RESUMEN
Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.
Asunto(s)
Dominios Proteicos , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Retículo Endoplásmico/metabolismo , Microscopía por Crioelectrón , Aparato de Golgi/metabolismo , Proproteína Convertasas/metabolismo , Proproteína Convertasas/genética , Colesterol/metabolismo , Animales , Células HEK293 , Transducción de SeñalRESUMEN
SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PAâC and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.
Asunto(s)
Proproteína Convertasas , Serina Endopeptidasas , Animales , Humanos , Ratones , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Hígado/metabolismo , Ratones Noqueados , Proproteína Convertasas/metabolismo , Proproteína Convertasas/genética , Proteolisis , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
The sterol regulatory element binding proteins (SREBPs) are transcription factors that govern cholesterol and fatty acid metabolism. We recently identified SPRING as a post-transcriptional regulator of SREBP activation. Constitutive or inducible global ablation of Spring in mice is not tolerated, and we therefore develop liver-specific Spring knockout mice (LKO). Transcriptomics and proteomics analysis reveal attenuated SREBP signaling in livers and hepatocytes of LKO mice. Total plasma cholesterol is reduced in male and female LKO mice in both the low-density lipoprotein and high-density lipoprotein fractions, while triglycerides are unaffected. Loss of Spring decreases hepatic cholesterol and triglyceride content due to diminished biosynthesis, which coincides with reduced very-low-density lipoprotein secretion. Accordingly, LKO mice are protected from fructose diet-induced hepatosteatosis. In humans, we find common genetic SPRING variants that associate with circulating high-density lipoprotein cholesterol and ApoA1 levels. This study positions SPRING as a core component of hepatic SREBP signaling and systemic lipid metabolism in mice and humans.
Asunto(s)
Metabolismo de los Lípidos , Hígado , Humanos , Femenino , Masculino , Animales , Ratones , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Metabolismo de los Lípidos/genética , Hepatocitos , Lipoproteínas HDLRESUMEN
PURPOSE OF REVIEW: The SREBP transcription factors are master regulators of lipid homeostasis owing to their role in controlling cholesterol and fatty acid metabolism. The core machinery required to promote their trafficking and proteolytic activation has been established close to 20âyears ago. In this review, we summarize the current understanding of a newly identified regulator of SREBP signaling, SPRING (formerly C12ORF49), its proposed mechanism of action, and its role in lipid metabolism. RECENT FINDINGS: Using whole-genome functional genetic screens we, and others, have recently identified SPRING as a novel regulator of SREBP signaling. SPRING is a Golgi-resident single-pass transmembrane protein that is required for proteolytic activation of SREBPs in this compartment. Mechanistic studies identified regulation of S1P, the protease that cleaves SREBPs, and control of retrograde trafficking of the SREBP chaperone SCAP from the Golgi to the ER as processes requiring SPRING. Emerging studies suggest an important role for SPRING in regulating circulating and hepatic lipid levels in mice and potentially in humans. SUMMARY: Current studies support the notion that SPRING is a novel component of the core SREBP-activating machinery. Additional studies are warranted to elucidate its role in cellular and systemic lipid metabolism.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Metabolismo de los Lípidos , Humanos , Ratones , Animales , Metabolismo de los Lípidos/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Colesterol/metabolismoRESUMEN
BACKGROUND: Galectins have numerous cellular functions in immunity and inflammation. Short-term galectin-2 (Gal-2) blockade in ischemia-induced arteriogenesis shifts macrophages to an anti-inflammatory phenotype and improves perfusion. Gal-2 may also affect other macrophage-related cardiovascular diseases. OBJECTIVES: This study aims to elucidate the effects of Gal-2 inhibition in atherosclerosis. METHODS: ApoE -/- mice were given a high-cholesterol diet (HCD) for 12 weeks. After 6 weeks of HCD, intermediate atherosclerotic plaques were present. To study the effects of anti-Gal-2 nanobody treatment on the progression of existing atherosclerosis, treatment with two llama-derived anti-Gal-2 nanobodies (clones 2H8 and 2C10), or vehicle was given for the remaining 6 weeks. RESULTS: Gal-2 inhibition reduced the progression of existing atherosclerosis. Atherosclerotic plaque area in the aortic root was decreased, especially so in mice treated with 2C10 nanobodies. This clone showed reduced atherosclerosis severity as reflected by a decrease in fibrous cap atheromas in addition to decreases in plaque size.The number of plaque resident macrophages was unchanged; however, there was a significant increase in the fraction of CD206+ macrophages. 2C10 treatment also increased plaque α-smooth muscle content, and Gal-2 may have a role in modulating the inflammatory status of smooth muscle cells. Remarkably, both treatments reduced serum cholesterol concentrations including reductions in very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein while triglyceride concentrations were unchanged. CONCLUSION: Prolonged and frequent treatment with anti-Gal-2 nanobodies reduced plaque size, slowed plaque progression, and modified the phenotype of plaque macrophages toward an anti-inflammatory profile. These results hold promise for future macrophage modulating therapeutic interventions that promote arteriogenesis and reduce atherosclerosis.
Asunto(s)
Aterosclerosis , Hiperlipidemias , Placa Aterosclerótica , Anticuerpos de Dominio Único , Animales , Antiinflamatorios/uso terapéutico , Apolipoproteínas E , Aterosclerosis/genética , Colesterol , Modelos Animales de Enfermedad , Galectina 2/farmacología , Galectina 2/uso terapéutico , Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoERESUMEN
The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.
Asunto(s)
Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Línea Celular , Desarrollo Embrionario/genética , Retículo Endoplásmico/metabolismo , Expresión Génica , Aparato de Golgi/metabolismo , Haploidia , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Placental inflammation and dysfunction during pregnancy are associated with short- and long-term adverse outcomes for the offspring. However, the mechanisms of vascular protection at the feto-placental interface are still poorly investigated. The high-density lipoprotein (HDL) associated sphingosine-1-phosphate (S1P) has been described as a powerful anti-inflammatory complex. This study aimed to elucidate the role of cord blood-derived HDL (nHDL) in feto-placental endothelial dysfunction. Here, we report that the exposure of primary fetal placental arterial endothelial cell (fPAEC) to healthy nHDL-S1P attenuated the ability of TNFα to activate NF-κB signaling and increase the expression of pro-inflammatory markers. Moreover, the angiotensin II (AngII)-induced reactive oxygen species (ROS) production was blunted in the presence of nHDL, whereas it was preserved when the cells were preincubated with S1P receptor antagonists, suggesting that S1P accounts for the vascular protective function of nHDL at the feto-placental unit. These results highlight the importance of HDL and S1P metabolism and signaling in pregnancy pathophysiology.