SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn(2+)). The present studies were performed to identify the processes responsible for this effect. One of the well-established mechanisms for protein-level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4-2 (neural precursor cells expressed developmentally downregulated 4-2), and that Mn(2+) increases both Nedd4-2 mRNA and protein levels. Additionally, we have found that Mn(2+) exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn(2+) effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid-inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4-2. Additional findings establish that Mn(2+) increases astrocytic caspase-like proteolytic proteasome activity and that the Mn(2+)-dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N-acetyl-leu-leu-norleucinal and lactacystin. Combined, these results demonstrate that Mn(2+)-induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin-mediated proteolytic system.

中文翻译：

---

锰诱导的星形胶质细胞谷氨酰胺转运蛋白 SNAT3 的下调涉及泛素介导的蛋白水解系统。

SNAT3 是星形胶质细胞中谷氨酰胺 (Gln) 流出的主要促进剂，为神经元合成神经递质提供 Gln。我们以前的调查表明，在原代皮质星形胶质细胞培养物中，SNAT3 蛋白在接触锰 (Mn(2+)) 后会降解。目前的研究是为了确定造成这种影响的过程。蛋白质水平调节的成熟机制之一是通过泛素化进行翻译后修饰，这导致蛋白质通过 26S 蛋白酶体途径快速降解。在这里，我们显示星形胶质细胞 SNAT3 直接与泛素连接酶、Nedd4-2（神经前体细胞表达发育下调 4-2）相互作用，并且 Mn(2+) 增加了 Nedd4-2 mRNA 和蛋白质水平。此外，我们发现Mn(2+) 暴露提高星形细胞泛素B mRNA 表达、 游离泛素蛋白水平和总蛋白泛素化。此外，Mn(2+) 有效降低星形胶质细胞 mRNA 表达以及血清和糖皮质激素诱导激酶的磷酸化，这是一种调节蛋白，处于活性磷酸化形式，负责 Nedd4-2 的磷酸化和随后的失活。其他研究结果表明，Mn(2+) 增加星形细胞半胱天冬酶样蛋白水解蛋白酶体活性，并且 SNAT3 蛋白的 Mn(2+) 依赖性降解被蛋白酶体抑制剂、N-乙酰-leu-leu-norleucinal 和lactacystin 阻止。结合，