a promising strategy to induce protective immunity against schistosomiasis, since both humoral and cellular immune responses are involved in parasite elimination. In this study, we evaluated the ability of Sm14 cDNA alone or in association with a plasmid expressing murine interleukin (IL)-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, Th1 type of immune response with high levels of interferon (IFN)-gamma and low levels of IL-4 in splenocyte culture supernatants and in bronchoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-gamma and tumor necrosis factor-alpha. PMID:16914349

  20. Urinary Markers of Tubular Injury in Early Diabetic Nephropathy

    PubMed Central

    Fiseha, Temesgen; Tamir, Zemenu

    2016-01-01

    Diabetic nephropathy (DN) is a common and serious complication of diabetes associated with adverse outcomes of renal failure, cardiovascular disease, and premature mortality. Early and accurate identification of DN is therefore of critical importance to improve patient outcomes. Albuminuria, a marker of glomerular involvement in early renal damage, cannot always detect early DN. Thus, more sensitive and specific markers in addition to albuminuria are needed to predict the early onset and progression of DN. Tubular injury, as shown by the detection of tubular injury markers in the urine, is a critical component of the early course of DN. These urinary tubular markers may increase in diabetic patients, even before diagnosis of microalbuminuria representing early markers of normoalbuminuric DN. In this review we summarized some new and important urinary markers of tubular injury, such as neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), N-acetyl-beta-glucosaminidase (NAG), alpha-1 microglobulin (A1M), beta 2-microglobulin (B2-M), and retinol binding protein (RBP) associated with early DN. PMID:27293888

  1. Urinary Markers of Tubular Injury in Early Diabetic Nephropathy.

    PubMed

    Fiseha, Temesgen; Tamir, Zemenu

    2016-01-01

    Diabetic nephropathy (DN) is a common and serious complication of diabetes associated with adverse outcomes of renal failure, cardiovascular disease, and premature mortality. Early and accurate identification of DN is therefore of critical importance to improve patient outcomes. Albuminuria, a marker of glomerular involvement in early renal damage, cannot always detect early DN. Thus, more sensitive and specific markers in addition to albuminuria are needed to predict the early onset and progression of DN. Tubular injury, as shown by the detection of tubular injury markers in the urine, is a critical component of the early course of DN. These urinary tubular markers may increase in diabetic patients, even before diagnosis of microalbuminuria representing early markers of normoalbuminuric DN. In this review we summarized some new and important urinary markers of tubular injury, such as neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), N-acetyl-beta-glucosaminidase (NAG), alpha-1 microglobulin (A1M), beta 2-microglobulin (B2-M), and retinol binding protein (RBP) associated with early DN. PMID:27293888

  2. FABP-1 gene ablation impacts brain endocannabinoid system in male mice.

    PubMed

    Martin, Gregory G; Chung, Sarah; Landrock, Danilo; Landrock, Kerstin K; Huang, Huan; Dangott, Lawrence J; Peng, Xiaoxue; Kaczocha, Martin; Seeger, Drew R; Murphy, Eric J; Golovko, Mikhail Y; Kier, Ann B; Schroeder, Friedhelm

    2016-08-01

    Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO

  3. Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency.

    PubMed

    Griffiths, Genevieve S; Miller, Kimberly A; Galileo, Deni S; Martin-DeLeon, Patricia A

    2008-03-01

    Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract. PMID:18299422

  4. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.

    PubMed

    Knott, Gavin J; Bond, Charles S; Fox, Archa H

    2016-05-19

    Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology. PMID:27084935

  5. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner. PMID:27198220

  6. Sodium nitrite potentiates renal oxidative stress and injury in hemoglobin exposed guinea pigs.

    PubMed

    Baek, Jin Hyen; Zhang, Xiaoyuan; Williams, Matthew C; Hicks, Wayne; Buehler, Paul W; D'Agnillo, Felice

