Regulator Of G Protein Signaling Research Articles

SymRK Regulates G-Protein Signaling During Nodulation in Soybean (Glycine max) by Modifying RGS Phosphorylation and Activity.

Molecular interspecies dialogue between leguminous plants and nitrogen-fixing rhizobia results in the development of symbiotic root nodules. This is initiated by several nodulation-related receptors present on the surface of root hair epidermal cells. We have shown previously that specific subunits of heterotrimeric G-proteins and their associated regulator of G-protein signaling (RGS) proteins act as molecular links between the receptors and downstream components during nodule formation in soybeans. Nod factor receptor 1 (NFR1) interacts with and phosphorylates RGS proteins to regulate the G-protein cycle. Symbiosis receptor-like kinases (SymRK) phosphorylate Gα to make it inactive and unavailable for Gβγ. We now show that like NFR1, SymRK also interacts with the RGS proteins to phosphorylate them. Phosphorylated RGS has higher activity for accelerating guanosine triphosphate (GTP) hydrolysis by Gα, which favors conversion of active Gα to its inactive form. Phosphorylation of RGS proteins is physiologically relevant, as overexpression of a phospho-mimic version of the RGS protein enhances nodule formation in soybean. These results reveal an intricate fine-tuning of the G-protein signaling during nodulation, where a negative regulator (Gα) is effectively deactivated by RGS due to the concerted efforts of several receptor proteins to ensure adequate nodulation. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

RGS10 Attenuates Systemic Immune Dysregulation Induced by Chronic Inflammatory Stress.

Regulator of G-protein signaling 10 (RGS10), a key homeostatic regulator of immune cells, has been implicated in multiple diseases associated with aging and chronic inflammation including Parkinson's Disease (PD). Interestingly, subjects with idiopathic PD display reduced levels of RGS10 in subsets of peripheral immune cells. Additionally, individuals with PD have been shown to have increased activated peripheral immune cells in cerebral spinal fluid (CSF) compared to age-matched healthy controls. However, it is unknown whether CSF-resident peripheral immune cells in individuals with PD also exhibit decreased levels of RGS10. Therefore, we performed an analysis of RGS10 levels in the proteomic database of the CSF from the Michael J. Fox Foundation Parkinson's Progression Markers Initiative (PPMI) study. We found that RGS10 levels are decreased in the CSF of individuals with PD compared to healthy controls and prodromal individuals. Moreover, we find that RGS10 levels decrease with age but not PD progression and that males have less RGS10 than females in PD. Importantly, studies have established an association between chronic systemic inflammation (CSI) and neurodegenerative diseases, such as PD, and known sources of CSI have been identified as risk factors for developing PD; however, the role of peripheral immune cell dysregulation in this process has been underexplored. As RGS10 levels are decreased in the CSF and circulating peripheral immune cells of individuals with PD, we hypothesized that RGS10 regulates peripheral immune cell responses to CSI prior to the onset of neurodegeneration. To test this, we induced CSI for 6 weeks in C57BL6/J mice and RGS10 KO mice to assess circulating and CNS-associated peripheral immune cell responses. We found that RGS10 deficiency synergizes with CSI to induce a bias for inflammatory and cytotoxic cell populations, a reduction in antigen presentation in peripheral blood immune cells, as well as in and around the brain that is most notable in males. These results highlight RGS10 as an important regulator of the systemic immune response to CSI and implicate RGS10 as a potential contributor to the development of immune dysregulation in PD.

