RESUMEN

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (CaV2.2) during presynaptic inhibition is a decades-old question. G proteins Gßγ bind to intracellular CaV2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gßγ acts by selectively inhibiting a subset of the four different CaV2.2 voltage-sensor domains (VSDs I to IV). During regular "willing" gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gßγ, CaV2.2 gating is "reluctant": pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gßγ inhibition of VSDs I and IV underlies reluctant CaV2.2 gating and subsequent presynaptic inhibition.

Asunto(s)

Canales de Calcio Tipo N , Activación del Canal Iónico , Canales de Calcio Tipo N/metabolismo , Animales , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Unión Proteica

RESUMEN

AIMS: In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4ß2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. MATERIALS AND METHODS: Loss-of-function strategies and mutants of arrestin2/PKCßII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. KEY FINDINGS: ERK activation via α4ß2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCßII, which exhibited mutual augmentation. Activation of PKCßII by α4ß2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin ß1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCßII signaling cascade, leading to the nuclear entry of PKCßII in a Gßγ-dependent manner. Upon nuclear localization, PKCßII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4ß2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCßII, which is reciprocally facilitated via positive feedback augmentation. SIGNIFICANCE: As α4ß2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4ß2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions.

Asunto(s)

Subunidades beta de la Proteína de Unión al GTP , Proteína Quinasa C beta , Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Proteína Quinasa C beta/metabolismo , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Animales , Transducción de Señal , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Núcleo Celular/metabolismo

RESUMEN

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

Asunto(s)

Microscopía por Crioelectrón , Guanosina Difosfato , Guanosina Trifosfato , Mutación , Humanos , Células HEK293 , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Unión Proteica , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética

RESUMEN

Seipin, a crucial protein for cellular lipid droplet (LD) assembly, oligomerizes at the interface between the endoplasmic reticulum and LDs to facilitate neutral lipid packaging. Using proximity labeling, we identified four proteins-Ldo45, Ldo16, Tgl4, and Pln1-that are recruited to the vicinity of yeast seipin, the Sei1-Ldb16 complex, exclusively when seipin function is intact, hence termed seipin accessory factors. Localization studies identified Tgl4 at the endoplasmic reticulum-LD contact site, in contrast to Ldo45, Ldo16, and Pln1 at the LD surface. Cells with compromised seipin function resulted in uneven distribution of these proteins with aberrant LDs, supporting a central role of seipin in orchestrating their association with the LD. Overexpression of any seipin accessory factor causes LD aggregation and affects a subset of LD protein distribution, highlighting the importance of their stoichiometry. Although single factor mutations show minor LD morphology changes, the combined mutations have additive effects. Lastly, we present evidence that seipin accessory factors assemble and interact with seipin in the absence of neutral lipids and undergo dynamical rearrangements during LD formation induction, with Ldo45 acting as a central hub recruiting other factors to interact with the seipin complex.

Asunto(s)

Subunidades gamma de la Proteína de Unión al GTP , Gotas Lipídicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Gotas Lipídicas/metabolismo , Retículo Endoplásmico/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/genética

RESUMEN

Lipodystrophy is a rare disorder which can be life-threatening. Here individuals fail to develop or maintain appropriate adipose tissue stores. This typically causes severe metabolic complications, including hepatic steatosis and lipoatrophic diabetes. There is no cure for lipodystrophy, and treatment options remain very limited. Here we evaluate whether tissue-selective adeno-associated virus (AAV) vectors can provide a targeted form of gene therapy for lipodystrophy, using a preclinical lipodystrophic mouse model of Bscl2 deficiency. We designed AAV vectors containing the mini/aP2 or thyroxine-binding globulin promoter to selectively target adipose or liver respectively. The AAV-aP2 vectors also contained the liver-specific microRNA-122 target sequence, restricting hepatic transgene expression. Systemic delivery of AAV-aP2 vectors overexpressing human BSCL2 restored adipose tissue development and metabolic health in lipodystrophic mice without detectable expression in the liver. High doses (1 × 1012 GCs) of liver-selective vectors led to off target expression and adipose tissue development, whilst low doses (1 × 1010 GCs) expressed selectively and robustly in the liver but did not improve metabolic health. This reveals that adipose tissue-selective, but not liver directed, AAV-mediated gene therapy is sufficient to substantially recover metabolic health in generalised lipodystrophy. This provides an exciting potential new avenue for an effective, targeted, and thereby safer therapeutic intervention.

