Hsp70/Hsc70 regulates the effect phosphorylation has on stabilizing ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an inherited neurodegenerative disorder. The mutation causing SCA1 is an expansion in the polyglutamine tract of the ATXN1 protein. Previous work demonstrated that phosphorylation of mutant ATXN1 at serine 776 (S776), a putative Akt phosphorylation site, is critical for pathogenesis. To examine this pathway further, we utilized a cell-transfection system that allowed the targeting of Akt to either the cytoplasm or the nucleus. In contrast to HeLa cells, we found that Akt targeted to the cytoplasm increased the degradation of ATXN1 in Chinese hamster ovary cells. However, Akt targeted to the cytoplasm failed to destabilize ATXN1 if Hsp70/Hsc70 was present. Thus, Hsp70/Hsc70 can regulate ATXN1 levels in concert with phosphorylation of ATXN1 at S776.     

Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy

Very-long-chain acyl–coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid β-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in β-oxidation–deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 μM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.     

Contrasting responses of phytoplankton and benthic algae to recent nutrient enrichment in Arctic tundra ponds

相似文献   

Laminin-rich blood vessels display activated growth factor signaling and act as the proliferation centers in Dupuytren’s contracture

IntroductionDupuytren’s contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches.MethodsWe studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR).ResultsWe found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types.ConclusionsBased on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0661-y) contains supplementary material, which is available to authorized users.     

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.     

CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.     

Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed.     

Deep-breath frequency in bronchoconstricted monkeys (Macaca fascicularis).

Deep-breath frequency has been shown to increase in spontaneously obstructed asthmatic subjects. Furthermore, deep breaths are known to be regulated by lung rapidly adapting receptors, yet the mechanism by which these receptors are stimulated is unclear. This study tested the hypothesis that deep-breath frequency increases during experimentally induced bronchoconstriction, and the magnitude of the increased deep-breath frequency is dependent on the method by which bronchoconstriction is induced. Nine cynomolgus monkeys (Macaca fascicularis) were challenged with methacholine (MCh), Ascaris suum (AS), histamine, or an external mechanical resistance. Baseline (BL) and challenge deep-breath frequency were calculated from the number of deep breaths per trial period. Airway resistance (Raw) and tissue compliance (Cti), as well as tidal volume, respiratory rate, and minute ventilation, were analyzed for BL and challenged conditions. Transfer impedance measurements were fit with the DuBois model to determine the respiratory parameters (Raw and Cti). The flow at the airway opening was measured and analyzed on a breath-by-breath basis to obtain the ventilatory parameters (tidal volume, respiratory rate, and minute ventilation). Deep-breath frequency resulting from AS and histamine challenges [0.370 (SD 0.186) and 0.467 breaths/min (SD 0.216), respectively] was significantly increased compared with BL, MCh, or external resistance challenges [0.61 (SD 0.046), 0.156 (SD 0.173), and 0.117 breaths/min (SD 0.082), respectively]. MCh and external resistance challenges resulted in insignificant changes in deep-breath frequency compared with BL. All four modalities produced similar levels of bronchoconstriction, as assessed through changes in Raw and Cti, and had similar effects on the ventilatory parameters except that non-deep-breath tidal volume was decreased in AS and histamine. We propose that increased deep-breath frequency during AS and histamine challenge is the result of increased vascular permeability, which acts to increase rapidly adapting receptor activity.