RGS5 balances macrophage vs vascular smooth muscle cell origin of foam cells in apoE-deficient plaques

Abstract Background G Protein Coupled Receptors and regulators of G-protein signalling (RGS), play a pivotal role in the signalling processes associated with atherosclerosis [1]. RGS5 is highly expressed in aortic tissue [2]. Development of atherosclerotic plaque is a complex process that involves foam cells originating from macrophages (Mϕ) and/or vascular smooth muscle cells (VSMCs). Differentiation of Mϕ and VSMC into various intermediary cells of varying inflammatory potency affects atherosclerotic plaque composition and vulnerability [3]. We hypothesize that RGS5 is involved in signalling of foam cell transition and thus, plaque biology. Purpose To evaluate the role of RGS5 on atherosclerotic plaque complexity and shifts of foam cell origin. Methods ApoE-/-RGS5+/+ and ApoE-/-RGS5-/- mice were fed a high fat Western Diet for 14 weeks. Cryosections bearing aortic valve cusps were used for staining. Lesion size was quantified on hematoxylin and eosin staining. Plaque composition was assessed by CD68, CD45, Oil red O and collagen staining. Triple staining was performed for CD45, CD68 and aSMA. Double immunofluorescent staining for CD45 and pro- and anti-inflammatory markers was carried out. Co-Localisation was analysed by Zeiss Zen. Data were analysed using Mann-Whitney-U test. Results Lesion size was significantly reduced in RGS5-deficient mice in comparison to control (in mm2: 0.398±0.02 vs 0.191±0.09, p=0.0001, n=15). Relative areas positive for Oil Red O and collagen did not differ between genotypes. Area positive for leukocyte marker CD45 increased significantly by 30.8% (p<0.0001, n=15), whereas area positive for CD68 was reduced by 10.3% (p=0.0292, n=15) in RGS5-deficient mice. Absence of RGS5 decreased co-localization of Mϕ-derived foam cells by 23% (p=0.03734, n=4-5) while leading to an 5.8% increase in aSMA-derived foam cells compared to control. Area positive for pro-inflammatory M1 Mϕ marker CD86 and iNOS increased by 36.3% and 36.8% after RGS5 knock-out (p<0.0001, n=15). No difference in M2 Mϕ positive area was observed when RGS5 was lacking. Conclusion RGS5 promotes plaque formation. The number of Mϕ-derived foam cells is increased by RGS5, while it reduces inflammatory M1 Mϕ. Interestingly, there were no differences in numbers of M2 Mϕ, indicating RGS5 inhibits VSMCs transition to M1 Mϕ resulting in an overall decrease of CD68-positive foam cells. Further study is required to determine molecular signalling of RGS5 that modulates the phenotype of Mϕ- and VSMC-derived cells. RGS5 could be useful as therapeutic target to suppress undesirable plaque phenotypes and to reduce acute cardiovascular events.

VPS26 Moonlights as a β-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana.

Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, β-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting β-arrestin, but it is not clear how. Arrestins are not encoded in the Arabidopsis thaliana genome, yet Arabidopsis cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.

RGS10 Deficiency Alleviated Intestinal Mucosal Inflammation Through Suppression of Th1/Th17 Cell Immune Responses in Ulcerative Colitis.

Regulator of G-protein signalling (RGS) 10 plays critical roles in several immune related diseases. However, whether RGS10 is involved in colonic inflammation of ulcerative colitis (UC) is still obscure. This study aimed to investigate the role of RGS10 in UC. In this study, RGS10 expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and immunofluorescent analysis. Single-cell RNA sequencing of intestinal mucosa was performed to identify key immune cells with differentially expressed RGS10. RGS10 knockout mice were generated and established dextran sulphate sodium (DSS)-induced colitis. Expression of inflammatory cytokines on mRNA and protein levels was detected by qRT-PCR, enzyme-linked immunosorbent assay, and flow cytometry. We found that RGS10 expression was significantly elevated in UC patients, especially in CD4+ T cells, compared with healthy subjects. Intriguingly, RGS10 deficiency markedly alleviated DSS-induced colitis and decreased the proportion of Th1 and Th17 cells in lamina propria mononuclear cells (LPMCs), peripheral blood (PB), spleens, and mesenteric lymph nodes (mLNs). Mechanistically, RGS10 deficiency blocked the differentiation of Th1 and Th17 cells by inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3. The co-immunoprecipitation analysis further showed that RGS10 could interact with protein tyrosine phosphatase non-receptor type 2 (PTPN2), and further regulated Th1 and Th17 cells differentiation of CD4+ T cells. In conclusion, RGS10 deficiency alleviated intestinal mucosal inflammation through inhibition of Th1/Th17 cell-mediated immune responses via interaction with PTPN2 in CD4+ T cells. Therefore, targeting RGS10 may represent a novel therapeutic approach for UC treatment.

