SUMMARY Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Over-expression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin−/− mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzyme’s affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro.

Cancer cells reprogram their metabolism to increase the synthesis of macromolecules for rapid proliferation. Compared to fatty acids, much less is known about the synthesis of phospholipids, which is essential for membrane biogenesis in cancer cells. We found that , which encodes lipin-1, a phosphatidic acid phosphatase (PAP) controlling the rate-limiting step in the phospholipid synthesis pathway, is highly up-regulated in basal-like triple-negative breast cancer (TNBC). Moreover, high expression correlates with the poor prognosis of these patients. Knockdown of increases apoptosis in basal-like TNBC cell lines, whereas it has minimal or less effect on normal human mammary gland epithelial cells (HMECs) and estrogen receptor-positive breast cancer cell lines. Fatty acid incorporation and lipidomics analyses showed that knockdown blocks phospholipid synthesis and changes membrane lipid compositions that ultimately induce the activation of 1 of the 3 branches of unfolded protein responses, the inositol-requiring enzyme-1α pathway. We also show for the first time, to our knowledge, that lipin-1 knockdown significantly inhibits tumor growth using an orthotopic xenograft breast mouse model. Our results suggest that lipin-1 is a potential target for cancer therapy.-He, J., Zhang, F., Tay, L. W. R., Boroda, S., Nian, W., Levental, K. R., Levental, I., Harris, T. E., Chang, J. T., Du, G. Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

Background: Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for DAG formation at the ER membrane during lipogenesis. Results: A combination of biochemical approaches is used to characterize lipin 2 phosphatase activity and regulation. Conclusion: The electrostatic charge of PA regulates activity, but phosphorylation does not.Significance: These findings demonstrate differential regulation of PAP activity within the lipin family.

Short guanine-rich sequences have a tendency to form quadruplexes that are stabilized by G-quartets with specific cation coordination. Quadruplexes are part of telomeres at the ends of chromosomes and play an important role in the regulation of gene expression. In addition, there is a strong interest in the therapeutic and biotechnological potential of quadruplex oligonucleotides. The HIV-integrase aptamer, d(GGGT)(4), demonstrated unusually favorable van't Hoff thermodynamics, and based on NMR studies the aptamer was proposed to fold into an antiparallel structure. Here we probed an apparent discrepancy between the NMR structure and the quadruplex topology suggested by circular dichroism (CD). Systematic thermodynamic analyses of d(GGGT)(4) and variants containing sequence modifications or missing specific nucleotides are consistent with a parallel quadruplex fold. CD studies carried out over a wide concentration range did not support a possible structural transition upon increasing strand concentration. Taken together, both optical and thermodynamic studies performed here strongly support a parallel fold for the d(GGGT)(4) aptamer.

Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.A dipose tissue plays an essential role in maintaining wholebody energy homeostasis by storing or releasing nutrients. This balance is controlled by opposing signaling pathways where anabolic processes are activated by insulin (INS) and catabolic actions are activated by catecholamines. An important unanswered question in adipose biology is how catecholamine-induced β-adrenergic signaling opposes insulin-stimulated glucose uptake (1-6). Surprisingly, the underlying mechanism for this wellestablished physiological response in adipocytes is still unknown.When nutrients are plentiful, insulin is released by the pancreas and stimulates the absorption of glucose and fatty acids in adipose tissue, where they are packaged and stored as triacylglycerol (TAG) in cellular lipid droplets. Insulin signaling in adipocytes is mediated by the phosphoinositide 3-kinase (PI3K)-Akt-mTOR pathway. mTOR is a highly conserved serine/threonine protein kinase that functions in either of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is defined primarily by the association of mTOR with raptor, whereas mTORC2 includes mTOR with rictor (7). Importantly, mTORC2 phosphorylation of Akt at S473 is required for Akt activity on AS160, which is necessary for glucose uptake in response to insulin (8-11). Of note, for both mTORC1 and mTORC2, the integrity of these protein complexes is essential for kinase substrate specificity and proper signaling (12, 13).During periods of fasting or stress, catecholamines are released by the sympathetic nervous system to activate lipolysis. Stimulation of the β-adrenergic receptor on adipocytes activates adenylyl cyclase (AC), leading to elevated cAMP and protein kinase A (PKA) activity. PKA initiates lipolysis by direct phosphorylation of hormone-sensitive lipase (HSL) and perilipin (14-16) and indirect activation of adipose triglyceride lipase (ATGL) (17-19). Lipolysis involves hydrolysis of TAG stored in the lipid droplet to produce diacylglycerol (DAG), mo...

Lipins 1, 2, and 3 are Mg-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation.