Calretinin interacts with huntingtin and reduces mutant huntingtin‐caused cytotoxicity

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats encoding for polyglutamine (polyQ) in the huntingtin (Htt) gene. Despite considerable effort, the mechanisms underlying the toxicity of the mutated Htt protein remains largely uncertain. To identify novel therapeutic targets, we recently employed the approach of tandem affinity purification and discovered that calretinin (Cr), a member of the EF‐hand family of calcium‐binding proteins, is preferentially associated with mHtt, although it also interacts with wild‐type Htt. These observations were supported by coimmunoprecipitation and by colocalization of Cr with mHtt in neuronal cultures. Over‐ expression of Cr reduced mHtt‐caused cytotoxicity in both non‐neuronal and neuronal cell models of HD, whereas knockdown of Cr expression in the cells enhanced mHtt‐caused neuronal cell death. In addition, over‐expression of Cr was also associated with reduction of intracellular free calcium and activation of Akt. These results suggest that Cr may be a potential therapeutic target for treatment of HD.     

Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.     

Conformational Selection and Substrate Binding Regulate the Monomer/Dimer Equilibrium of the C-terminal domain of Escherichia coli Enzyme I

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the β3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ～1500 s(-1)) between major open (～97%) and minor closed (～3%) conformations. Upon PEP binding, the β3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Optimisation of imidazole compounds as selective TAAR1 agonists: Discovery of RO5073012

A series of imidazole compounds has been identified which affords potent and selective partial and full agonists of the TAAR1 receptor. Starting from 2-benzyl-imidazoline screening hits, a series of structurally related 2-benzyl- and 4-benzyl-imidazoles was investigated first, but it proved highly challenging to obtain compounds having sufficient selectivity against the adrenergic alpha 2 receptor. This issue could be successfully addressed by modification of the linker region and SAR exploration led to the discovery of highly selective isopropyl-substituted 4-aminomethyl-imidazole compounds. The work culminated in the identification of the selective TAAR1 partial agonist RO5073012 (4-chlorophenyl)-(1H-imidazol-4-ylmethyl)-isopropyl-amine, 24), which has a good pharmacokinetic profile after oral administration in rodents. RO5073012 has been found to be active in a behavioural rat model which is considered indicative for schizophrenia.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Relation of left ventricular mass to QTc in normotensive severely obese patients

Prolongation of the corrected QT interval (QTc) has been described in obese subjects. This study assesses the relation of left ventricular (LV) mass to QTc in normotensive severely obese subjects. Fifty normotensive patients whose BMI was ≥40 kg/m(2) (mean age: 38 ± 7 years) were studied. QTc was derived using Bazett's formula. LV mass was calculated using the formula of Devereux et al. and was indexed to height(2.7). Mean QTc was 428.8 ± 19.0 ms and was significantly longer in those with than in those without LV hypertrophy (P < 0.01) QTc correlated positively and significantly with BMI (r = 0.392, P < 0.025), LV mass/height(2.7) (r = 0.793, P < 0.0005), systolic blood pressure (r = 0.742, P < 0.001), LV end - systolic wall stress (r = 0.746, P < 0.001) and LV internal dimension in diastole (r = 0.788, P < 0.0005). Among five variables tested, LV mass/height(2.7) was identified as the sole predictor of QTc by multivariate analysis. In conclusion, LV mass and loading conditions that may affect LV mass are important determinants of QTc in normotensive severely obese subjects.     

Anti-type II collagen antibodies are associated with early radiographic destruction in rheumatoid arthritis

Introduction

We have previously reported that high levels of antibodies specific for native human type II collagen (anti-CII) at the time of RA diagnosis were associated with concurrent but not later signs of inflammation. This was associated with CII/anti-CII immune complex (IC)-induced production of pro-inflammatory cytokines in vitro. In contrast, anti-cyclic citrullinated peptide antibodies (anti-CCP) were associated both with late inflammation and late radiological destruction in the same RA cohort. We therefore hypothesized that anti-CII are also associated with early erosions.

Methods

Two-hundred-and-fifty-six patients from an early RA cohort were included. Baseline levels of anti-CII, anti-CCP and anti-mutated citrullinated vimentin were analyzed with ELISA, and rheumatoid factor levels were determined by nephelometry. Radiographs of hands and feet at baseline, after one and after two years were quantified using the 32-joints Larsen erosion score.

Results

Levels of anti-CII were bimodally distributed in the RA cohort, with a small (3.1%, 8/256) group of very high outliers with a median level 87 times higher than the median for the healthy control group. Using a cut-off discriminating the outlier group that was associated with anti-CII IC-induced production of proinflammatory cytokines in vitro, baseline anti-CII antibodies were significantly (p = 0.0486) associated with increased radiographic damage at the time of diagnosis. Anti-CII-positive patient had also significantly increased HAQ score (p = 0.0303), CRP (p = 0.0026) and ESR (p = 0.0396) at the time of diagnosis but not during follow-up. The median age among anti-CII-positive subjects was 12 years higher than among the anti-CII-negative patients.

Conclusion

In contrary to anti-CCP, anti-CII-positive patients with RA have increased joint destruction and HAQ score at baseline. Anti-CII thus characterizes an early inflammatory/destructive phenotype, in contrast to the late appearance of an inflammatory/destructive phenotype in anti-CCP positive RA patients. The anti-CII phenotype might account for part of the elderly acute onset RA phenotype with rather good prognosis.     

Improbable but true: the invasive inbreeding ambrosia beetle Xylosandrus morigerus has generalist genotypes

The wide distribution and dominance of invasive inbreeding species in many forest ecosystems seems paradoxical in face of their limited genetic variation. Successful establishment of invasive species in new areas is nevertheless facilitated by clonal reproduction: parthenogenesis, regular self-fertilization, and regular inbreeding. The success of clonal lineages in variable environments has been explained by two models, the frozen niche variation (FNV) model and the general-purpose genotype (GPG) model. We tested these models on a widely distributed forest pest that has been recently established in Costa Rica-the sibling-mating ambrosia beetle Xylosandrus morigerus. Two deeply diverged mitochondrial haplotypes coexist at multiple sites in Costa Rica. We find that these two haplotypes do not differ in their associations with ecological factors. Overall the two haplotypes showed complete overlap in their resource utilization; both genotypes have broad niches, supporting the GPG model. Thus, probable or not, our findings suggest that X. morigerus is a true ecological generalist. Clonal aspects of reproduction coupled with broad niches are doubtless important factors in the successful colonization of new habitats in distant regions.