Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

Solution properties of targacanthin (water-soluble part of gum tragacanth exudate from Astragalus gossypinus)

Solution properties of tragacanthin (the water-soluble part of gum tragacanth) were studied by gel permeation chromatography (GPC) combined with multi-angle light scattering and viscometry at 25 degrees C. Photon correlation spectroscopy was used to determine the hydrodynamic radius. Ultrasonic degradation was applied to obtain biopolymer fractions of different molecular weights. The dependence of intrinsic viscosity [eta] and radius of gyration (s2)z(1/2) on weight average molecular mass M(w) for this biopolymer were found to be [eta] = 9.077 x 10(-5) M(w)(0.87) (dL g(-1)) and (s2)z(1/2) in the range of M(w) from 1.8 x 10(5) to 1.6 x 10(6). The conformational parameters of tragacanthin were calculated to be 1111 nm for molar mass per unit contour length (M(L)), 26 nm for persistence length (q) and 1.87 ratio of R(g)/R(h). It was found that the Smidsr?d parameter B, the empirical stiffness parameter was 0.013, which is lower than that of several polysaccharides indicating the stiff backbone for tragacanthin. The rheological behavior of aqueous solutions of gum tragacanth and its insoluble and soluble fractions (bassorin and tragacanthin, respectively) were studied. For concentrations equal to 1%, at 25 degrees C and in the absence of salt, bassorin solution showed the highest viscosity and shear thinning behaviour. Power law and Williamson models were used to describe the rheological behaviour of bassorin and tragacanthin, respectively. Oscillatory shear experiments showed a gel like structure for the bassorin but for tragacanthin the oscillatory data were as would be expected for semi-dilute to concentrated solution of entangled, random coil polymers. NaCl changed the steady and oscillatory rheological properties of both fractions and in this way the final viscosity of bassorin was even less than tragacanthin. The calculated activation energy for bassorin and tragacanthin indicated a more rapid decrease in viscosity with temperature for tragacanthin. The plot of eta(sp,0) versus C[eta] revealed that the transition from dilute to semi-dilute regime occurs at C*[eta] = 2.82 for tragacanthin.     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Genetic analysis of an F2 intercross between two strains of Japanese quail provided evidence for quantitative trait loci affecting carcass composition and internal organs

The purpose of this study was to identify genomic regions, quantitative trait loci (QTL), affecting carcass traits on chromosome 1 in an F2 population of Japanese quail. For this purpose, two white and wild strains of Japanese quail (16 birds) were crossed reciprocally and F1 generation (34 birds) was created. The F2 generation was produced by intercrossing of the F1 birds. Phenotypic data including carcass weight, internal organs and carcass parts were collected on F2 animals (422 birds). The total mapping population (472 birds) was genotyped for 8 microsatellite markers on chromosome 1. QTL analysis was performed with interval mapping method applying the line-cross model. Significant QTL were identified for breast weight at 0 (P < 0.01), 172 (P < 0.05) and 206 (P < 0.01), carcass weight at 91 (P < 0.05), carcass fatness at 0 (P < 0.01), pre-stomach weight at 206 (P < 0.01) and uropygial weight gland at 197 (P < 0.01) cM on chromosome 1. There was also evidence for imprinted QTL affecting breast weight (P < 0.01) on chromosome 1. The proportion of the F2 phenotypic variation explained by the significant additive, dominance and imprinted QTL effects ranged from 1.0 to 7.3 %, 1.2 to 3.3 % and 1.4 to 2.2 %, respectively.     

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.     

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.