The role of genetic and epigenetic GNAS alterations in the development of early-onset obesity.

BACKGROUND: GNAS (guanine nucleotide-binding protein, alpha stimulating) is an imprinted gene that encodes Gsα, the α subunit of the heterotrimeric stimulatory G protein. This subunit mediates the signalling of a diverse array of G protein-coupled receptors (GPCRs), including the melanocortin 4 receptor (MC4R) that serves a pivotal role in regulating food intake, energy homoeostasis, and body weight. Genetic or epigenetic alterations in GNAS are known to cause pseudohypoparathyroidism in its different subtypes and have been recently associated with isolated, early-onset, severe obesity. Given the diverse biological functions that Gsα serves, multiple molecular mechanisms involving various GPCRs, such as MC4R, ß2- and ß3-adrenoceptors, and corticotropin-releasing hormone receptor, have been implicated in the pathophysiology of severe, early-onset obesity that results from genetic or epigenetic GNAS changes. SCOPE OF REVIEW: This review examines the structure and function of GNAS and provides an overview of the disorders that are caused by defects in this gene and may feature early-onset obesity. Moreover, it elucidates the potential molecular mechanisms underlying Gsα deficiency-induced early-onset obesity, highlighting some of their implications for the diagnosis, management, and treatment of this complex condition. MAJOR CONCLUSIONS: Gsα deficiency is an underappreciated cause of early-onset, severe obesity. Therefore, screening children with unexplained, severe obesity for GNAS defects is recommended, to enhance the molecular diagnosis and management of this condition.

Functional Characterization of Novel MC4R Variants Identified in Two Unrelated Patients with Morbid Obesity in Qatar.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

STIM1 signals through NFAT1 independently of Orai1 and SOCE to regulate breast cancer cell migration.

Store-operated calcium entry (SOCE) contributes to several physiological and pathological conditions including transcription, secretion, immunodeficiencies, and cancer. SOCE has been shown to be important for breast cancer cell migration where knockdown of SOCE components (STIM1 or Orai1) decreases cancer metastasis. Here we show unexpectedly that complete knockout of STIM1 (STIM1-KO) using gene editing in metastatic MDA-MB-231 breast cancer cells results in faster migration and enhanced invasion capacity. In contrast, Orai1-KO cells, which have similar levels of SOCE inhibition as STIM1-KO, migrate slower than the parental cell line. This shows that the enhanced migration phenotype of STIM1-KO cells is not due to the loss of Ca2+ entry through SOCE, rather it involves transcriptional remodeling as elucidated by RNA-seq analyses. Interestingly, NFAT1 is significantly downregulated in STIM1-KO cells and overexpression of NFAT1 reversed the enhanced migration of STIM1-KO cells. STIM1 knockout in other breast cancer cells, independent of their metastatic potential, also enhanced cell migration while reducing NFAT1 expression. These data argue that in breast cancer cells STIM1 modulates NFAT1 expression and cell migration independently of its role in SOCE.

Phosphorylation of STIM1 at ERK/CDK sites is dispensable for cell migration and ER partitioning in mitosis.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway required for multiple physiological functions including cell motility. SOCE is triggered in response to depletion of intracellular Ca2+ stores following the activation of the endoplasmic reticulum (ER) Ca2+ sensor STIM1, which recruits the plasma membrane (PM) Ca2+ channel Orai1 at ER-PM junctions. STIM1 is phosphorylated dynamically, and this phosphorylation has been implicated in several processes including SOCE inactivation during M-phase, maximal SOCE activation, ER segregation during mitosis, and cell migration. Human STIM1 has 10 Ser/Thr residues in its cytosolic domain that match the ERK/CDK consensus phosphorylation. We recently generated a mouse knock-in line where wild-type STIM1 was replaced by a non-phosphorylatable STIM1 with all ten S/Ts mutated to Ala (STIM1-10A). Here, we generate mouse embryonic fibroblasts (MEF) from the STIM1-10A mouse line and a control MEF line (WT) that express wild-type STIM1 from a congenic mouse strain. These lines offer a unique model to address the role of STIM1 phosphorylation at endogenous expression levels in contrast to previous studies that relied mostly on overexpression. We show that STIM1 phosphorylation at ERK/CDK sites is not required for SOCE activation, cell migration, or ER partitioning during mitosis. These results rule out STIM1 phosphorylation as a regulator of SOCE, migration, and ER distribution in mitosis.

