Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction

Contains fulltext : 138516.pdf (publisher's version ) (Open Access)Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled

Association of Plasminogen Activator Inhibitor (PAI)-1 (SERPINE1) SNPs With Myocardial Infarction, Plasma PAI-1, and Metabolic Parameters

Objective— The purpose of this study was to investigate the effects of plasminogen activator inhibitor-1 (PAI-1) gene (SERPINE1) single nucleotide polymorphisms (SNPs) on the risk of myocardial infarction (MI), on PAI-1 levels, and factors related to the metabolic syndrome. Methods and Results— Eleven SNPs capturing the common genetic variation of the SERPINE1 gene were genotyped in the HIFMECH study. In the 510 male cases and their 543 age-matched controls, a significant gene-smoking interaction was observed. In nonsmokers, the rs7242-G allele was more frequent in cases than in controls (0.486 versus 0.382, P =0.013) whereas the haplotype derived from the rs2227631 (−844A>G)-G and rs2227683-A alleles was ≈3-fold lower in cases than in controls (0.042 versus 0.115, P =0.006). SERPINE1 haplotypes explained 3.5% ( P =0.007) of the variability of PAI-1 levels, which was attributable to the combined effects of 3 SNPs, −844A>G, rs2227666, and rs2227694. The rs6092 (Ala15Thr) and rs7242 SNPs acted additively to explain 4.4% of the variability of plasma insulin levels and 1.6% of the variability of BMI ( P <10 −3 and P =0.023, respectively). Conclusions— SERPINE1 haplotypes are mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They are also associated with insulin levels and BMI

Vemurafenib treatment of pleomorphic xanthoastrocytoma in a child with Down syndrome

Brain tumors are the most common solid neoplasms of childhood, but they are very rarely reported in children with Down Syndrome (DS), who develop more commonly different types of malignancies. In particular, we hereby report the case of an 8-years-old child with DS that presented to our attention for neurological and endocrinological issues. Brain imaging revealed the presence of a mass that was partially resected revealing a histological diagnosis of Pleomorphic Xanthoastrocytoma (PXA), a rare WHO grade II tumor extending from the diencephalic region into the surrounding brain tissue. These tumors can harbor the BRAF mutation p.V600E, targetable by the specific inhibitor Vemurafenib. After confirming the presence of the mutation in the tumor, the patient was treated with Vemurafenib. The treatment proved to be effective, leading to a partial response and a stabilization of the disease. Usually, in patients with DS a reduction of the dose of chemotherapeutic drugs is necessary. Vemurafenib was instead well-tolerated as the only observed adverse effect was grade I skin toxicity. This is, to our knowledge, the first case of a PXA reported in a child with DS and the first DS patient treated with Vemurafenib

Accelerating Polarized Protons to 250 GEV

The Relativistic Heavy Ion Collider (RHIC) as the first high energy polarized proton collider was designed t o provide polarized proton collisions a t a maximum beam energy of 250 GeV. I t has been providing collisions a t a beam energy of 100 Gel' since 2001. Equipped with two full Siberian snakes in each ring, polarization is preserved during the acceleration from injection to 100 GeV with careful control of the betatron tunes and the vertical orbit distortions. However, the intrinsic spin resonances beyond 100 GeV are about a factor of two stronger than those below 100 GeV? making it important t o examine the impact of these strong intrinsic spin resonances on polarization survival and the tolerance for vertical orbit distortions. Polarized protons were accelerated t o the record energy of 250 GeV in RHIC with a polarization of 46% measured a t top energy in 2006. The polarization measurement as a function of beam energy also shows some polarization loss around 136 GeV, the first strong intrinsic resonance above 100 GeV. This paper presents the results and discusses the sensitivity of the polarization survival t o orbit distortions

Establishment of a national network of cetacean monitoring within the marine strategy

CONISMA, CNR and CIRCE, involved Italian research units (RUs) working on cetaceans to joina National Network answering the Marine Strategy Framework Directive (MSFD) requirements by sharing monitoring data. Data obtained during the 2016 monitoring campaigns by 13 RUs are presented here

Role of Interleukin 17 in arthritis chronicity through survival of synoviocytes via regulation of synoviolin expression