    2015-07-01

    Methemoglobin-forming drugs, such as sodium nitrite (NaNO2), may exacerbate oxidative toxicity under certain chronic or acute hemolytic settings. In this study, we evaluated markers of renal oxidative stress and injury in guinea pigs exposed to extracellular hemoglobin (Hb) followed by NaNO2 at doses sufficient to simulate clinically relevant acute methemoglobinemia. NaNO2 induced rapid and extensive oxidation of plasma Hb in this model. This was accompanied by increased renal expression of the oxidative response effectors nuclear factor erythroid 2-derived-factor 2 (Nrf-2) and heme oxygenase-1 (HO-1), elevated non-heme iron deposition, lipid peroxidation, interstitial inflammatory cell activation, increased expression of tubular injury markers kidney injury-1 marker (KIM-1) and liver-fatty acid binding protein (L-FABP), podocyte injury, and cell death. Importantly, these indicators of renal oxidative stress and injury were minimal or absent following infusion of Hb or NaNO2 alone. Together, these results suggest that the exposure to NaNO2 in settings associated with increased extracellular Hb may potentiate acute renal toxicity via processes that are independent of NaNO2 induced erythrocyte methemoglobinemia. PMID:25891524

  7. Urinary biomarkers for early diabetic nephropathy in type 2 diabetic patients.

    PubMed

    Fiseha, Temesgen

    2015-01-01

    Diabetic nephropathy (DN) is a serious complication of diabetes associated with increased risk of mortality, and cardiovascular and renal outcomes. Diagnostic markers to detect DN at early stage are important as early intervention can slow loss of kidney function and improve patient outcomes. Urinary biomarkers may be elevated in diabetic patients even before the appearance of microalbuminuria, and can be used as useful marker for detecting nephropathy in patients with normoalbuminuria (early DN). We reviewed some new and important urinary biomarkers, such as: Neutrophil gelatinase associated lipocalin (NGAL), N-acetyl-beta-glucosaminidase (NAG), Cystatin C, alpha 1-microglobulin, immunoglobulin G or M, type IV collagen, nephrin, angiotensinogen and liver-type fatty acid-binding protein (L-FABP) associated with early DN in type 2 diabetic patients. Our search identified a total of 42 studies that have been published to date. Urinary levels of these biomarkers were elevated in type 2 diabetic patients compared with non-diabetic controls, including in patients who had no signs indicating nephropathy (without microalbuminuria), and showed positive correlation with albuminuria. Despite the promise of these new urinary biomarkers, further large, multicenter prospective studies are still needed to confirm their clinical utility as a screening tool for early type 2 DN in every day practice. PMID:26146561

  8. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels

    PubMed Central

    Zhu, Yi-bing; Huang, Rong-dong; Lu, Qing-Qing; Lin, Xu

    2015-01-01

    Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = —0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans. PMID:26439934

  9. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

    PubMed

    Peng, Xian-E; Wu, Yun-Li; Zhu, Yi-Bing; Huang, Rong-Dong; Lu, Qing-Qing; Lin, Xu

    2015-01-01

    Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans. PMID:26439934

  10. Folding dynamics of a family of beta-sheet proteins

    NASA Astrophysics Data System (ADS)

    Rousseau, Denis

    2008-03-01

    Fatty acid binding proteins (FABP) consist of ten anti-parallel beta strands and two small alpha helices. The beta strands are arranged into two nearly orthogonal five-strand beta sheets that surround the interior cavity, which binds unsaturated long-chain fatty acids. In the brain isoform (BFABP), these are very important for the development of the central nervous system and neuron differentiation. Furthermore, BFABP is implicated in the pathogenesis of a variety of human diseases including cancer and neuronal degenerative disorders. In this work, site-directed spin labeling combined with EPR techniques have been used to study the folding mechanism of BFABP. In the first series of studies, we labeled the two Cys residues at position 5 and 80 in the wild type protein with an EPR spin marker; in addition, two singly labeled mutants at positions 5 and 80 in the C80A and C5A mutants, respectively, were also produced and used as controls. The changes in the distances between the two residues were examined by a pulsed EPR method, DEER (Double Electron Electron Resonance), as a function of guanidinium hydrochloride concentration. The results were compared with those from CW EPR, circular dichroism and fluorescence measurements, which provide the information regarding sidechain mobility, secondary structure and tertiary structure, respectively. The results will be discussed in the context of the folding mechanism of the family of fatty acid binding proteins.

