Cell-free protein synthesis as a tool to study RXFP3- Relaxin-3 protein interactions

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more “biologically relevant” setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cellfree protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies

Folding pathway of an Ig domain is conserved on and off the ribosome.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-β Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus

The development and initial evaluation of the Diarrhoea Management Diary (DMD) in patients with metastatic breast cancer

Purpose Chemotherapy-induced diarrhoea (CID) is a common, but often underreported problem in patients with breast cancer that has a profound effect on quality of life. It is best measured from a patient’s perspective, but tools are limited. The aim of this study was to develop and evaluate the Diarrhoea Management Diary (DMD), a self-report measure to assess CID, use of self-management strategies and treatment adherence. Methods The DMD was constructed using an iterative process of instrument development: concept elicitation (literature review), item generation and reduction (cognitive debriefing), and pilot testing in the target population. After translation into eight languages, the DMD was used in an international randomised trial for women receiving lapatinib and capecitabine for metastatic breast cancer with or without prophylactic octreotide. Patterns of missing data and sensitivity to change were examined. Results The understandability and completeness of the 8-item DMD was confirmed in cognitive interviews and pilot testing. Practicability of the DMD was evaluated in 62 women with metastatic breast cancer (median age 57). Up to 68% reported CID at any given time-point, and 19% had diarrhoea at each time-point. Patients also described efficacy of different strategies for diarrhoea management. Missing data were associated with study discontinuation. DMD missing item response was 0.9%. Sensitivity to change was good at most assessment points. Conclusions Although further psychometric testing is recommended, initial evaluation of the DMD showed good content validity and practicability in international research with cancer patients

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Bipolar multiplex families have an increased burden of common risk variants for psychiatric disorders.

Multiplex families with a high prevalence of a psychiatric disorder are often examined to identify rare genetic variants with large effect sizes. In the present study, we analysed whether the risk for bipolar disorder (BD) in BD multiplex families is influenced by common genetic variants. Furthermore, we investigated whether this risk is conferred mainly by BD-specific risk variants or by variants also associated with the susceptibility to schizophrenia or major depression. In total, 395 individuals from 33 Andalusian BD multiplex families (166 BD, 78 major depressive disorder, 151 unaffected) as well as 438 subjects from an independent, BD case/control cohort (161 unrelated BD, 277 unrelated controls) were analysed. Polygenic risk scores (PRS) for BD, schizophrenia (SCZ), and major depression were calculated and compared between the cohorts. Both the familial BD cases and unaffected family members had higher PRS for all three psychiatric disorders than the independent controls, with BD and SCZ being significant after correction for multiple testing, suggesting a high baseline risk for several psychiatric disorders in the families. Moreover, familial BD cases showed significantly higher BD PRS than unaffected family members and unrelated BD cases. A plausible hypothesis is that, in multiplex families with a general increase in risk for psychiatric disease, BD development is attributable to a high burden of common variants that confer a specific risk for BD. The present analyses demonstrated that common genetic risk variants for psychiatric disorders are likely to contribute to the high incidence of affective psychiatric disorders in the multiplex families. However, the PRS explained only part of the observed phenotypic variance, and rare variants might have also contributed to disease development

The genetics of the mood disorder spectrum:genome-wide association analyses of over 185,000 cases and 439,000 controls

Background Mood disorders (including major depressive disorder and bipolar disorder) affect 10-20% of the population. They range from brief, mild episodes to severe, incapacitating conditions that markedly impact lives. Despite their diagnostic distinction, multiple approaches have shown considerable sharing of risk factors across the mood disorders. Methods To clarify their shared molecular genetic basis, and to highlight disorder-specific associations, we meta-analysed data from the latest Psychiatric Genomics Consortium (PGC) genome-wide association studies of major depression (including data from 23andMe) and bipolar disorder, and an additional major depressive disorder cohort from UK Biobank (total: 185,285 cases, 439,741 controls; non-overlapping N = 609,424). Results Seventy-three loci reached genome-wide significance in the meta-analysis, including 15 that are novel for mood disorders. More genome-wide significant loci from the PGC analysis of major depression than bipolar disorder reached genome-wide significance. Genetic correlations revealed that type 2 bipolar disorder correlates strongly with recurrent and single episode major depressive disorder. Systems biology analyses highlight both similarities and differences between the mood disorders, particularly in the mouse brain cell-types implicated by the expression patterns of associated genes. The mood disorders also differ in their genetic correlation with educational attainment – positive in bipolar disorder but negative in major depressive disorder. Conclusions The mood disorders share several genetic associations, and can be combined effectively to increase variant discovery. However, we demonstrate several differences between these disorders. Analysing subtypes of major depressive disorder and bipolar disorder provides evidence for a genetic mood disorders spectrum

Determinants of Ligand Subtype-Selectivity at α-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR

α- and α-adrenoceptors (α-AR and α-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α-AR and α-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α-AR and α-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α-AR over α-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design