Asunto(s)

Tejido Adiposo , Dependovirus , Terapia Genética , Vectores Genéticos , Lipodistrofia Generalizada Congénita , Hígado , Animales , Terapia Genética/métodos , Ratones , Dependovirus/genética , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Hígado/metabolismo , Humanos , Lipodistrofia Generalizada Congénita/terapia , Lipodistrofia Generalizada Congénita/genética , Lipodistrofia Generalizada Congénita/metabolismo , Tejido Adiposo/metabolismo , Modelos Animales de Enfermedad , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo

RESUMEN

Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαßγ proteins, followed by the recruitment of GPCR kinases and ßarrestin (ßarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both ßarr and Gßγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)-ßarr complex scaffolds Gßγ at the plasma membrane through a direct interaction with ßarr, enabling its transport to endosomes. Gßγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of ßarr in mediating sustained G protein signaling.

Asunto(s)

Endosomas , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Transporte de Proteínas , Receptores de Vasopresinas , beta-Arrestinas , Humanos , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/genética , Transducción de Señal

RESUMEN

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.

Asunto(s)

Quinasas de Receptores Acoplados a Proteína-G , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fosforilación , Animales , Transducción de Señal

RESUMEN

Seipin is one key mediator of lipid metabolism that is highly expressed in adipose tissues as well as in the brain. Lack of Seipin gene, Bscl2, leads to not only severe lipid metabolic disorders but also cognitive impairments and motor disabilities. Myelin, composed mainly of lipids, facilitates nerve transmission and is important for motor coordination and learning. Whether Seipin deficiency-leaded defects in learning and motor coordination is underlined by lipid dysregulation and its consequent myelin abnormalities remains to be elucidated. In the present study, we verified the expression of Seipin in oligodendrocytes (OLs) and their precursors, oligodendrocyte precursor cells (OPCs), and demonstrated that Seipin deficiency compromised OPC differentiation, which led to decreased OL numbers, myelin protein, myelinated fiber proportion and thickness of myelin. Deficiency of Seipin resulted in impaired spatial cognition and motor coordination in mice. Mechanistically, Seipin deficiency suppressed sphingolipid metabolism-related genes in OPCs and caused morphological abnormalities in lipid droplets (LDs), which markedly impeded OPC differentiation. Importantly, rosiglitazone, one agonist of PPAR-gamma, substantially restored phenotypes resulting from Seipin deficiency, such as aberrant LDs, reduced sphingolipids, obstructed OPC differentiation, and neurobehavioral defects. Collectively, the present study elucidated how Seipin deficiency-induced lipid dysregulation leads to neurobehavioral deficits via impairing myelination, which may pave the way for developing novel intervention strategy for treating metabolism-involved neurological disorders.

Asunto(s)

Diferenciación Celular , Disfunción Cognitiva , Subunidades gamma de la Proteína de Unión al GTP , Vaina de Mielina , Células Precursoras de Oligodendrocitos , Animales , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Ratones , Células Precursoras de Oligodendrocitos/metabolismo , Vaina de Mielina/metabolismo , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Disfunción Cognitiva/genética , Metabolismo de los Lípidos , Oligodendroglía/metabolismo , Oligodendroglía/patología , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , PPAR gamma/genética , Ratones Noqueados , Masculino , Rosiglitazona/farmacología