  11. ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We are reviewing here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the double-stranded RNA and Z-DNA binding domains. PMID:21728134

  12. Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch

    PubMed Central

    Berbasova, Tetyana; Santos, Elizabeth M.; Nosrati, Meisam; Vasileiou, Chrysoula; Geiger, James H.; Borhan, Babak

    2016-01-01

    Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa, leading to protonation of the imine and subsequent “turn-on” of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa, leading to loss of color because the imine is not protonated. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance. PMID:26684483

  13. Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

    PubMed Central

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  14. Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate†

    PubMed Central

    Zornetzer, Gregory A.; Fox, Brian G.; Markley, John L.

    2008-01-01

    Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo- and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0- and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0- and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners. PMID:16618110

  15. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    PubMed

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity. PMID:26853627

  16. First Post-Operative Urinary Kidney Injury Biomarkers and Association with the Duration of AKI in the TRIBE-AKI Cohort

    PubMed Central

    Coca, Steven G.; Nadkarni, Girish N.; Garg, Amit X.; Koyner, Jay; Thiessen-Philbrook, Heather; McArthur, Eric; Shlipak, Michael G.; Parikh, Chirag R.

    2016-01-01

    Background We previously demonstrated that assessment of the duration of AKI, in addition to magnitude of rise in creatinine alone, adds prognostic information for long-term survival. We evaluated whether post-operative kidney injury biomarkers in urine collected immediately after cardiac surgery associate with duration of serum creatinine elevation. Methods We studied 1199 adults undergoing cardiac surgery in a prospective cohort study (TRIBE-AKI) and examined the association between the levels of five urinary biomarkers individually at 0–6 hours after surgery: interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver fatty acid binding protein (L-FABP) and albumin with duration of serum creatinine-based AKIN criteria for AKI (0 (no AKI), 1–2, 3–6, ≥7 days). Results Overall, 407 (34%) patients had at least stage 1 AKI, of whom 251 (61.7%) had duration of 1–2 days, 118 (28.9%) had duration 3–6 days, and 38 (9.3%) had duration of ≥7 days. Higher concentrations of all biomarkers (per log increase) were independently associated with a greater odds of a longer duration of AKI; odds ratios and 95% confidence intervals using ordinal logistic regression were the following: IL-18: 1.22, 1.13–1.32; KIM-1: 1.36, 1.21–1.52; albumin 1.20, 1.09–1.32; L-FABP 1.11, 1.04–1.19; NGAL 1.06, 1.00–1.14). AKI duration of 7 days or longer was associated with a 5-fold adjusted risk of mortality at 3 years. Conclusions There was an independent dose-response association between urinary levels of injury biomarkers immediately after cardiac surgery and longer duration of AKI. Duration of AKI was also associated with long term mortality. Future studies should explore the potential utility of these urinary kidney injury biomarkers to enrich enrollment of patients at risk for longer duration of AKI into trials of interventions to prevent or treat post-operative AKI. PMID:27537050

  17. Comparative analysis and molecular characterization of genomic sequences and proteins of FABP4 and FABP5 from the giant panda (Ailuropoda melanoleuca).

    PubMed

    Song, B; Hou, Y L; Ding, X; Wang, T; Wang, F; Zhong, J C; Xu, T; Zhong, J; Hou, W R; Shuai, S R

    2014-01-01

    Fatty acid binding proteins (FABPs) are a family of small, highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. In this study, cDNA and genomic sequences of FABP4 and FABP5 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using reverse transcription polymerase chain reaction (RT-PCR) technology and touchdown-PCR. The cDNAs of FABP4 and FABP5 cloned from the giant panda were 400 and 413 bp in length, containing an open reading frame of 399 and 408 bp, encoding 132 and 135 amino acids, respectively. The genomic sequences of FABP4 and FABP5 were 3976 and 3962 bp, respectively, which each contained four exons and three introns. Sequence alignment indicated a high degree of homology with reported FABP sequences of other mammals at both the amino acid and DNA levels. Topology prediction revealed seven protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, two N-myristoylation sites, and one cytosolic fatty acid-binding protein signature in the FABP4 protein, and three N-glycosylation sites, three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one N-myristoylation site, one amidation site, and one cytosolic fatty acid-binding protein signature in the FABP5 protein. The FABP4 and FABP5 genes were overexpressed in Escherichia coli BL21 and they produced the expected 16.8- and 17.0-kDa polypeptides. The results obtained in this study provide information for further in-depth research of this system, which has great value of both theoretical and practical significance. PMID:24634121

