Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

Epizootic cutaneous papillomatosis, cortisol and male ornamentation during and after breeding in the roach Rutilus rutilus

The prevalence of epidermal papillomatosis in roach is known to peak during the spawning period and to be higher in males than in females. The high occurrence of papillomatosis in polluted waters suggests that stress may contribute to the outbreak of the disease. However, little is known about breeding-induced stress in fish and its relationship with diseases. In this study, plasma cortisol concentration, hematocrit and the relative size of the spleen were determined in healthy and diseased male and female roach Rutilus rutilus during and shortly after spawning in a wild population. In addition, the sexual ornamentation (breeding tubercles on the lateral sides and on the frontal) of male roach during spawning was examined. Plasma cortisol concentration was higher during than after the spawning period, and higher in males than in females during spawning, indicating a spawning-induced stress and higher spawning stress among males. There was no correlation between cortisol concentration and the intensity of papillomatosis (number of scales under papilloma tumors) among the diseased fish. However, the significant interaction sex x disease status revealed by ANCOVA suggested that diseased males could be more prone to increased cortisol levels than diseased females or healthy males. Hematocrit values (ratio of the volume of red blood cells to total volume of blood) but not condition factor were lowered in papilloma-diseased fish after spawning. The relative size of the spleen was greater in males than in females. The number of frontal breeding tubercles correlated negatively with the intensity of papillomatosis. Experimental studies are needed to investigate the association of papillomatosis with stress and cortisol.     

High resolution crystal structures of unliganded and liganded human liver ACBP reveal a new mode of binding for the acyl-CoA ligand

The acyl-CoA binding protein (ACBP) is essential for the fatty acid metabolism, membrane structure, membrane fusion, and ceramide synthesis. Here high resolution crystal structures of human cytosolic liver ACBP, unliganded and liganded with a physiological ligand, myristoyl-CoA are described. The binding of the acyl-CoA molecule induces only few structural differences near the binding pocket. The crystal form of the liganded ACBP, which has two ACBP molecules in the asymmetric unit, shows that in human ACBP the same acyl-CoA binding pocket is present as previously described for the bovine and Plasmodium falciparum ACBP and the mode of binding of the 3'-phosphate-AMP moiety is conserved. Unexpectedly, in one of the acyl-CoA binding pockets the acyl moiety is bound in a reversed mode as compared with the bovine and P. falciparum structures. In this binding mode, the myristoyl-CoA molecule is fully ordered and bound across the two ACBP molecules of the crystallographic asymmetric unit: the 3'-phosphate-AMP moiety is bound in the binding pocket of one ACBP molecule and the acyl chain is bound in the pocket of the other ACBP molecule. The remaining binding pocket cavities of these two ACBP molecules are filled by other ligand fragments. This novel binding mode shows that the acyl moiety can flip out of its classical binding pocket and bind elsewhere, suggesting a mechanism for the acyl-CoA transfer between ACBP and the active site of a target enzyme. This mechanism is of possible relevance for the in vivo function of ACBP.     

Effects of Ethanolic Extract of Cynara cardunculus (Artichoke) Leaves on Neuroinflammatory and Neurochemical Parameters in a Diet-Induced Mice Obesity Model

Neurochemical Research - This study aimed to evaluate the effect of Cynara cardunculus leaf ethanol extract on inflammatory and oxidative stress parameters in the hypothalamus, prefrontal cortex,...     

Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.     

C-reactive protein binds to the 3beta-OH group of cholesterol in LDL particles

C-reactive protein (CRP) has been suggested to contribute to the development of atherosclerosis. We previously found binding of CRP to cholesterol in modified low density lipoprotein (LDL) particles. Here, we characterize the interaction between CRP and cholesterol in more detail. When lipids of native LDL were separated by thin-layer chromatography, CRP bound only to cholesterol. When various cholesterol analogues were compared for their ability to bind CRP, we found that any modification of the 3beta-OH group blocked binding of CRP to cholesterol. Similarly, enrichment of LDL with cholesterol but not with its analogues triggered the binding of CRP to LDL. Finally, with the aid of anti-CRP monoclonal antibodies and by molecular modeling, we obtained evidence for involvement of the phosphorylcholine-binding site of CRP in cholesterol binding. Thus, CRP can bind to cholesterol, and the interaction is mediated by the phosphorylcholine-binding site of CRP and the 3beta-hydroxyl group of cholesterol.     

Type 2 diabetes as a lipid disorder

Diabetic dyslipidemia is a cluster of plasma lipid and lipoprotein abnormalities that are metabolically interrelated. The recognition that the elevation of large VLDL 1 particles initiates a sequence of events that leads to the formation of small dense LDL and HDL species has focused the assembly of VLDL particles on the spotlight as a potential culprit of dyslipidemia. Notably dyslipidemia is associated with insulin resistance, visceral obesity and liver fat content. Insulin resistance is associated with excessive flux of substrates for VLDL assembly to the liver as well as the upregulation of the machinery generating large VLDL particles in excess. The regulation of different molecular steps in this cascade of events are complex and so far poorly understood. The disordered crosstalk between adipose tissue and the liver results in an imbalance of the machinery that orchestrates the regulation of VLDL production. A number of studies indicates that adipocytokines in particular adiponectin may be a seminal player in the regulation of fat metabolism in the liver. Future discoveries hopefully will delineate the regulatory steps to allow more targeted treatment of diabetic dyslipidemia.