RESUMEN

Seipin is a key regulator of lipid metabolism, the deficiency of which leads to severe lipodystrophy. Hypothalamus is the pivotal center of brain that modulates appetite and energy homeostasis, where Seipin is abundantly expressed. Whether and how Seipin deficiency leads to systemic metabolic disorders via hypothalamus-involved energy metabolism dysregulation remains to be elucidated. In the present study, we demonstrated that Seipin-deficiency induced hypothalamic inflammation, reduction of anorexigenic pro-opiomelanocortin (POMC), and elevation of orexigenic agonist-related peptide (AgRP). Importantly, administration of rosiglitazone, a thiazolidinedione antidiabetic agent, rescued POMC and AgRP expression, suppressed hypothalamic inflammation, and restored energy homeostasis in Seipin knockout mice. Our findings offer crucial insights into the mechanism of Seipin deficiency-associated energy imbalance and indicates that rosiglitazone could serve as potential intervening agent towards metabolic disorders linked to Seipin.

Asunto(s)

Proteína Relacionada con Agouti , Metabolismo Energético , Subunidades gamma de la Proteína de Unión al GTP , Homeostasis , Hipotálamo , Ratones Noqueados , Proopiomelanocortina , Rosiglitazona , Animales , Ratones , Hipotálamo/metabolismo , Metabolismo Energético/efectos de los fármacos , Proopiomelanocortina/genética , Proopiomelanocortina/biosíntesis , Proteína Relacionada con Agouti/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Rosiglitazona/farmacología , Masculino , Enfermedades Neuroinflamatorias/etiología , Ratones Endogámicos C57BL , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Neuropéptidos/genética , Neuropéptidos/deficiencia , Regulación de la Expresión Génica/efectos de los fármacos

RESUMEN

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Asunto(s)

Fosfatidilinositol 3-Quinasa Clase I , Proteína de Unión al GTP rac1 , Proteínas de Unión al GTP rho , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Microscopía Fluorescente , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/química , Transducción de Señal , Fosfatidilinositol 3-Quinasa Clase I/química , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Asunto(s)

Analgésicos Opioides , Tolerancia a Medicamentos , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Ratones Endogámicos C57BL , Morfina , Nocicepción , Transducción de Señal , Animales , Morfina/farmacología , Tolerancia a Medicamentos/fisiología , Transducción de Señal/efectos de los fármacos , Ratones , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Masculino , Analgésicos Opioides/farmacología , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Nocicepción/efectos de los fármacos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/agonistas , Fosfolipasa C beta/metabolismo , Xantenos

RESUMEN

Lipid droplets (LDs) are dynamic organelles essential for cellular lipid homeostasis. Assembly of LDs occurs in the endoplasmic reticulum (ER), and the conserved ER membrane protein seipin emerged as a key player in this process. Here, we review recent advances provided by structural, biochemical, and in silico analysis that revealed mechanistic insights into the molecular role of the seipin complexes and led to an updated model for LD biogenesis. We further discuss how other ER components cooperate with seipin during LD biogenesis. Understanding the molecular mechanisms underlying seipin-mediated LD assembly is important to uncover the fundamental aspects of lipid homeostasis and organelle biogenesis and to provide hints on the pathogenesis of lipid storage disorders.

Asunto(s)

Retículo Endoplásmico , Subunidades gamma de la Proteína de Unión al GTP , Gotas Lipídicas , Gotas Lipídicas/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Humanos , Retículo Endoplásmico/metabolismo , Animales , Metabolismo de los Lípidos