  18. Soluble expression of proteins correlates with a lack of positively-charged surface

    NASA Astrophysics Data System (ADS)

    Chan, Pedro; Curtis, Robin A.; Warwicker, Jim

    2013-11-01

    Prediction of protein solubility is gaining importance with the growing use of protein molecules as therapeutics, and ongoing requirements for high level expression. We have investigated protein surface features that correlate with insolubility. Non-polar surface patches associate to some degree with insolubility, but this is far exceeded by the association with positively-charged patches. Negatively-charged patches do not separate insoluble/soluble subsets. The separation of soluble and insoluble subsets by positive charge clustering (area under the curve for a ROC plot is 0.85) has a striking parallel with the separation that delineates nucleic acid-binding proteins, although most of the insoluble dataset are not known to bind nucleic acid. Additionally, these basic patches are enriched for arginine, relative to lysine. The results are discussed in the context of expression systems and downstream processing, contributing to a view of protein solubility in which the molecular interactions of charged groups are far from equivalent.

  19. Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch.

    PubMed

    Berbasova, Tetyana; Santos, Elizabeth M; Nosrati, Meisam; Vasileiou, Chrysoula; Geiger, James H; Borhan, Babak

    2016-03-01

    Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa , leading to protonation of the imine and subsequent "turn-on" of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa , leading to loss of color because the imine is not protonated. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance. PMID:26684483

  20. Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

    PubMed Central

    Laulumaa, Saara; Nieminen, Tuomo; Lehtimäki, Mari; Aggarwal, Shweta; Simons, Mikael; Koza, Michael M.; Vattulainen, Ilpo; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations. PMID:26068118

  1. The Nucleocapsid Protein of Human Coronavirus NL63

    PubMed Central

    Zuwała, Kaja; Golda, Anna; Kabala, Wojciech; Burmistrz, Michał; Zdzalik, Michal; Nowak, Paulina; Kedracka-Krok, Sylwia; Zarebski, Mirosław; Dobrucki, Jerzy; Florek, Dominik; Zeglen, Sławomir; Wojarski, Jacek; Potempa, Jan; Dubin, Grzegorz; Pyrc, Krzysztof

    2015-01-01

    Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV. PMID:25700263

  2. Crystal structure of a novel Sm-like protein of putative cyanophage origin at 2.60 Å resolution

    SciTech Connect

    Das, Debanu; Kozbial, Piotr; Axelrod, Herbert L.; Miller, Mitchell D.; McMullan, Daniel; Krishna, S. Sri; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Burra, Prasad; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Ernst, Dustin; Farr, Carol; Feuerhelm, Julie; Grzechnik, Anna; Grzechnik, Slawomir K.; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Murphy, Kevin D.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Sudek, Sebastian; Tien, Henry; Trame, Christine; Trout, Christina V.; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-08-28

    ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome-specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA-binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid-binding capabilities and suggests a role in RNA and/or DNA processing.

  3. Crystal Structure and RNA Binding of the Tex Protein from Pseudomonas aeruginosa

    SciTech Connect

    Johnson,S.; Close, D.; Robinson, H.; Vallet-Gely, I.; Dove, S.; Hill, C.

