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.

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.

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.

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.

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.

Gßγ-SNAP25 exocytotic brake removal enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity.

Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gßγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gßγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gßγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gßγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gßγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gßγ signaling axes in yeast.

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gßγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gßγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gßγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gßγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gßγ signaling axes.

A self-inactivating invertebrate opsin optically drives biased signaling toward Gßγ-dependent ion channel modulation.

Animal opsins, light-sensitive G protein-coupled receptors, have been used for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Gα and Gßγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Gα- and Gßγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Gα and Gßγ Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for Gßγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gßγ-dependent and Gα-dependent responses. The opsin-induced transient Gi/o activation preferentially causes activation of the kinetically fast Gßγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gßγ-biased signaling properties were observed in a self-inactivating vertebrate visual pigment, Platynereis c-opsin1 requires fewer retinal molecules to evoke cellular responses. Furthermore, the Gßγ-biased signaling properties of Platynereis c-opsin1 are enhanced by genetically fusing with RGS8 protein, which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gßγ-dependent ion channel modulation.

A Gγ protein regulates alkaline sensitivity in crops.

The use of alkaline salt lands for crop production is hindered by a scarcity of knowledge and breeding efforts for plant alkaline tolerance. Through genome association analysis of sorghum, a naturally high-alkaline-tolerant crop, we detected a major locus, Alkaline Tolerance 1 (AT1), specifically related to alkaline-salinity sensitivity. An at1 allele with a carboxyl-terminal truncation increased sensitivity, whereas knockout of AT1 increased tolerance to alkalinity in sorghum, millet, rice, and maize. AT1 encodes an atypical G protein γ subunit that affects the phosphorylation of aquaporins to modulate the distribution of hydrogen peroxide (H2O2). These processes appear to protect plants against oxidative stress by alkali. Designing knockouts of AT1 homologs or selecting its natural nonfunctional alleles could improve crop productivity in sodic lands.

Multiple potassium channel tetramerization domain (KCTD) family members interact with Gßγ, with effects on cAMP signaling.

G protein-coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gßγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gßγ. However, comprehensive details on the KCTD interaction with Gßγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gßγ. In this study, we screened Gßγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer-based assay, we find that roughly half of KCTD proteins interact with Gßγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gßγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gßγ interaction. Finally, we demonstrated the functional consequence of the KCTD-Gßγ interaction by examining sensitization of the adenylyl cyclase-cAMP pathway in live cells. We found that Gßγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gßγ to shape GPCR signal transmission.

Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Disruption of the interaction between mutationally activated Gαq and Gßγ attenuates aberrant signaling.

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gßγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gßγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gßγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gßγ binding, overexpression of the G protein Gαo to sequester Gßγ, and siRNA depletion of Gß subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gßγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gßγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.

Features of BSCL2 related congenital generalized lipodystrophy in China: long-term follow-up of three patients and literature review.

OBJECTIVES: Congenital generalized lipodystrophy (CGL) is a group of rare autosomal inherited diseases characterized by a widespread loss of adipose tissue. The main purpose of this study was to evaluate the features of Chinese patients with CGL2. METHODS: Three patients diagnosed with CGL2 from our center were reviewed. Data on clinical features, results of laboratory analyses, and previous treatments were retrospectively collected. This study also reviewed studies that reported patients diagnosed with CGL2 in the last 30 years. RESULTS: All patients presented a lack of subcutaneous fat, hypertriglyceridemia, reversed triangular faces, acanthosis nigricans, and hepatomegaly within the first six months of life. All three patients developed splenomegaly, and mental retardation in later life. Dietary control dramatically lowered triglyceride levels in all patients. One patient presented with diabetes mellitus at 1 year-old. Although combined therapy with low fat diet and metformin maintained normal levels of blood lipid and glucose, this patient developed hypertrophic cardiomyopathy at the age of three. By a literature review on all Chinese cases with CGL2, it is known that classic manifestations such as hypertriglyceridemia, hepatomegaly and diabetes mellitus can occur shortly after birth, and early diagnosis and treatment can improve quality of life. In this cohort, the most frequent variations are c.782dupG and c.974dup in the BSCL2 gene. However, the same genotype may have different clinical phenotypes in patients with CGL2. CONCLUSIONS: This study not only described the clinical and genetic features of three patients with CGL2 in China, but also reviewed literature about CGL2 around the world.

Metreleptin Treatment in a Boy with Congenital Generalized Lipodystrophy due to Homozygous c.465_468delGACT (p.T156Rfs*8) Mutation in the BSCL2 Gene: Results From the First-year

Congenital generalized lipodystrophy (CGL) is a rare, autosomal recessive disorder characterized by an almost complete absence of body fat. In CGL, patients may have hyperphagia due to leptin deficiency. Recombinant human leptin (metreleptin) has been suggested as an effective treatment option. We present successful treatment with metreleptin in a boy with CGL and results from the first year of follow-up. An eight-month-old boy presented with excessive hair growth and a muscular appearance. On examination he had hypertrichosis, decreased subcutaneous adipose tissue over the whole body and hepatomegaly. Laboratory investigations revealed hypertriglyceridemia, hyperinsulinemia, elevated liver transaminases and low leptin levels. Molecular genetic analysis detected a homozygous, c.465_468delGACT (p.T156Rfs*8) mutation in the BSCL2 gene. A diagnosis of CGL type 2 was considered. Despite dietary intervention, exercise, and treatment with additional omega-3 and metformin, the hypertriglyceridemia, hyperinsulinemia, and elevated liver transaminase levels worsened. Metreleptin treatment was started and after one year hyperphagia had disappeared, and there was dramatic improvement in levels of insulin, hemoglobin A1c, triglycerides and liver transaminases. Hepatosteatosis was lessened and hepatosplenomegaly was much improved. Metreleptin appears to be an effective treatment option in children with CGL that remarkably improved metabolic complications in the presented case. Initiation of metreleptin treatment in the early period may decrease mortality and morbidity, and increase the quality of life in children with CGL.