RESUMEN

Seipin plays a crucial role in lipid metabolism and is involved in neurological disorders. However, the function and mechanism of action of seipin in acute ischemic stroke have not yet been elucidated. Here, we aimed to investigate the effect of seipin on neuroinflammation induced by oxygen-glucose deprivation/reoxygenation (OGD/R) and further explore the molecular mechanism by functional experiments. Our results revealed a significant decrease in seipin mRNA levels, accompanied by enhanced expression of TNF-α in patients with AIS, and a significant negative correlation between seipin and TNF-α was observed. Additionally, there was a negative correlation between seipin levels and the National Institutes of Health Stroke Scale (NIHSS) score. Furthermore, seipin levels were also decreased in middle cerebral artery occlusion/reperfusion (MCAO/R) mice and OGD/R-treated BV2 cells. RNA sequencing analysis showed that seipin knockdown altered the Toll-like receptor 3 (TLR3) signaling pathway. It was further confirmed in vitro that seipin knockdown caused significantly increased secretion of inflammatory factors including TNF-α, interleukin (IL)-1ß, and interferon (IFN)-ß. Meanwhile, seipin knockdown activated the Tlr3 signal pathway while this effect could be reversed by Tlr3 inhibitor in OGD/R treated BV2 cells. Furthermore, neuroinflammation induced by OGD/R was significantly reduced by seipin overexpression. Overall, our study demonstrate that seipin deficiency aggravates neuroinflammation by activating the TLR3/TRAF3/NF-κB signaling pathway after OGD/R stimuli, and suggest that seipin may be a potential therapeutic target for AIS.

Asunto(s)

Glucosa , FN-kappa B , Enfermedades Neuroinflamatorias , Oxígeno , Transducción de Señal , Factor 3 Asociado a Receptor de TNF , Receptor Toll-Like 3 , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Línea Celular , Modelos Animales de Enfermedad , Glucosa/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/inmunología , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/inmunología , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/metabolismo , FN-kappa B/metabolismo , Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética

RESUMEN

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.

Asunto(s)

Canales de Calcio Tipo T , Subunidades beta de la Proteína de Unión al GTP , Animales , Humanos , Ratas , Canales de Calcio Tipo R , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/química , Proteínas de Transporte de Catión , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Células HEK293 , Cinética , Técnicas de Placa-Clamp , Unión Proteica

RESUMEN

The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gßγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gßγ complexes in the presence of substrates/analogs, revealing two Gßγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gßγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gßγ binding sites and interdomain contacts altered by Gßγ binding suggest that Gßγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gßγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.

Asunto(s)

Fosfatidilinositol 3-Quinasa Clase Ib , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/química , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Sitios de Unión , Pez Cebra , Unión Proteica , Neutrófilos/metabolismo , Modelos Moleculares , Activación Enzimática , Conformación Proteica , Regulación Alostérica

RESUMEN

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gßγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gßγ and a dimeric form of AC5 that could be involved in its regulation. Gßγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gßγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gßγ and show reduced conditional activation by Gßγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gßγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

Asunto(s)

Adenilil Ciclasas , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/química , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/química , Modelos Moleculares , Células HEK293 , Multimerización de Proteína , Unión Proteica , Animales , Mutación , Conformación Proteica

RESUMEN

The global prevalence of nonalcoholic fatty liver disease (NAFLD) defined by the increased number of lipid droplets (LDs) in hepatocytes, have risen continuously in parallel with the obesity. LDs and related proteins are known to affect cellular metabolism and signaling. Seipin, one of the most important LD-related proteins, plays a critical role in LD biogenesis. Although the role of adipose tissue-specific Seipin silencing is known, hepatocyte-specific silencing upon cholesterol-mediated lipid accumulation has not been investigated. In our study, we investigated the effect of Seipin on endoplasmic reticulum (ER) stress and lipophagy in cholesterol accumulated mouse hepatocyte cells. In this direction, cholesterol accumulation was induced by cholesterol-containing liposome, while Seipin mRNA and protein levels were reduced by siRNA. Our findings show that cholesterol containing liposome administration in hepatocytes increases both Seipin protein and number of large LDs. However Seipin silencing reduced the increase of cholesterol mediated large LDs and Glucose-regulated protein 78 (GRP78) mRNA. Additionally, lysosome-LD colocalization increased only in cells treated with cholesterol containing liposome, while the siRNA against Seipin did not lead any significant difference. According to our findings, we hypothesize that Seipin silencing in hepatocytes reduced cholesterol mediated LD maturation as well as GRP78 levels, but not lipophagy.

