Characterization of ankyrin repeat domain 54 (ANKRD54) and its role on the regulation and subcellular localization of Bruton’s tyrosine kinase (BTK)

Bruton's tyrosine kinase (BTK) is an important cytoplasmic signaling protein, where the kinase activity plays a pivotal role in the development, proliferation and differentiation of B-cell lineages. Ankyrin repeat domain 54 (ANKRD54) is a nuclear-resident adaptor protein, where the ankyrin domain repeats are critical for specific protein-protein interaction, while the NLS and NES motifs control the nucleo-cytoplasmic shuttling ability. We have identified and characterized ANKRD54 as a novel functional (paper I), interaction-partner for BTK using proteomics analysis. ANKRD54 is the first protein identified that specially influences the nuclear export of both BTK and TXK/RLK, in a Crm-1 dependent manner. Further, we mapped the interaction site to the C -terminus of BTK-SH3 domain, by using a synthetic peptide of BTK, covering the following region: C- ARDKNGQEEGYIPSNYVTEAEDS. In addition, tyrosine phosphorylation of BTK was investigated in the presence of increased amount of ANKRD54 and selectively the phosphorylation of BTK was down regulated. We have presented a second novel interaction-partner and regulator of BTK (paper II), the 14-3-3 ζ protein, which is also identified by proteomics strategy. In this work, we have mapped the interaction sites on BTK to phospho-serine pS51 in the (RGRRGpS)-motif in the PH-domain and phospho-threonine pT495 in the (RHRFQpT)-motif in the kinase domain. Additionally, a newly characterized 14-3-3 inhibitor (BV02) interfered binding with BTK and siRNA knockdown of 14-3-3ζ increased the nuclear translocation of BTK, while overexpression of 14-3-3ζ resulted in accumulation of BTK in the perinuclear region. We have generated single ankryin domain deletions of ANKRD54 and subsequently characterized their binding capacity and also their influence on the sub-cellular localization of BTK (paper III). In this work, we report that three out of four ankyrin repeats are required for the interaction and nucleo-cytoplasmic shuttling of BTK. Inhibition of Crm-1 nuclear export pathway influences differently the nuclear shuttling; rapid-ANKRD54 versus slow-BTK nuclear accumulation. Furthermore, we have determined that the interaction between BTK and ANKRD54 establishes in the nuclear compartment. We have classified ANKRD54 as a prime interactor to the SH3-domain of BTK (paper IV). In this study, we utilized a screening strategy based on phage display libraries of the complete human “SH3-domainome” as a possible binding-target for ANKRD54. The aim is to identify the target spectrum and specificity of ANKRD54 for SH3 domain library, containing all the 296 human SH3 domains. The novel finding is that BTK is not only binding to ANKRD54, but also stands out as the preferred interactor, being highly dominant over all other human SH3 domains. However, other lower colony-score candidates for SH3-domain interactions were found, but without any further in vivo/in vitro validation

Potential to Enhance the Prescribing of Generic Drugs in Patients with Mental Health Problems in Austria; Implications for the Future

Background: Scrutiny over pharmaceutical expenditure is increasing leading to multiple reforms. This includes Austria with measures to lower generic prices and enhance their utilization. However the situation for newer antidepressants and atypical antipsychotic medicines (AAPs) is different to PPIs, statins, and renin-angiotensin inhibitor drugs with greater tailoring of therapy and no wish to switch products in stable patients. Authorities welcome generics though given the high costs particularly of single-sourced AAPs. Objective: Assess (a) changes in utilization of venlafaxine versus other newer antidepressants before and after availability of generics, (b) utilization of generic versus originator venlafaxine, (c) price reductions of venlafaxine over time and their influence on total expenditure, (d) utilization of risperidone versus other AAPs, (e) suggest potential additional reforms that could be introduced if pertinent to further enhance the use of generics. Methodology: A quasi-experimental study design with a segmented time series and an observational study. Utilization measured in defined daily doses (DDDs) and total expenditure per DDD and over time. Results: No appreciable changes in the utilization of venlafaxine and risperidone after generics. The reduction in expenditure/DDD for venlafaxine decreased overall expenditure on newer antidepressants by 5% by the end of the study versus just before generics despite a 37% increase in utilization. Expenditure will further decrease if reduced prescribing of duloxetine. Conclusion: Depression, schizophrenia, and bipolar diseases are complex diseases. As a result, specific measures are needed to encourage the prescribing of generic risperidone and venlafaxine when multiple choices are appropriate. Authorities cannot rely on a “Hawthorne” effect between classes to enhance the use of generics. Measures may include prescribing restrictions for duloxetine. No specific measures planned for AAPs with more multiple-sourced AAPs becoming available

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.Peer reviewe

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients

Testing gravitational-wave searches with numerical relativity waveforms: Results from the first Numerical INJection Analysis (NINJA) project

The Numerical INJection Analysis (NINJA) project is a collaborative effort between members of the numerical relativity and gravitational-wave data analysis communities. The purpose of NINJA is to study the sensitivity of existing gravitational-wave search algorithms using numerically generated waveforms and to foster closer collaboration between the numerical relativity and data analysis communities. We describe the results of the first NINJA analysis which focused on gravitational waveforms from binary black hole coalescence. Ten numerical relativity groups contributed numerical data which were used to generate a set of gravitational-wave signals. These signals were injected into a simulated data set, designed to mimic the response of the Initial LIGO and Virgo gravitational-wave detectors. Nine groups analysed this data using search and parameter-estimation pipelines. Matched filter algorithms, un-modelled-burst searches and Bayesian parameter-estimation and model-selection algorithms were applied to the data. We report the efficiency of these search methods in detecting the numerical waveforms and measuring their parameters. We describe preliminary comparisons between the different search methods and suggest improvements for future NINJA analyses.Comment: 56 pages, 25 figures; various clarifications; accepted to CQ

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Minimum wages in 2021 : annual review

Aquesta publicació s'elabora a partir de les contribucions de cadascú dels membres nacionals que integren la Network of Eufound Correspondent. Pel cas d'Espanya la contribució ha estat realitzada per l'Oscar MolinaThis report summarises how minimum wage rates for 2021 were set during 2020 - the year marked by the COVID-19 pandemic. It reviews the difficulties faced by national decision-makers and how they reacted to the challenges of the economic and social fall-out of the pandemic when making decisions regarding the minimum wage. It maps the extent to which minimum wages were referred to in COVID-19-related support measures. It discusses advances made on the EU initiative on adequate minimum wages and maps the reactions of the EU-level social partners and national decision-makers. The report is accompanied by two complementary working papers: one providing an analysis of developments for low-paid employees and minimum wage workers over the past decade; the other summarising the most recent research on minimum wages in EU countries, Norway and the UK

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis