Liu Lily, Wang Jinhai, Zheng Xianrui, Zhang Qin
College of Biological and Food Engineering, Southwest Forestry University, Kunming, 650224, China.
The Roslin Institute, University of Edinburgh, Edinburgh, EH25 9RG, UK.
Sci Rep. 2024 Dec 28;14(1):31310. doi: 10.1038/s41598-024-82774-0.
VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs) and is involved in ubiquitination. Ubiquitination is a critical system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in the regulation of lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell line (MAC-T) and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results showed that VPS28 knockdown significantly upregulated the levels of the fatty acid transporter CD36 molecule (CD36) and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, along with elevated ubiquitin (UB) levels, while reducing proteasome activity. In contrast, VPS28 overexpression increased CD36 levels while not significantly affecting ADFP or TG levels, with a trend toward reduced lipid droplets and increased UB expression and proteasome activity. In addition, inhibition of the ubiquitin-proteasome system and the endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, further increased CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo in a mouse model, where VPS28 knockdown increased mammary CD36, ADFP, UB expression, TG content, and lipid droplets without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine cell model.
VPS28（液泡蛋白分选28）是内体转运所需分选复合体（ESCRTs）的一个亚基，参与泛素化过程。泛素化是真核生物中蛋白质降解的关键系统。鉴于最近关于泛素化在脂质代谢调节中作用的研究结果，我们推测VPS28可能会影响参与乳脂肪合成的基因表达。为了验证这一假设，我们利用慢病毒介导的技术调节了牛乳腺上皮细胞系（MAC-T）中VPS28的表达，并测量了其对甘油三酯（TG）合成的影响。结果表明，VPS28基因敲低显著上调了脂肪酸转运蛋白CD36分子（CD36）和脂肪分化相关蛋白（ADFP）的水平，导致TG和脂肪酸生成增加，同时泛素（UB）水平升高，而蛋白酶体活性降低。相反，VPS28过表达增加了CD36水平，但对ADFP或TG水平没有显著影响，脂质滴有减少趋势，UB表达和蛋白酶体活性增加。此外，分别使用环氧霉素和氯喹抑制泛素-蛋白酶体系统和内体-溶酶体途径，进一步增加了CD36、ADFP和TG水平，从而提高了细胞活力。这些体外研究结果在小鼠模型中得到了体内验证，在该模型中，VPS28基因敲低增加了乳腺中CD36、ADFP、UB表达、TG含量和脂质滴，乳腺组织无病理变化，血液TG也无改变。这些结果证实了VPS28在通过泛素化途径调节TG合成中的关键作用，为牛细胞模型中乳脂肪生成的分子机制提供了新的见解。