    2008-01-01

    Tex is a highly conserved bacterial protein that likely functions in a variety of transcriptional processes. Here, we describe two crystal structures of the 86-kDa Tex protein from Pseudomonas aeruginosa at 2.3 and 2.5 Angstroms resolution, respectively. These structures reveal a relatively flat and elongated protein, with several potential nucleic acid binding motifs clustered at one end, including an S1 domain near the C-terminus that displays considerable structural flexibility. Tex binds nucleic acids, with a preference for single-stranded RNA, and the Tex S1 domain is required for this binding activity. Point mutants further demonstrate that the primary nucleic acid binding site corresponds to a surface of the S1 domain. Sequence alignment and modeling indicate that the eukaryotic Spt6 transcription factor adopts a similar core structure. Structural analysis further suggests that the RNA polymerase and nucleosome interacting regions of Spt6 flank opposite sides of the Tex-like scaffold. Therefore, the Tex structure may represent a conserved scaffold that binds single-stranded RNA to regulate transcription in both eukaryotic and prokaryotic organisms.

  4. Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects.

    PubMed

    Ceccon, Alberto; Busato, Mirko; Pérez Santero, Silvia; D'Onofrio, Mariapina; Musiani, Francesco; Giorgetti, Alejandro; Assfalg, Michael

    2015-12-01

    Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP-lysozyme complex was obtained by data-driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment. PMID:26449487

  5. Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

    PubMed Central

    Michalska, Karolina; Chang, Changsoo; Mack, Jamey C.; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R.

    2013-01-01

    In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein–ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure–function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino-acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequencebased methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria. PMID:22925578

  6. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    SciTech Connect

    Zanzoni, Serena; D'Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  7. Circulating TNF receptors 1 and 2 are associated with the severity of renal interstitial fibrosis in IgA nephropathy.

    PubMed

    Sonoda, Yuji; Gohda, Tomohito; Suzuki, Yusuke; Omote, Keisuke; Ishizaka, Masanori; Matsuoka, Joe; Tomino, Yasuhiko

    2015-01-01

    The current study aimed to examine whether the levels of TNF receptors 1 and 2 (TNFR1 and TNFR2) in serum and urine were associated with other markers of kidney injury and renal histological findings, including TNFR expression, in IgA nephropathy (IgAN). The levels of the parameters of interest were measured by immunoassay in 106 biopsy-proven IgAN patients using samples obtained immediately before renal biopsy and in 34 healthy subjects. Renal histological findings were evaluated using immunohistochemistry. The levels of serum TNFRs were higher in IgAN patients than in healthy subjects. The levels of both TNFRs in serum or urine were strongly correlated with each other (r > 0.9). Serum TNFR levels were positively correlated with the urinary protein to creatinine ratio (UPCR) and four markers of tubular damage of interest (N-acetyl-β-D-glucosaminidase [NAG], β2 microglobulin [β2m], liver-type fatty acid-binding protein [L-FABP], and kidney injury molecule-1 [KIM-1]) and negatively correlated with estimated glomerular filtration rate (eGFR). Patients in the highest tertile of serum TNFR levels showed more severe renal interstitial fibrosis than did those in the lowest or second tertiles. The tubulointerstitial TNFR2-, but not TNFR1-, positive area was significantly correlated with the serum levels of TNFRs and eGFR. Stepwise multiple regression analysis revealed that elevated serum TNFR1 or TNFR2 levels were a significant determinant of renal interstitial fibrosis after adjusting for eGFR, UPCR, and other markers of tubular damage. In conclusion, elevated serum TNFR levels were significantly associated with the severity of renal interstitial fibrosis in IgAN patients. However, the source of TNFRs in serum and urine remains unclear. PMID:25860248

  8. [Acute kidney injury in children].