Regulator of G-Protein Signaling 2 Knockout in CD4+ T Cells Promotes Anti-Inflammatory T Cells, Enhancing Ovulation, and Oocyte Yield.

To determine the downstream effects on ovarian function and immune cell differentiation in the ovary and uterus using a model in which RGS2 was knocked out specifically in CD4+ T cells. Laboratory based experiments with female mice. Female congenic (fully backcrossed) and non-congenic (mixed strain) mice with CD4 T cell-specific RGS2 knockout. Four-week-old female CD4 RGS2 knockout (CD4 RGS2 KO ) mice and their littermate controls (CD4 RGS2 CTL ) were subjected to superovulation using pregnant mare serum gonadotropins. Oocyte numbers, lymphocyte populations in the ovary and uterus, and serum estradiol and progesterone concentrations. In non-congenic (mixed strain) mice, CD4 RGS2 knockout (KO) promoted higher oocyte ovulation and increased uterine total leukocyte numbers. Similarly, congenic (fully backcrossed strain) mice showed higher oocyte numbers and increased uterine total leukocytes in the CD4 RGS2 KO mice compared to CD4 RGS2 CTL mice. Pro-inflammatory CD4+ T helper (T H ) 1 and T H 17 cell frequencies in the ovary and uterus were unchanged, while Treg and T H 2 cell frequencies were elevated, along with increased concentrations of estradiol and progesterone in the serum of CD4 RGS2 KO mice. Our study highlights the important role of RGS2 in CD4+ T cells within the context of reproduction. The dysregulation of immune responses due to RGS2 knockout in CD4+ T cells appears to enhance oocyte production. Further research is warranted to elucidate the precise mechanisms by which RGS2 influences reproductive outcomes, including its impact on fecundability, endometrial receptivity, and successful implantation.

In Silico Design of Novel RGS2-Galpha-q Interaction Inhibitors with Anticancer Activity.

Regulators of G-protein signaling (RGS) are a family of approximately 30 proteins that bind to and deactivate the alpha subunits of G-proteins (Gα) by accelerating their GTP hydrolysis rates, which terminates G-protein coupled receptor (GPCR) signaling. Thus, RGS proteins are essential in regulating GPCR signaling, and most members are implicated as critical nodes in human diseases such as hypertension, depression, and others. Regulator of G-protein signaling 2 (RGS2), a member of the R4 family of RGS proteins, is overexpressed in many solid breast cancers, and its levels in prostate cancer significantly correlate with the metastatic stage and poor prognosis. We sought to develop RGS2 inhibitors as potential chemotherapeutic agents utilizing structure-based drug design approaches. Available structures of the RGS2-Gα complex were used to extract a pharmacophore model for searching chemical databases. Docking of identified hits to RGS2 as well as other RGS structures was used to screen the hits for potent and selective RGS2 inhibitors. Whole cell assays showed the top 10 ranking compounds, AJ-1-AJ-10, to inhibit RGS2-Gαq interactions. Differential scanning fluorimetry showed AJ-3 to bind RGS2 but not Gαq. All 10 compounds inhibited the growth of several RGS2 expressing cancers in cell culture assays. In addition, AJ-3 inhibited the migration of LNCaP prostate cancer cells in wound healing assays. This is the first group of RGS2 inhibitors identified by structure-based approaches and that show anticancer activity. These results highlight the potential RGS2 inhibitors have to be a new class of chemotherapeutic agents.

