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1.
Mol Cell Biol ; 44(4): 123-137, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38747374

RESUMEN

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 S1PA→C 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)
Aparato de Golgi , Ratones Noqueados , Proproteína Convertasas , Animales , Ratones , Aparato de Golgi/metabolismo , Humanos , Proproteína Convertasas/metabolismo , Proproteína Convertasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal , Células HEK293 , Hígado/metabolismo , Proteolisis , Retículo Endoplásmico/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética
2.
Dev Cell ; 58(21): 2195-2205.e5, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37647897

RESUMEN

Brown adipose tissue (BAT) is a thermogenic organ that protects animals against hypothermia and obesity. BAT derives from the multipotent paraxial mesoderm; however, the identity of embryonic brown fat progenitor cells and regulators of adipogenic commitment are unclear. Here, we performed single-cell gene expression analyses of mesenchymal cells during mouse embryogenesis with a focus on BAT development. We identified cell populations associated with the development of BAT, including Dpp4+ cells that emerge at the onset of adipogenic commitment. Immunostaining and lineage-tracing studies show that Dpp4+ cells constitute the BAT fascia and contribute minorly as adipocyte progenitors. Additionally, we identified the transcription factor GATA6 as a marker of brown adipogenic progenitor cells. Deletion of Gata6 in the brown fat lineage resulted in a striking loss of BAT. Together, these results identify progenitor and transitional cells in the brown adipose lineage and define a crucial role for GATA6 in BAT development.


Asunto(s)
Adipocitos Marrones , Dipeptidil Peptidasa 4 , Animales , Ratones , Adipocitos Marrones/metabolismo , Adipogénesis , Tejido Adiposo Pardo/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Obesidad/metabolismo , Termogénesis/genética
3.
Nat Commun ; 11(1): 1128, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-32111832

RESUMEN

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/genética
4.
Atherosclerosis ; 281: 137-142, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30658189

RESUMEN

BACKGROUND AND AIMS: Cholesterol is an essential lipid for cellular function and membrane integrity, and hence its cellular levels and distribution must be tightly regulated. Biosynthesis of cholesterol is ramped when its cellular levels are low. Herein, the ER-resident and rate-limiting enzymes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) play a prominent role. We have recently reported that MARCH6, an E3 ubiquitin ligase, specifically promotes cholesterol-stimulated ubiquitylation and subsequent proteasomal degradation of SQLE, but not of HMGCR. To further delineate how post-translational regulation of SQLE and HMGCR is differentially achieved, we hypothesized that their sterol-dependent degradation machinery makes use of distinct E2 ubiquitin conjugating enzymes. METHODS: To study this possibility, we therefore used a CRISPR/Cas9-based approach to screen for ER-associated degradation (ERAD)-associated E2 enzymes that are essential for MARCH6-dependent degradation of SQLE. RESULTS: We report here the identification of UBE2J2 as the primary E2 ubiquitin conjugating enzyme essential for this process in mammalian cells, in contrast to UBE2G2, which is essential for sterol-stimulated degradation of HMGCR. We demonstrate that ablating UBE2J2 disturbs cholesterol-accelerated SQLE degradation in multiple human cell types, including cells of hepatic origin, and that the ability of UBE2J2 to support SQLE degradation critically depends on its enzymatic activity. CONCLUSIONS: Our findings establish UBE2J2 as an important partner of MARCH6 in cholesterol-stimulated degradation of SQLE, thereby contributing to the complex regulation of cellular cholesterol homeostasis.


Asunto(s)
Colesterol/biosíntesis , Hepatocitos/enzimología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Proteínas de la Membrana/metabolismo , Escualeno-Monooxigenasa/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Estabilidad de Enzimas , Células HEK293 , Células Hep G2 , Humanos , Proteínas de la Membrana/genética , Proteolisis , Factores de Tiempo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
5.
PLoS One ; 12(11): e0187424, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29095935

RESUMEN

Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to specific NRTIs has predominantly been assigned to mitochondrial polymerase-γ inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of first generation NRTIs, which are rarely discussed in the literature, include inhibition of oxygen consumption, decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that these effects can be normalized by addition of the anti-oxidant N-acetylcysteine (NAC), which suggests that ROS likely influence the onset and severity of adverse events upon drug exposure.


Asunto(s)
Fármacos Anti-VIH/farmacología , ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/envenenamiento , Animales , Caenorhabditis elegans/efectos de los fármacos , Humanos , Especies Reactivas de Oxígeno/metabolismo
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