    PubMed

    Amira-Peco-Antić; Paripović, Dusan

    2014-01-01

    Acute kidney injury (AKI) is a clinical condition considered to be the consequence of a sudden decrease (> 25%) or discontinuation of renal function. The term AKI is used instead of the previous term acute renal failure, because it has been demonstrated that even minor renal lesions may cause far-reaching consequences on human health. Contemporary classifications of AKI (RIFLE and AKIN) are based on the change of serum creatinine and urinary output. In the developed countries, AKI is most often caused by renal ischemia, nephrotoxins and sepsis, rather than a (primary) diffuse renal disease, such as glomerulonephritis, interstitial nephritis, renovascular disorder and thrombotic microangiopathy. The main risk factors for hospital AKI are mechanical ventilation, use of vasoactive drugs, stem cell transplantation and diuretic-resistant hypervolemia. Prerenal and parenchymal AKI (previously known as acute tubular necrosis) jointly account for 2/3 of all AKI causes. Diuresis and serum creatinine concentration are not early diagnostic markers of AKI. Potential early biomarkers of AKI are neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, kidney injury molecule-1 (KIM-1), interleukins 6, 8 and 18, and liver-type fatty acid-binding protein (L-FABP). Early detection of kidney impairment, before the increase of serum creatinine, is important for timely initiated therapy and recovery. The goal of AKI treatment is to normalize the fluid and electrolyte status, as well as the correction of acidosis and blood pressure. Since a severe fluid overload resistant to diuretics and inotropic agents is associated with a poor outcome, the initiation of dialysis should not be delayed. The mortality rate of AKI is highest in critically ill children with multiple organ failure and hemodynamically unstable patients. PMID:25033598

  9. Comparison of Plasma and Urine Biomarker Performance in Acute Kidney Injury

    PubMed Central

    Schley, Gunnar; Köberle, Carmen; Manuilova, Ekaterina; Rutz, Sandra; Forster, Christian; Weyand, Michael; Formentini, Ivan; Kientsch-Engel, Rosemarie; Eckardt, Kai-Uwe; Willam, Carsten

    2015-01-01

    Background New renal biomarkers measured in urine promise to increase specificity for risk stratification and early diagnosis of acute kidney injury (AKI) but concomitantly may be altered by urine concentration effects and chronic renal insufficiency. This study therefore directly compared the performance of AKI biomarkers in urine and plasma. Methods This single-center, prospective cohort study included 110 unselected adults undergoing cardiac surgery with cardiopulmonary bypass between 2009 and 2010. Plasma and/or urine concentrations of creatinine, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), liver fatty acid-binding protein (L-FABP), kidney injury molecule 1 (KIM1), and albumin as well as 15 additional biomarkers in plasma and urine were measured during the perioperative period. The primary outcome was AKI defined by AKIN serum creatinine criteria within 72 hours after surgery. Results Biomarkers in plasma showed markedly better discriminative performance for preoperative risk stratification and early postoperative (within 24h after surgery) detection of AKI than urine biomarkers. Discriminative power of urine biomarkers improved when concentrations were normalized to urinary creatinine, but urine biomarkers had still lower AUC values than plasma biomarkers. Best diagnostic performance 4h after surgery had plasma NGAL (AUC 0.83), cystatin C (0.76), MIG (0.74), and L-FAPB (0.73). Combinations of multiple biomarkers did not improve their diagnostic power. Preoperative clinical scoring systems (EuroSCORE and Cleveland Clinic Foundation Score) predicted the risk for AKI (AUC 0.76 and 0.71) and were not inferior to biomarkers. Preexisting chronic kidney disease limited the diagnostic performance of both plasma and urine biomarkers. Conclusions In our cohort plasma biomarkers had higher discriminative power for risk stratification and early diagnosis of AKI than urine biomarkers. For preoperative risk stratification of AKI clinical models showed

  10. Utility of anion and cation combinations for phasing of protein structures.

    PubMed

    Sharma, Ashwani; Yogavel, Manickam; Sharma, Amit

    2012-09-01

    We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination. PMID:22562242

  11. A delicate balance between functionally required flexibility and aggregation risk in a β-rich protein

    PubMed Central

    Ferrolino, Mylene C.; Zhuravleva, Anastasia; Budyak, Ivan L.; Krishnan, Beena; Gierasch, Lila M.