Store Operated Calcium Entry in Cell Migration and Cancer Metastasis.

Ca2+ signaling is ubiquitous in eukaryotic cells and modulates many cellular events including cell migration. Directional cell migration requires the polarization of both signaling and structural elements. This polarization is reflected in various Ca2+ signaling pathways that impinge on cell movement. In particular, store-operated Ca2+ entry (SOCE) plays important roles in regulating cell movement at both the front and rear of migrating cells. SOCE represents a predominant Ca2+ influx pathway in non-excitable cells, which are the primary migrating cells in multicellular organisms. In this review, we summarize the role of Ca2+ signaling in cell migration with a focus on SOCE and its diverse functions in migrating cells and cancer metastasis. SOCE has been implicated in regulating focal adhesion turnover in a polarized fashion and the mechanisms involved are beginning to be elucidated. However, SOCE is also involved is other aspects of cell migration with a less well-defined mechanistic understanding. Therefore, much remains to be learned regarding the role and regulation of SOCE in migrating cells.

Correction: Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

[This corrects the article DOI: 10.1371/journal.pbio.3000901.].

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis.

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260-275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

Structure-activity relationship of piperine and its synthetic amide analogs for therapeutic potential to prevent experimentally induced ER stress in vitro.

Endoplasmic reticulum (ER) is the key organelle involved in protein folding and maturation. Emerging studies implicate the role of ER stress in the development of chronic kidney disease. Thus, there is an urgent need for compounds that could ameliorate ER stress and prevent CKD. Piperine and its analogs have been reported to exhibit multiple pharmacological activities; however, their efficacy against ER stress in kidney cells has not been studied yet. Hence, the goal of this study was to synthesize amide-substituted piperine analogs and screen them for pharmacological activity to relieve ER stress using an in vitro model of tunicamycin-induced ER stress using normal rat kidney (NRK-52E) cells. Five amide-substituted piperine analogs were synthesized and their chemical structures were elucidated by pertinent spectroscopic techniques. An in vitro model of ER stress was developed using tunicamycin, and the compounds of interest were screened for their effect on cell viability, and the expression of ER chaperone GRP78, the pro-apoptotic ER stress marker CHOP, and apoptotic caspases 3 and 12 (via western blotting). Our findings indicate that exposure to tunicamycin (0.5 µg/mL) for 2 h induces the expression of GRP78 and CHOP, and apoptotic markers (caspase-3 and caspase-12) and causes a significant reduction in renal cell viability. Pre-treatment of cells with piperine and its cyclohexylamino analog decreased the tunicamycin-induced upregulation of GRP78 and CHOP and cell death. Taken together, our findings demonstrate that piperine and its analogs differentially regulate ER stress, and thus represent potential therapeutic agents to treat ER stress-related renal disorders. Graphical Abstract Piperine (PIP) reduces the expression of ER stress markers (GRP78 and CHOP) induced by pathologic stimuli and consequently decreases the activation of apoptotic caspase-12 and caspase-3; all of which contributes to its chemical chaperone and cytoprotective properties to protect renal cells against ER stress and ER stress-induced cell death, and would ultimately prevent the development of chronic kidney disease.

Pharmacy students' attitudes toward pharmaceutical care in Qatar.

OBJECTIVES: The study objectives were to investigate Qatar pharmacy students' attitudes toward pharmaceutical care (PC), to identify the factors that influence their attitudes, and to recognize their perceived barriers for PC provision. METHODS: A cross-sectional and online survey of Qatar pharmacy students was conducted. RESULTS: Over 4 weeks, 46 surveys were submitted (88% response rate). All respondents agreed that the pharmacist's primary responsibility is to prevent and resolve medication therapy problems. Most respondents believed that PC provision is professionally rewarding and that all pharmacists should provide PC (93% and 91% of respondents, respectively). Highly perceived barriers for PC provision included lack of access to patient information (76%), inadequate drug information sources (55%), and time constraints (53%). Professional year and practical experience duration were inversely significantly associated with four and five statements, respectively, out of the 13 Standard Pharmaceutical Care Attitudes Survey statements, including the statements related to the value of PC, and its benefit in improving patient health and pharmacy practitioners' careers. CONCLUSION: Qatar pharmacy students had positive attitudes toward PC. Efforts should be exerted to overcome their perceived barriers.