Background: The use of TNF inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond. In addition, response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. Therefore we studied the capacity of IL-17 to regulate synoviolin, an E3 ubiquitin ligase implicated in synovial hyperplasia in human rheumatoid arthritis (RA) FLS and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis.&lt;p&gt;&lt;/p&gt; Methodology/Principal Findings: Chronic reactivated SCW-induced arthritis was examined in IL-17R deficient and wild-type mice. Synoviolin expression was analysed by real-time RT-PCR, Western Blot or immunostaining in RA FLS and tissue, and p53 assessed by Western Blot. Apoptosis was detected by annexin V/propidium iodide staining, SS DNA apoptosis ELISA kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved by small interfering RNA (siRNA) or neutralizing antibodies. IL-17 induced sustained synoviolin expression in RA FLS. Sodium nitroprusside (SNP)-induced RA FLS apoptosis was associated with reduced synoviolin expression and was rescued by IL-17 treatment with a corresponding increase in synoviolin expression. IL-17RC or IL-17RA RNA interference increased SNP-induced apoptosis, and decreased IL-17-induced synoviolin. IL-17 rescued RA FLS from apoptosis induced by synoviolin knockdown. IL-17 and TNF had additive effects on synoviolin expression and protection against apoptosis induced by synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis severity was characterized by increased synovial apoptosis, reduced proliferation and a marked reduction in synoviolin expression. A distinct absence of synoviolin expressing germinal centres in IL-17R deficient mice contrasted with synoviolin positive B cells and Th17 cells in synovial germinal centre-like structures.&lt;p&gt;&lt;/p&gt; Conclusion/Significance: IL-17 induction of synoviolin may contribute at least in part to RA chronicity by prolonging the survival of RA FLS and immune cells in germinal centre reactions. These results extend the role of IL-17 to synovial hyperplasia.&lt;p&gt;&lt;/p&gt

ACCELERATING POLARIZED PROTONS TO HIGH ENERGY.

The Relativistic Heavy Ion Collider (RHIC) is designed to provide collisions of high energy polarized protons for the quest of understanding the proton spin structure. Polarized proton collisions at a beam energy of 100 GeV have been achieved in RHIC since 2001. Recently, polarized proton beam was accelerated to 250 GeV in RHIC for the first time. Unlike accelerating unpolarized protons, the challenge for achieving high energy polarized protons is to fight the various mechanisms in an accelerator that can lead to partial or total polarization loss due to the interaction of the spin vector with the magnetic fields. We report on the progress of the RHIC polarized proton program. We also present the strategies of how to preserve the polarization through the entire acceleration chain, i.e. a 200 MeV linear accelerator, the Booster, the AGS and RHIC

Genomic analysis of the function of the transcription factor gata3 during development of the Mammalian inner ear

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKB beta, a dramatic increase in Akt1/PKB alpha protein and relocation of Akt1/PKB alpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome

Multiparametric magnetic resonance imaging and frozen-section analysis efficiently predict upgrading, upstaging, and extraprostatic extension in patients undergoing nerve-sparing robotic-assisted radical prostatectomy

To evaluate the role of multiparametric magnetic resonance imaging (mpMRI) in predicting upgrading, upstaging, and extraprostatic extension in patients with low-risk prostate cancer (PCa). MpMRI may reduce positive surgical margins (PSM) and improve nerve-sparing during robotic-assisted radical prostatectomy (RARP) for localized prostate cancer PCa.This was a retrospective, monocentric, observational study. We retrieved the records of patients undergoing RARP from January 2012 to December 2013 at our Institution. Inclusion criteria were: PSA <10\u200ang/mL; clinical stage <T3a; biopsy Gleason score <7; prostate mpMRI performed preoperatively at our Institution; intraoperative FSA of the posterolateral aspects of the specimen.All the identified lesions were scored according to the Prostate Imaging Reporting and Data System (PIRADS). We considered the lesion with the highest PIRADS score as index lesion. All the included patients underwent nerve-sparing RARP. During surgery, the specimen was sent for FSA of the posterolateral aspects. The surgeon, according to the localization scheme provided by the mpMRI, inked the region of the posterolateral aspect of the prostate that had to be submitted to FSA.We evaluated association between clinical features and PSM, upgrading, upstaging, and presence of unfavorable disease.Two hundred fifty-four patients who underwent nerve-sparing RARP were included. PSM rate was 29.13% and 15.75% at FSA and final pathology respectively. Interestingly, the use of FSA reduced PSM rate in pT3 disease (25.81%). Higher PIRADS scores demonstrated to be related to high probability of upgrading and upstaging. This significativity remains even when considering PIRADS 2-3 versus 4 versus 5 and PIRADS 2-3 versus 4-5. Also PSM at FSA were associated with higher probability of upgrading and upstaging.PIRADS score and FSA resulted to be strictly related to grading and staging, thus being able to predict upgrading and/or upstaging at final pathology

Leading indicators of fiscal distress: evidence from extreme bounds analysis

Early warning systems (EWSs) are widely used to assess a country’s vulnerability to fiscal distress. A fiscal distress episode is identified as a period when government experiences extreme funding difficulties. Most EWSs employ a specific set of only fiscal leading indicators predetermined by the researchers, which casts doubt on their robustness. We revisit this issue using extreme bounds analysis, which allows identifying robust leading indicators of fiscal distress from a large set. A robust leading indicator’s effect does not strongly depend on the model specification. Consistent with the theoretical predictions of latest generation crisis models, we find that both fiscal and non-fiscal leading indicators are robust. In addition, we find that a fiscal vulnerability indicator based on fiscal and non-fiscal leading indicators offers a 29% gain in predictive power compared to a traditional one based only on fiscal leading indicators. This suggests that both fiscal and non-fiscal leading indicators should be taken into account when assessing country’s vulnerability to fiscal distress