Asunto(s)

Autofagia , Colesterol , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Subunidades gamma de la Proteína de Unión al GTP , Hepatocitos , Gotas Lipídicas , Estrés del Retículo Endoplásmico/fisiología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico/metabolismo , Animales , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Ratones , Gotas Lipídicas/metabolismo , Colesterol/metabolismo , Hepatocitos/metabolismo , Autofagia/fisiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas de Choque Térmico/metabolismo

RESUMEN

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gßγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gßγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

Asunto(s)

Péptido Relacionado con Gen de Calcitonina , Vasos Coronarios , AMP Cíclico , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Vasodilatación , Humanos , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Masculino , Persona de Mediana Edad , AMP Cíclico/metabolismo , Vasodilatación/efectos de los fármacos , Femenino , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Transducción de Señal/efectos de los fármacos , Técnicas In Vitro , Vasodilatadores/farmacología

RESUMEN

Adipose dysfunction in lipodystrophic SEIPIN deficiency is associated with multiple metabolic disorders and increased risks of developing cardiovascular diseases, such as atherosclerosis, cardiac hypertrophy, and heart failure. Recently, adipose transplantation has been found to correct adipose dysfunction and metabolic disorders in lipodystrophic Seipin knockout mice; however, whether adipose transplantation could improve lipodystrophy-associated cardiovascular consequences is still unclear. Here, we aimed to explore the effects of adipose transplantation on lipodystrophy-associated metabolic cardiovascular diseases in Seipin knockout mice crossed into atherosclerosis-prone apolipoprotein E (Apoe) knockout background. At 2 months of age, lipodystrophic Seipin/Apoe double knockout mice and nonlipodystrophic Apoe knockout controls were subjected to adipose transplantation or sham operation. Seven months later, mice were euthanized. Our data showed that although adipose transplantation had no significant impact on endogenous adipose atrophy or gene expression, it remarkably increased plasma leptin but not adiponectin concentration in Seipin/Apoe double knockout mice. This led to significantly reduced hyperlipidemia, hepatic steatosis, and insulin resistance in Seipin/Apoe double knockout mice. Consequently, atherosclerosis burden, intraplaque macrophage infiltration, and aortic inflammatory gene expression were all attenuated in Seipin/Apoe double knockout mice with adipose transplantation. However, adipocyte morphology, macrophage infiltration, or fibrosis of the perivascular adipose tissue was not altered in Seipin/Apoe double knockout mice with adipose transplantation, followed by no significant improvement of vasoconstriction or relaxation. In conclusion, we demonstrate that adipose transplantation could alleviate lipodystrophy-associated metabolic disorders and atherosclerosis but has an almost null impact on perivascular adipose abnormality or vascular dysfunction in lipodystrophic Seipin/Apoe double knockout mice.NEW & NOTEWORTHY Adipose transplantation (AT) reverses multiply metabolic derangements in lipodystrophy, but whether it could improve lipodystrophy-related cardiovascular consequences is unknown. Here, using Seipin/Apoe double knockout mice as a lipodystrophy disease model, we showed that AT partially restored adipose functionality, which translated into significantly reduced atherosclerosis. However, AT was incapable of reversing perivascular adipose abnormality or vascular dysfunction. The current study provides preliminary experimental evidence on the therapeutic potential of AT on lipodystrophy-related metabolic cardiovascular diseases.

Asunto(s)

Tejido Adiposo , Aterosclerosis , Subunidades gamma de la Proteína de Unión al GTP , Lipodistrofia , Ratones Noqueados , Animales , Ratones , Tejido Adiposo/metabolismo , Tejido Adiposo/trasplante , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Subunidades gamma de la Proteína de Unión al GTP/deficiencia , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Resistencia a la Insulina , Leptina/sangre , Leptina/metabolismo , Lipodistrofia/metabolismo , Lipodistrofia/genética , Lipodistrofia/patología , Ratones Endogámicos C57BL

RESUMEN

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

Asunto(s)

Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP Heterotriméricas , Lipodistrofia Generalizada Congénita , Lipodistrofia , Animales , Humanos , Lipodistrofia Generalizada Congénita/genética , Lipodistrofia Generalizada Congénita/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Lipodistrofia/genética