A study of congenital generalized lipodystrophy (CGL) caused by BSCL2 gene mutation.

Congenital generalized lipodystrophy (CGL) is an extremely rare genetic disease mainly characterized by absence of whole-body adipose tissue and metabolic dysfunctions such as insulin resistance, diabetes mellitus, hypertriglyceridemia, hepatic steatosis, and acanthosis nigricans. In this study, we reported a novel case of a young woman patient with CGL. The patient came to the hospital for early-onset lipodystrophy and diabetes. She was 19-year-old with a height of 160 cm, a weight of 46 kg, BMI of 17.9 kg/m2, and a serum leptin level of 0.14 µg/L. Genomic DNA was extracted from blood samples of the patient and her family members, including her mother, father and brother. Genetic analysis revealed compound heterozygous mutations of the BSCL2 gene (c.560A>G and c.565G>T) in the patient. Her father carried a heterozygous mutation (c.565G>T), and her mother carried a heterozygous mutation (c.560A>G) in the BSCL2 gene. The mutant p.Y187C plasmid was transfected into HEK293T cells. The protein expression of SEIPIN and its interaction with glycerol-3-phosphate acyltransferase (GPAT3) were observed to be reduced. In addition, based on primary cultured skin fibroblasts from the patient, SEIPIN protein was decreased, and lipid droplets were much smaller when fatty acid was stimulated compared with those observed from healthy subject controls. However, histone deacetylase inhibitors (HDACis) was found capable of rescuing SEIPIN protein in fibroblasts of the patient. In addition, we further summarized and discussed gene mutations of BSCL2 reported in the current literature. Collectively, these findings have expanded the clinical phenotype and pathogenic gene spectrum of CGL, which might help clinicians to achieve better management of lipodystrophy.

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization.

Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.

G protein gamma subunit, a hidden master regulator of GPCR signaling.

Heterotrimeric G proteins (αßγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound ßγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation ßγ complexes translocate reversibly to internal membranes. The translocation kinetics of ßγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some ßγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses.

Causative links between ER stress and oxidative damage in a yeast model of human N88S seipinopathy.

Seipin is encoded by the gene Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) and FLD1/SEI1 in yeast. The gain-of-function N88S mutation in the BSCL2 gene was identified in a cohort of autosomal dominant motor neuron diseases (MNDs) collectively known as seipinopathies. Previous work has shown that this mutation disrupts N-glycosylation, leading to the formation of inclusion bodies (IBs) and contributing to severe Endoplasmic Reticulum (ER) stress and cell death. In this work, we established a humanized yeast model of N88S seipinopathy that recapitulated the formation of IBs and activation of the unfolded protein response (UPR) observed in mammalian systems. Autophagy and the Hrd1-mediated endoplasmic reticulum-associated degradation (ERAD) were fully functional in cells expressing mutant homomers and WT-mutant heteromers of seipin, discarding the possibility that mutant seipin accumulate due to impaired protein quality control systems. Importantly, the N88S seipin form IBs that appear to induce changes in ER morphology, in association with Kar2 chaperone and the Hsp104 disaggregase. For the first time, we have determined that N88S homo-oligomers expressing cells present reduced viability, decreased antioxidant activity and increased oxidative damage associated with loss of mitochondrial membrane potential, higher reactive oxygen species (ROS) levels and lipid peroxidation. This was correlated with the activation of oxidative stress sensor Yap1. Moreover, activation of ERAD and UPR quality control mechanisms were essential for proper cell growth, and crucial to prevent excessive accumulation of ROS in cells expressing N88S homomers solely. Overall, this study provides new insights into the molecular underpinnings of these rare diseases and offers novel targets for potential pharmacological intervention.

[Novel MFN2, BSCL2 and LRSAM1 variants in a cohort of Chinese patients with Charcot-Marie-Tooth disease].

Objective: Charcot-Marie-Tooth disease (CMT) comprises a group of clinically and genetically heterogeneous inherited neuropathies with an estimated prevalence of 1 in 2500. This study aimed to analyze the clinical and mutational characteristics of Chinese CMT patients with MFN2, BSCL2 and LRSAM1 variants. Methods: In this study, genetic analysis was performed in 206 Chinese patients at Chinese PLA General Hospital from December 2012 to March 2020 with clinical diagnosis of CMT, and reported variants of MFN2, BSCL2 and LRSAM1 related to CMT2. Results: We reported ten MFN2 mutations in ten unrelated patients (7 male, 3 female), two of whom had positive family history. Three novel mutations were detected including c.475-2A>G (splicing); c.687dupA (p.E230Rfs*16) and c.558dupT (p.S186fs). We reported three BSCL2 mutations of four unrelated patients, including c.461C>G (p.S154W), c.461C>T(p.S154L), and novel variants of c.1309G>C (p.A437P) and c.845C>T (p.A282V). Furthermore, two novel variants of LRSAM1, including c.1930G>T (p.G644C) and c.1178T>A (p.L393Q) were detected in two unrelated patients. Conclusion: Mutational spectrum of MFN2-, BSCL2-and LRSAM1-related CMT disease is expanded with the identification of novel variants in Chinese patients.

Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits.

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.