    2014-01-01

    Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail: cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein’s natural tendency to fold, ‘breathe’, and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability, and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk. PMID:24236614

  12. Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

    SciTech Connect

    Fan Xiuzhen; Wang Jianling; Soman, Kizhake V.; Ansari, G.A.S.; Khan, M. Firoze

    2011-08-15

    Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6 kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity. - Highlights: > Proteomic approaches are used to identify nitrated proteins in the spleen. > Twenty five nitrated proteins were found only in the spleen of aniline-treated rats. > Aniline exposure led to increased iNOS mRNA and protein expression in

  13. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  14. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

    Frank, Martin; Hähnlein-Schick, Irmgard; Ekström, Jens-Ola; Arnberg, Niklas; Stehle, Thilo

    2014-01-01

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC. PMID:25329320

  15. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  16. Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

    PubMed

    Talukder, Poulami; Dedkova, Larisa M; Ellington, Andrew D; Yakovchuk, Petro; Lim, Jaebum; Anslyn, Eric V; Hecht, Sidney M

    2016-09-15

    Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail. PMID:27452282

  17. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold

    PubMed Central

    Knott, Gavin J.; Bond, Charles S.; Fox, Archa H.

    2016-01-01

    Nuclear proteins are often given a concise title that captures their function, such as ‘transcription factor,’ ‘polymerase’ or ‘nuclear-receptor.’ However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein–protein and protein–nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology. PMID:27084935

  18. RNA-LIM: a novel procedure for analyzing protein/single-stranded RNA propensity data with concomitant estimation of interface structure.

    PubMed

    Hall, Damien; Li, Songling; Yamashita, Kazuo; Azuma, Ryuzo; Carver, John A; Standley, Daron M

    2015-03-01

    RNA-LIM is a procedure that can analyze various pseudo-potentials describing the affinity between single-stranded RNA (ssRNA) ribonucleotides and surface amino acids to produce a coarse-grained estimate of the structure of the ssRNA at the protein interface. The search algorithm works by evolving an ssRNA chain, of known sequence, as a series of walks between fixed sites on a protein surface. Optimal routes are found by application of a set of minimal "limiting" restraints derived jointly from (i) selective sampling of the ribonucleotide amino acid affinity pseudo-potential data, (ii) limited surface path exploration by prior determination of surface arc lengths, and (iii) RNA structural specification obtained from a statistical potential gathered from a library of experimentally determined ssRNA structures. We describe the general approach using a NAST (Nucleic Acid Simulation Tool)-like approximation of the ssRNA chain and a generalized pseudo-potential reflecting the location of nucleic acid binding residues. Minimum and maximum performance indicators of the methodology are established using both synthetic data, for which the pseudo-potential defining nucleic acid binding affinity is systematically degraded, and a representative real case, where the RNA binding sites are predicted by the amplified antisense RNA (aaRNA) method. Some potential uses and extensions of the routine are discussed. RNA-LIM analysis programs along with detailed instructions for their use are available on request from the authors. PMID:25479604

  19. Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

    SciTech Connect

    Robach, Jessica G.; Lamb, Robert A.

    2010-10-25

    The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

  20. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  1. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  2. Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity[W

    PubMed Central

    Wang, Ching-I A.; Gunčar, Gregor; Forwood, Jade K.; Teh, Trazel; Catanzariti, Ann-Maree; Lawrence, Gregory J.; Loughlin, Fionna E.; Mackay, Joel P.; Schirra, Horst Joachim; Anderson, Peter A.; Ellis, Jeffrey G.; Dodds, Peter N.; Kobe, Boštjan

    2007-01-01

    The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities. PMID:17873095

  3. Total protein

    MedlinePlus

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  4. Storage Proteins

    PubMed Central

    Fujiwara, Toru; Nambara, Eiji; Yamagishi, Kazutoshi; Goto, Derek B.; Naito, Satoshi

    2002-01-01

    Plants accumulate storage substances such as starch, lipids and proteins in certain phases of development. Storage proteins accumulate in both vegetative and reproductive tissues and serve as a reservoir to be used in later stages of plant development. The accumulation of storage protein is thus beneficial for the survival of plants. Storage proteins are also an important source of dietary plant proteins. Here, we summarize the genome organization and regulation of gene expression of storage protein genes in Arabidopsis. PMID:22303197

  5. The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs

    PubMed Central

    Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

    2014-01-01

    The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs—the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3–RRM4 block is the main platform mediating the stable association with the H12–H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP–RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. PMID:24748666

