Homologous cardiac calcium pump regulators phospholamban and sarcolipin adopt distinct oligomeric states in the membrane.

Phospholamban (PLN) and Sarcolipin (SLN) are homologous membrane proteins that belong to the family of proteins that regulate the activity of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA). PLN and SLN share highly conserved leucine zipper motifs that control self-association; consequently, it has been proposed that both PLN and SLN assemble into stable pentamers in the membrane. In this study, we used molecular dynamics (MD) simulations and Western blot analysis to investigate the precise molecular architecture of the PLN and SLN oligomers. Analysis showed that the PLN pentamer is the predominant oligomer present in mouse ventricles and ventricle-like human iPSC-derived cardiomyocytes, in agreement with the MD simulations showing stable leucine zipper interactions across all protomer-protomer interfaces and MD replicates. Interestingly, we found that the PLN pentamer populates an asymmetric structure of the transmembrane region, which is likely an intrinsic feature of the oligomer in a lipid bilayer. The SLN pentamer is not favorably formed across MD replicates and species of origin; instead, SLN from human and mouse atria primarily populate coexisting dimeric and trimeric states. In contrast to previous studies, our findings indicate that the SLN pentamer is not the predominant oligomeric state populated in the membrane. We conclude that despite their structural homology, PLN and SLN adopt distinct oligomeric states in the membrane. We propose that the distinct oligomeric states populated by PLN and SLN may contribute to tissue-specific SERCA regulation via differences in protomer-oligomer exchange, oligomer-SERCA dynamics, and noise filtering during ß-adrenergic stimulation in the heart.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins.

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Gene expression alterations in bipolar disorder postmortem brains.

OBJECTIVES: Bipolar disorder (BD) is a mental illness of unknown neuropathology and has several genetic associations. Antipsychotics are effective for the treatment of acute mania, psychosis, or mixed states in individuals with BD. We aimed to identify gene transcripts differentially expressed in postmortem brains from antipsychotics-exposed individuals with BD (hereafter the 'exposed' group), non-exposed individuals with BD (hereafter the 'non-exposed' group), and controls. METHODS: We quantified the abundance of gene transcripts in postmortem brains from seven exposed individuals, seven non-exposed individuals, and 12 controls with the Affymetrix U133P2 GeneChip microarrays and technologies. We applied a q-value of ≤0.005 to identify statistically significant transcripts with mean abundance differences between the exposed, non-exposed and control groups. RESULTS: We identified 2191 unique genes with significantly altered expression levels in non-exposed brains compared to those in the control and exposed groups. The expression levels of these genes were not significantly different between exposed brains and controls, suggesting a normalization effect of antipsychotics on the expression of these genes. Gene ontology (GO) enrichment analysis showed significant (Bonferroni p ≤ 0.05) clustering of subgroups of the 2191 genes under many GO terms; notably, the protein products of genes enriched are critical to the function of synapses, affecting, for example, intracellular trafficking and synaptic vesicle biogenesis, transport, release and recycling, as well as organization and stabilization of the node of Ranvier. CONCLUSIONS: These results support a hypothesis of synaptic and intercellular communication impairment in BD. The apparent normalization of expression patterns with exposure to antipsychotic medication may represent a physiological process that relates both to etiology and improvement patterns of the disorder.

CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR.

BACKGROUND: Patients with cystic fibrosis (CF) manifest a multisystem disease due to deleterious mutations in each gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). However, the role of dysfunctional CFTR is uncertain in individuals with mild forms of CF (ie, pancreatic sufficiency) and mutation in only one CFTR gene. METHODS: Eleven pancreatic sufficient (PS) CF patients with only one CFTR mutation identified after mutation screening (three patients), mutation scanning (four patients) or DNA sequencing (four patients) were studied. Bi-directional sequencing of the coding region of CFTR was performed in patients who had mutation screening or scanning. If a second CFTR mutation was not identified, CFTR mRNA transcripts from nasal epithelial cells were analysed to determine if any PS-CF patients harboured a second CFTR mutation that altered RNA expression. RESULTS: Sequencing of the coding regions of CFTR identified a second deleterious mutation in five of the seven patients who previously had mutation screening or mutation scanning. Five of the remaining six patients with only one deleterious mutation identified in the coding region of one CFTR gene had a pathologic reduction in the amount of RNA transcribed from their other CFTR gene (8.4-16% of wild type). CONCLUSIONS: These results show that sequencing of the coding region of CFTR followed by analysis of CFTR transcription could be a useful diagnostic approach to confirm that patients with mild forms of CF harbour deleterious alterations in both CFTR genes.