RGS Proteins in Sympathetic Nervous System Regulation: Focus on Adrenal RGS4.

The sympathetic nervous system (SNS) consists largely of two different types of components: neurons that release the neurotransmitter norepinephrine (NE, noradrenaline) to modulate homeostasis of the innevrvated effector organ or tissue and adrenal chromaffin cells, which synthesize and secrete the hormone epinephrine (Epi, adrenaline) and some NE into the blood circulation to act at distant organs and tissues that are not directly innervated by the SNS. Like almost every physiological process in the human body, G protein-coupled receptors (GPCRs) tightly modulate both NE release from sympathetic neuronal terminals and catecholamine (CA) secretion from the adrenal medulla. Regulator of G protein Signaling (RGS) proteins, acting as guanosine triphosphatase (GTPase)-activating proteins (GAPs) for the Gα subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins), play a central role in silencing G protein signaling from a plethora of GPCRs. Certain RGS proteins and, in particular, RGS4, have been implicated in regulation of SNS activity and of adrenal chromaffin cell CA secretion. More specifically, recent studies have implicated RGS4 in regulation of NE release from cardiac sympathetic neurons by means of terminating free fatty acid receptor (FFAR)-3 calcium signaling and in regulation of NE and Epi secretion from the adrenal medulla by means of terminating cholinergic calcium signaling in adrenal chromaffin cells. Thus, in this review, we provide an overview of the current literature on the involvement of RGS proteins, with a particular focus on RGS4, in these two processes, i.e., NE release from sympathetic nerve terminals & CA secretion from adrenal chromaffin cells. We also highlight the therapeutic potential of RGS4 pharmacological manipulation for diseases characterized by sympathetic dysfunction or SNS hyperactivity, such as heart failure and hypertension.

RGS22 maintains the physiological function of ependymal cells to prevent hydrocephalus.

Ependymal cells line the wall of cerebral ventricles and ensure the unidirectional cerebrospinal fluid (CSF) flow by beating their motile cilia coordinately. The ependymal denudation or ciliary dysfunction causes hydrocephalus. Here, we report that the deficiency of regulator of G-protein signaling 22 (RGS22) results in severe congenital hydrocephalus in both mice and rats. Interestingly, RGS22 is specifically expressed in ependymal cells within the brain. Using conditional knock-out mice, we further demonstrate that the deletion of Rgs22 exclusively in nervous system is sufficient to induce hydrocephalus. Mechanistically, we show that Rgs22 deficiency leads to the ependymal denudation and impaired ciliogenesis. This phenomenon can be attributed to the excessive activation of lysophosphatidic acid receptor (LPAR) signaling under Rgs22-/- condition, as the LPAR blockade effectively alleviates hydrocephalus in Rgs22-/- rats. Therefore, our findings unveil a previously unrecognized role of RGS22 in the central nervous system, and present RGS22 as a potential diagnostic and therapeutic target for hydrocephalus.

A gain of function variant in RGS18 candidate for a familial mild bleeding syndrome

BackgroundInherited platelet diseases are bleeding disorders characterized by either defects in platelet count or platelet function, the latter being less common and very heterogeneous. Numerous gene variants associated with abnormal receptors, granules, and signaling pathways have been reported. Despite significant advancements in our understanding, many patients still lack a precise diagnosis. ObjectivesTo identify the genetic basis of a novel mild bleeding syndrome in a family exhibiting a selective defect of platelet aggregation. MethodsOur study included 6 family members across 3 generations who displayed reduced platelet aggregation in response to adenosine diphosphate, PAR1-AP, arachidonic acid, and epinephrine but not collagen. Platelet morphology, granule content, and expression of major surface glycoproteins were all found to be normal. Whole exome sequencing was performed for affected and nonaffected family members. ResultsWe identified RGS18, which encodes the regulator of G protein signaling (RGS) 18, as a candidate gene for the platelet function defect observed in this family. The RGS18 protein serves as a crucial negative regulator of G protein-coupled receptor signaling and coordinates the signaling pathways of natural platelet inhibitors. The heterozygous RGS18 c.643C>T, p.Arg215∗ variant was found to cosegregate among all 6 affected subjects. ConclusionTruncation at Arg215 removes the S216 and S218 phosphorylation sites, which are crucial regulatory domains for RGS18 activation. The impaired platelet function is thought to arise from excessive platelet downregulation due to constitutive activation of RGS18, resulting from a loss of association of the truncated form with the 14-3-3 protein.

Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice.

Addictive drugs hijack the neuronal mechanisms of learning and memory in motivation and emotion processing circuits to reinforce their own use. Regulator of G-protein Signaling 14 (RGS14) is a natural suppressor of post-synaptic plasticity underlying learning and memory in the hippocampus. The present study used immunofluorescence and RGS14 knockout mice to assess the role of RGS14 in behavioral plasticity and reward learning induced by chronic cocaine in emotional-motivational circuits. We report that RGS14 is strongly expressed in discrete regions of the ventral striatum and extended amygdala in wild-type mice, and is co-expressed with D1 and D2 dopamine receptors in neurons of the nucleus accumbens (NAc). Of note, we found that RGS14 is upregulated in the NAc in mice with chronic cocaine history following acute cocaine treatment. We found significantly increased cocaine-induced locomotor sensitization, as well as enhanced conditioned place preference and conditioned locomotor activity in RGS14-deficient mice compared to wild-type littermates. Together, these findings suggest that endogenous RGS14 suppresses cocaine-induced plasticity in emotional-motivational circuits, implicating RGS14 as a protective agent against the maladaptive neuroplastic changes that occur during addiction.

Distinct and overlapping RGS14 and RGS12 actions regulate NPT2A-mediated phosphate transport

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control serum phosphate levels by downregulating the renal Na-phosphate transporter NPT2A, thereby decreasing phosphate absorption and augmenting urinary excretion. This mechanism requires NHERF1, a PDZ scaffold protein, and is governed by the regulator of G protein signaling-14 (RGS14), which harbors a carboxy-terminal PDZ ligand that binds NHERF1. RGS14 is part of a triad of structurally related RGS proteins that includes RGS12 and RGS10. Like RGS14, RGS12 contains a class 1 PDZ ligand. However, unlike RGS14, the larger RGS12 contains an upstream PDZ-binding domain. The studies outlined here examined and characterized the binding of RGS12 with NHERF1 and NPT2A and its function on hormone-regulated phosphate transport. Immunoblotting experiments revealed RGS12 C-terminal PDZ ligand binding to NHERF1. Further structural analysis disclosed that NPT2A engaged full-length RGS12 and the upstream fragment containing the PDZ domain. Neither the downstream RGS12 portion nor RGS14 interacted with NPT2A. PTH and FGF23 profoundly inhibited phosphate uptake in opossum kidney proximal tubule cells. Transfection with human RGS14, or human RGS12, abolished hormone-sensitive phosphate transport as reported for human proximal tubule cells. RGS12 inhibitory activity resides in the downstream region and is comparable to RGS14. The carboxy-terminal RGS12(667–1447) splice variant is prominently expressed in the kidney and may contribute to regulating hormone-sensitive phosphate transport.

Selective optogenetic inhibition of Gαq or Gαi signaling by minimal RGS domains disrupts circuit functionality and circuit formation

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.

RGS10 deficiency facilitates distant metastasis by inducing epithelial–mesenchymal transition in breast cancer

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial–mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.

RGS10 deficiency facilitates distant metastasis by inducing epithelial-mesenchymal transition in breast cancer.

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial-mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.