  6. Binding of a Streptococcus mutans cationic protein to kidney in vitro.

    PubMed Central

    Choi, S H; Stinson, M W

    1991-01-01

    An 8-kDa protein, with binding activity for heparin and heparan sulfate of basal laminae of animal tissues, was isolated from Streptococcus mutans MT703 and purified to homogeneity. Binding of radioiodinated 8-kDa protein to rabbit kidney tissue in vitro showed a high degree of specificity, as indicated by saturation kinetics, time dependence, and competitive inhibition by unlabeled protein. Binding activity for kidney tissue was competitively inhibited by selected glycosaminoglycans and polyanions in the following order: heparin greater than dextran sulfate greater than heparan sulfate greater than chondroitin sulfate greater than lipoteichoic acid greater than keratan sulfate greater than hyaluronic acid. Binding of the streptococcal protein to rabbit kidney tissue was also strongly inhibited by protamine sulfate, polylysine, and a random copolymer of lysine and alanine. Among the monosaccharides tested at 50 mM, glucosamine 2,3- or 2,6-disulfate, glucuronic acid, glucose 6-phosphate, and glucose 6-sulfate inhibited 50% or more of the binding activity, whereas N-acetylglucosamine 3-sulfate, glucosamine 6-sulfate, N-acetyl-glucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and a selection of neutral sugars were not inhibitory. The heparin-binding protein was detected on the cell wall of S. mutans and in the culture medium following growth. Several other species of streptococci produce an immunologically related protein of similar size. Images PMID:1987071

  7. Crystal structure of CspA, the major cold shock protein of Escherichia coli.

    PubMed

    Schindelin, H; Jiang, W; Inouye, M; Heinemann, U

    1994-05-24

    The major cold shock protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major cold shock protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common cold shock domain. PMID:8197194

  8. Visualizing Protein Movement on DNA at the Single-molecule Level using DNA Curtains

    PubMed Central

    Silverstein, Timothy D.; Gibb, Bryan; Greene, Eric C.

    2014-01-01

    A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively. PMID:24598576

  9. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    PubMed

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off. PMID:26260520

  10. Change of uterine histroph proteins during follicular and luteal phase in pigs.

    PubMed

    Lee, Sang-Hee; Song, Eun-Ji; Hwangbo, Yong; Lee, Seunghyung; Park, Choon-Keun

    2016-05-01

    The aim of this study was to examine protein expression patterns of uterine histroph (UH) during the follicular phase (FP) and luteal phase (LP) in pigs. Forty-nine common proteins were identified from FP and LP samples; five were significantly down-regulated (>1.5-fold), while 15 were significantly up-regulated (>1.5-fold) in LPUH compared with FPUH (P<0.05). The 20 differentially-expressed proteins are involved in cell proliferation, cell responses, translation, transport, and metabolism and their molecular functions include nucleic acid binding, oxygen activity, enzymatic activity, growth activity, iron binding, and redox binding. Protein expression of vascular endothelial growth factor D (VEGFD), coatomer subunit gamma-2 (G2COP), collagen alpha 4 chain (COL4), cysteine rich protein 2 (CRP2), myoglobin (MYG), and galactoside 3-L-fucosyltransferase 4 (FUT4) was analyzed by Western blotting. These proteins were significantly higher in LPUH compared to FPUH (P<0.05). These data expand our understanding of changes in the intrauterine environment during the pre-implantation period in pigs. PMID:26968245

  11. Extracellular and circulating redox- and metalloregulated eRNA and eRNP: copper ion-structured RNA cytokines (angiotropin ribokines) and bioaptamer targets imparting RNA chaperone and novel biofunctions to S100-EF-hand and disease-associated proteins.

    PubMed

    Wissler, Josef H

    2004-06-01

    Bioassays for cellular differentiation and tissue morphogenesis were used to design methods for isolation of bioactive redox- and metalloregulated nucleic acids and copper ion complexes with proteins from extracellular, circulating, wound, and supernatant fluids of cultured cells. In extracellular biospheres, diversities of nucleic acids were found to be secreted by cells upon activation. They may reflect nucleic acid biolibraries with molecular imprints of cellular history. After removal of protein components, eRNA prototypes exuded by activated cells were sequenced. They are small, endogenous, highly modified and edited