Department of Veterans' Affairs, VA-Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA.
J Biol Chem. 2023 Apr;299(4):103071. doi: 10.1016/j.jbc.2023.103071. Epub 2023 Feb 25.
Lipid droplets (LDs) are fat-storing organelles enclosed by a phospholipid monolayer, which harbors membrane-associated proteins that regulate distinct LD functions. LD proteins are degraded by the ubiquitin-proteasome system (UPS) and/or by lysosomes. Because chronic ethanol (EtOH) consumption diminishes the hepatic functions of the UPS and lysosomes, we hypothesized that continuous EtOH consumption slows the breakdown of lipogenic LD proteins targeted for degradation, thereby causing LD accumulation. Here, we report that LDs from livers of EtOH-fed rats exhibited higher levels of polyubiquitylated-proteins, linked at either lysine 48 (directed to proteasome) or lysine 63 (directed to lysosomes) than LDs from pair-fed control rats. MS proteomics of LD proteins, immunoprecipitated with UB remnant motif antibody (K-ε-GG), identified 75 potential UB proteins, of which 20 were altered by chronic EtOH administration. Among these, hydroxysteroid 17β-dehydrogenase 11 (HSD17β11) was prominent. Immunoblot analyses of LD fractions revealed that EtOH administration enriched HSD17β11 localization to LDs. When we overexpressed HSD17β11 in EtOH-metabolizing VA-13 cells, the steroid dehydrogenase 11 became principally localized to LDs, resulting in elevated cellular triglycerides (TGs). Ethanol exposure augmented cellular TG, while HSD17β11 siRNA decreased both control and EtOH-induced TG accumulation. Remarkably, HSD17β11 overexpression lowered the LD localization of adipose triglyceride lipase. EtOH exposure further reduced this localization. Reactivation of proteasome activity in VA-13 cells blocked the EtOH-induced rises in both HSD17β11 and TGs. Our findings indicate that EtOH exposure blocks HSD17β11 degradation by inhibiting the UPS, thereby stabilizing HSD17β11 on LD membranes, to prevent lipolysis by adipose triglyceride lipase and promote cellular LD accumulation.
脂滴（LDs）是由磷脂单层包裹的脂肪储存细胞器，其中含有调节不同 LD 功能的膜相关蛋白。LD 蛋白通过泛素蛋白酶体系统（UPS）和/或溶酶体降解。由于慢性乙醇（EtOH）消耗会降低 UPS 和溶酶体的肝功能，我们假设持续的 EtOH 消耗会减缓针对降解的生脂 LD 蛋白的分解，从而导致 LD 积累。在这里，我们报告说，来自 EtOH 喂养大鼠肝脏的 LD 表现出更高水平的多泛素化蛋白，与赖氨酸 48（定向于蛋白酶体）或赖氨酸 63（定向于溶酶体）连接，而不是与配对喂养的对照大鼠的 LD 相比。用 UB 残留基序抗体（K-ε-GG）免疫沉淀的 LD 蛋白的 MS 蛋白质组学鉴定了 75 种潜在的 UB 蛋白，其中 20 种被慢性 EtOH 给药改变。其中，羟甾类 17β-脱氢酶 11（HSD17β11）很突出。LD 级分的免疫印迹分析显示，EtOH 给药使 HSD17β11 向 LD 的定位富集。当我们在 EtOH 代谢的 VA-13 细胞中过表达 HSD17β11 时，类固醇脱氢酶 11 主要定位到 LD 上，导致细胞内三酰基甘油（TG）升高。乙醇暴露增加了细胞内 TG，而 HSD17β11 siRNA 降低了对照和 EtOH 诱导的 TG 积累。值得注意的是，HSD17β11 过表达降低了脂肪甘油三酯脂肪酶在 LD 上的定位。EtOH 暴露进一步降低了这种定位。VA-13 细胞中蛋白酶体活性的再激活阻止了 EtOH 诱导的 HSD17β11 和 TG 的升高。我们的发现表明，乙醇暴露通过抑制 UPS 来阻止 HSD17β11 的降解，从而稳定 HSD17β11 在 LD 膜上，防止脂肪甘油三酯脂肪酶的脂解作用，并促进细胞 LD 积累。
