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.

Beyond the G protein α subunit: investigating the functional impact of other components of the Gαi3 heterotrimers.

BACKGROUND: Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gß and Gγ subunits and the effects of different Gßγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi3-based heterotrimers with different combinations of Gß and Gγ subunits. METHODS: Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gß1 or Gß2 heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi3-based heterotrimers in the plasma membrane, specifically with the dopamine D2 receptor (D2R). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes. RESULTS: Our results show that Gαi3 heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gß and Gγ subunits. Heterotrimers containing Gß2 showed slightly lower membrane localization compared to those containing Gß1, but certain combinations, such as Gαi3ß2γ8 and Gαi3ß2γ10, deviated from this trend. Examination of the spatial arrangement of Gαi3 in relation to D2R and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gß1 and Gγ8 or Gγ10 subunit. Deprivation of Gαi3 lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels. CONCLUSION: Our studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gß and Gγ subunits in G-protein signaling pathways. Video Abstract.

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.

Yeast Chaperone Hsp70-Ssb Modulates a Variety of Protein-Based Heritable Elements.

Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-ß ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb is known (and confirmed here) to modulate formation and propagation of the prion form of the Sup35 protein [PSI+]. Our new data show that both formation and mitotic transmission of the stress-inducible prion form of the Lsb2 protein ([LSB+]) are also significantly increased in the absence of Ssb. Notably, heat stress leads to a massive accumulation of [LSB+] cells in the absence of Ssb, implicating Ssb as a major downregulator of the [LSB+]-dependent memory of stress. Moreover, the aggregated form of Gγ subunit Ste18, [STE+], behaving as a non-heritable mnemon in the wild-type strain, is generated more efficiently and becomes heritable in the absence of Ssb. Lack of Ssb also facilitates mitotic transmission, while lack of the Ssb cochaperone Hsp40-Zuo1 facilitates both spontaneous formation and mitotic transmission of the Ure2 prion, [URE3]. These results demonstrate that Ssb is a general modulator of cytosolic amyloid aggregation, whose effect is not restricted only to [PSI+].

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 signaling regulates microtubule-dependent control of Golgi integrity.

Gßγ subunits regulate several non-canonical functions at distinct intracellular organelles. Previous studies have shown that Gßγ signaling at the Golgi is necessary to mediate vesicular protein transport function and to regulate mitotic Golgi fragmentation. Disruption of Golgi structure also occurs in response to microtubule depolymerizing agents, such as nocodazole. In this study, we use siRNA against Gß1/2 or specific Gγ subunits to deplete their expression, and show that their knockdown causes a significant reduction in nocodazole-induced Golgi fragmentation. We establish that knockdown of Gßγ or inhibition of Gßγ with gallein resulted in decreased activation of protein kinase D (PKD) in response to nocodazole treatment. We demonstrate that restricting the amount of free Gßγ available for signaling by either inhibiting Gαi activation using pertussis toxin or by knockdown of the non-GPCR GEF, Girdin/GIV protein, results in a substantial decrease in nocodazole-induced Golgi fragmentation and PKD phosphorylation. Our results also indicate that depletion of Gßγ or inhibition with gallein or pertussis toxin significantly reduces the microtubule disruption-dependent Golgi fragmentation phenotype observed in cells transfected with mutant SOD1, a major causative protein in familial amyotrophic lateral sclerosis (ALS). These results provide compelling evidence that Gßγ signaling is critical for the regulation of Golgi integrity.

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.

Exosomes derived from M1 macrophages inhibit the proliferation of the A549 and H1299 lung cancer cell lines via the miRNA-let-7b-5p-GNG5 axis.