MicroRNA expression changes in lymphoblastoid cell lines in response to lithium treatment.

Lithium (Li) is commonly used in the treatment of bipolar disorder (BD). However, the molecular mechanism of its action is not completely understood. MicroRNAs (miRNAs), a class of small RNA species are recognized as important regulators in post-transcription gene expression. To explore the role of miRNAs in Li's action, we quantitatively analysed the expression patterns of 13 miRNAs in 20 lymphoblastoid cell lines (LCLs) with or without Li treatment in culture. Using paired t statistics in the analysis, we identified significant changes in seven of the 13 miRNAs tested in LCLs sampled at treatment day 4 (p<0.05). Four of the seven significant miRNAs, miR-34a, miR-152, miR-155, and miR-221 consistently changed expression in the same LCLs at a longer treatment time-point, day 16 (Bonferroni p<0.05). Interestingly, miR-221 and miR-34a also changed expression in rat hippocampus in response to Li treatment (Zhou et al. 2008), although in the opposite direction. We focused on the predicted target mRNAs of miR-221 and miR-34a, and identified 29 and ten targets that were strongly and inversely correlated to expression with the two miRNAs, respectively. Our results suggest that miRNAs are excellent candidates for the study of the molecular basis of Li's treatment action in cell systems such as lymphocytes given limited access to the human brain.

Discriminating single-base difference miRNA expressions using microarray Probe Design Guru (ProDeG).

MicroRNAs (miRNA) are endogenous tissue-specific short RNAs that regulate gene expression. Discriminating each let-7 family member expression is especially important due to let-7's abundance and connection with development and cancer. However, short lengths (22 nt) and similarities between multiple sequences have prevented identification of individual members. Here, we present ProDeG, a computational algorithm which designs imperfectly matched sequences (previously yielding only noise levels in microarray experiments) for genome-wide microarray "signal" probes to discriminate single nucleotide differences and to improve probe qualities. Our probes for the entire let-7 family are both homogeneous and specific, verified using microarray signals from fluorescent dye-tagged oligonucleotides corresponding to the let-7 family, demonstrating the power of our algorithm. In addition, false let-7c signals from conventional perfectly-matched probes were identified in lymphoblastoid cell-line samples through comparison with our probe-set signals, raising concerns about false let-7 family signals in conventional microarray platform.

Lack of association of common cystic fibrosis transmembrane conductance regulator gene mutations with primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease of uncertain etiology. However, the histologic features of PSC liver disease can resemble those in cystic fibrosis (CF), an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to determine if PSC patients have a higher frequency of common CF alleles than disease controls. METHODS: DNA was extracted from peripheral lymphocytes of patients with end-stage liver disease. Samples were obtained before liver transplantation from 59 PSC patients and from three groups of control patients (20 each with primary biliary cirrhosis, autoimmune hepatitis, or hepatitis C). DNA samples were genotyped for 32 common CF mutations, the intron 8 T tract variants, and the M470V variant. RESULTS: One of 59 PSC patients (1.7%) had the common CF mutation (DeltaF508) in one CFTR gene. Two controls (3.3%) carried a single CF mutation (DeltaF508 in one primary biliary cirrhosis patient; W1282X in one hepatitis C patient). These rates do not differ from expected in the general population. The frequency of CFTR variants (5T and M470V) was also similar between PSC patients and controls. CONCLUSIONS: Despite anatomical similarities between CF liver disease and PSC, we could not confirm that PSC patients carried common CF mutations or common CFTR variants in higher than expected frequencies. These data suggest that CFTR dysfunction does not influence the pathogenesis of PSC.