A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.

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.

GRK specificity and Gßγ dependency determines the potential of a GPCR for arrestin-biased agonism.

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.

Role of the V2R-ßarrestin-Gßγ complex in promoting G protein translocation to endosomes.

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.

Seipin Deficiency Leads to Energy Dyshomeostasis via Inducing Hypothalamic Neuroinflammation and Aberrant Expression of Neuropeptides.

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.

G protein ß subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus.

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.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

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.

A unifying mechanism for seipin-mediated lipid droplet formation.

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.

Seipin is involved in oxygen-glucose deprivation/reoxygenation induced neuroinflammation by regulating the TLR3/TRAF3/NF-κB pathway.

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.

Seipin deficiency-induced lipid dysregulation leads to hypomyelination-associated cognitive deficits via compromising oligodendrocyte precursor cell differentiation.

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.

Structure of adenylyl cyclase 5 in complex with Gßγ offers insights into ADCY5-related dyskinesia.

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.

Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans.

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.

Adipose transplantation improves metabolism and atherosclerosis but not perivascular adipose tissue abnormality or vascular dysfunction in lipodystrophic Seipin/Apoe null mice.

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.

Effects of Seipin on Mouse Mesenchymal Stem Cell Osteo-Adipogenic Balance.

Seipin deficiency is an important cause of type 2 Berardinelli-Seip congenital dyslipidemia (BSCL2). BSCL2 is a severe lipodystrophy syndrome with lack of adipose tissue, hepatic steatosis, insulin resistance, and normal or higher bone mineral density. Bone marrow mesenchymal stem cells (BMSCs) are believed to maintain bone and fat homeostasis by differentiating into osteoblasts and adipocytes. We aimed to explore the role of seipin in the osteogenic/adipogenic differentiation balance of BMSCs. Seipin loxP/loxP mice are used to explore metabolic disorders caused by seipin gene mutations. Compared with wild-type mice, subcutaneous fat deficiency and ectopic fat accumulation were higher in seipin knockout mice. Microcomputed tomography of the tibia revealed the increased bone content in seipin knockout mice. We generated seipin-deficient BMSCs in vitro and revealed that lipogenic genes are downregulated and osteogenic genes are upregulated in seipin-deficient BMSCs. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) signaling is reduced in seipin-deficient BMSCs, while using the PPARγ activator increased the lipogenic differentiation and decreased osteogenic differentiation of seipin-deficient BMSCs. Our findings indicated that bone and lipid metabolism can be regulated by seipin through modulating the differentiation of mesenchymal stem cells. Thus, a new insight of seipin mutations in lipid metabolism disorders was revealed, providing a prospective strategy for MSC transplantation-based treatment of BSCL2.

Targeted massively parallel sequencing for congenital generalized lipodystrophy

ABSTRACT Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). Subjects and methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. Conclusions: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.