Background: Almost all cells are capable of secreting exosomes (Exos) for intercellular communication and regulation. Therefore, Exos can be used as a natural therapeutic platform to regulate genes or deliver drugs to treat diseases. M1 macrophages inhibit tumor growth by releasing pro-inflammatory factors. This study explored the applicability of M1 macrophage exosomes (M1-Exos) as gene carriers and the effects on GNG5 protein, and further examined whether macrophage repolarization could inhibit tumor activity. Methods: M0 macrophages were polarized toward M1 using vitexin. Exos were obtained from M1 macrophages by ultra-centrifugation. The transwell non-contact co-culture system was used to co-culture M1 macrophages with HLF-α human lung epithelial cells or A549 or H1299 lung cancer cells. MTT, scratch, and transwell assays were used to detect the cell viability, migration, and invasion ability of cells in the four groups. Flow cytometry was used to detect the apoptosis rate of each group, and western blot (WB) analysis was performed to detect the change in the expression of proliferation- and apoptosis-related proteins. We screened the differentially expressed microRNAs using quantitative polymerase chain reaction technology. Luciferase reporter analysis was performed to explore the interaction between miRNA and protein. We used Xenografted A549 tumors in nude mice to study the effect of M1-Exos on tumor cell growth in vivo. Results: The results showed that, under the M1 macrophage co-culture system, lung cancer cell viability, invasion, and migration ability decreased, and the number of apoptotic cells increased, will all indicators being statistically significant (P < 0.05). The expression levels of PCNA, KI67, and Bcl-2 decreased significantly, but that of Bax increased (P < 0.05). Exosomes can have the same effect on tumor cells as M1 macrophages. Exosomes can transport miR-let-7b-5p to tumor cells, and miR-let-7b-5p can inhibit tumor cell proliferation and promote tumor cell apoptosis by regulating the GNG5 protein level. Conclusions: M1-Exos inhibit the proliferation, invasion, and metastasis of lung cancer cells through miRNA-let-7b-5p and GNG5 signaling pathways and inhibit the anti-apoptotic ability of lung cancer cells.

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.

Accelerated epigenetic aging and DNA methylation alterations in Berardinelli-Seip congenital lipodystrophy.

Berardinelli-Seip congenital lipodystrophy type 2 (CGL2) is a very rare human genetic disorder with potential significance to the understanding of the pathobiology of aging. CGL2 patients display characteristic progeroid features and suffer from type 2 diabetes, insulin resistance and fatty liver. In this study, we profiled genome-wide DNA methylation levels in CGL2 patients with BSCL2 mutations to study epigenetic age acceleration and DNA methylation alterations. This analysis revealed significant age acceleration in blood DNA of CGL2 patients using both first- and second-generation epigenetic clocks. We also observed a shortened lifespan of Caenorhabditis elegans following knockdown of the BSCL2 homolog seip-1 on a daf-16/forkhead box, class O mutant background. DNA methylation analysis revealed significant differentially methylated sites enriched for lyase activity, kinase regulator activity, protein kinase regulator activity and kinase activator activity. We could also observe significant hypomethylation in the promoter of the dual specificity phosphatase 22 gene when comparing CGL2 patients versus controls. We conclude that in line with the observed progeroid features, CGL2 patients exhibit significant epigenetic age acceleration and DNA methylation alterations that might affect pathways/genes of potential relevance to the disease.

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.

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.

Non-canonical Golgi-compartmentalized Gßγ signaling: mechanisms, functions, and therapeutic targets.

G protein Gßγ subunits are key mediators of G protein-coupled receptor (GPCR) signaling under physiological and pathological conditions; their inhibitors have been tested for the treatment of human disease. Conventional wisdom is that the Gßγ complex is activated and subsequently exerts its functions at the plasma membrane (PM). Recent studies have revealed non-canonical activation of Gßγ at intracellular organelles, where the Golgi apparatus is a major locale, via translocation or local activation. Golgi-localized Gßγ activates specific signaling cascades and regulates fundamental cell processes such as membrane trafficking, proliferation, and migration. More recent studies have shown that inhibiting Golgi-compartmentalized Gßγ signaling attenuates cardiomyocyte hypertrophy and prostate tumorigenesis, indicating new therapeutic targets. We review novel activation mechanisms and non-canonical functions of Gßγ at the Golgi, and discuss potential therapeutic interventions by targeting Golgi-biased Gßγ-directed signaling.

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.