Regulator of G-protein signaling expression in human intestinal enteroendocrine cells and potential role in satiety hormone secretion in health and obesity

Gut L-type enteroendocrine cells (EECs) are intestinal chemosensory cells that secrete satiety hormones GLP-1 and PYY in response to activation of G-protein coupled receptors (GPCRs) by luminal components of nutrient digestion and microbial fermentation. Regulator of G-protein Signaling (RGS) proteins are negative regulators of GPCR signaling. The expression profile of RGS in EECs, and their potential role in satiety hormone secretion and obesity is unknown. Transcriptomic profiling of RGS was completed in native colonic EECs was completed using single-cell RNA sequencing (scRNA-Seq) in lean and obesity, and human jejunal EECs with data obtained from a publicly available RNAseq dataset (GSE114853). RGS validation studies were completed using whole mucosal intestinal tissue obtained during endoscopy in 61 patients (n=42 OB, n=19 Lean); a subset of patients' postprandial plasma was assayed for GLP-1 and PYY. Exvivo human intestinal cultures and invitro NCI-H716cells overexpressing RGS9 were exposed to GLP-1 secretagogues in conjunction with a nonselective RGS-inhibitor and assayed for GLP-1 secretion. Transcriptomic profiling of colonic and jejunal enteroendocrine cells revealed a unique RGS expression profile in EECs, and further within GLP-1+ L-type EECs. In obesity the RGS expression profile was altered in colonic EECs. Human gut RGS9 expression correlated positively with BMI and negatively with postprandial GLP-1 and PYY. RGS inhibition in human intestinal cultures increased GLP-1 release from EECs exvivo. NCI-H716cells overexpressing RGS9 displayed defective nutrient-stimulated GLP-1 secretion. This study introduces the expression profile of RGS in human EECs, alterations in obesity, and suggests a role for RGS proteins as modulators of GLP-1 and PYY secretion from intestinal EECs. AA is supported by the NIH(C-Sig P30DK84567, K23 DK114460), a Pilot Award from the Mayo Clinic Center for Biomedical Discovery, and a Translational Product Development Fund from The Mayo Clinic Center for Clinical and Translational Science Office of Translational Practice in partnership with the University of Minnesota Clinical and Translational Science Institute.

Hsa-miR-874-3p Reduces Endogenous Expression of RGS4-1 Isoform In Vitro.

The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3'untranslated regions (3'UTRs) are known to regulate the levels of their mRNA splice variants. We constructed recombinant pmir-GLO vectors with a truncated 3' regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. The dual-luciferase reporter assay found that regions +401-+789, +789-+1152, and +1562-+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789-+1152 region in the 3'UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853-+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853-+859.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.

MGST3 regulates BACE1 protein translation and amyloidogenesis by controlling the RGS4-mediated AKT signaling pathway

Microsomal glutathione transferase 3 (MGST3) regulates eicosanoid and glutathione metabolism. These processes are associated with oxidative stress and apoptosis, suggesting that MGST3 might play a role in the pathophysiology of Alzheimer's disease (AD). Here, we report that knockdown (KD) of MGST3 in cell lines reduced the protein level of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the resulting amyloidogenesis. Interestingly, MGST3 KD did not alter intracellular ROS level but selectively reduced the expression of apoptosis indicators which could be associated with the receptor of cysteinyl leukotrienes (cysLTs), the downstream metabolites of MGST3 in arachidonic acid pathway. We then showed that the effect of MGST3 on BACE1 was independent of cysLTs but involved a translational mechanism. Further RNA-seq analysis identified that regulator of G-protein signaling 4 (RGS4) was a target gene of MGST3. Silencing of RGS4 inhibited BACE1 translation and prevented MGST3 KD-mediated reduction of BACE1. The potential mechanism was related to AKT activity, as the protein level of phosphorylated AKT (p-AKT) was significantly reduced by silencing of MGST3 and RGS4, and the AKT inhibitor abolished the effect of MGST3/RGS4 on p-AKT and BACE1. Together, MGST3 regulated amyloidogenesis by controlling BACE1 protein expression, which was mediated by RGS4 and downstream AKT signaling pathway.