Istanbul Medipol University, School of Medicine, Dept. of Physiology, Istanbul, Turkey; Istanbul Medipol University, Regenerative and Restorative Medical Research Center, Istanbul, Turkey.
Istanbul Medipol University, Regenerative and Restorative Medical Research Center, Istanbul, Turkey; Imperial College London, Division of Computational and Systems Medicine, Dept. of Surgery and Cancer, London, UK.
Toxicol Appl Pharmacol. 2019 Sep 15;379:114686. doi: 10.1016/j.taap.2019.114686. Epub 2019 Jul 17.
Indolamine melatonin structurally resembles non-covalent proteasome inhibitors; however, the role of ubiquitin proteasome system (UPS) in neuronal survival and how melatonin carries out UPS inhibition remain largely unknown. With the use of melatonin treated cells, we evaluated the expression of Nedd4-1, an E3 ligase, how melatonin regulates its activity and its relationship with neuronal survival. Nedd4-1 was upregulated in the hypoxic condition in both control and Nedd4-1 overexpressed cells and melatonin treatment reversed its expression in both normoxic and hypoxic conditions, which was associated with increased cellular survival. Melatonin had no effect on the expression of Nedd4-1 at mRNA level. However, when melatonin was administered along with protein synthesis inhibitor cycloheximide, protein level of Nedd4-1 was further reduced, indicating that melatonin possibly downregulates Nedd4-1 after its synthesis. Notably, co-immunoprecipitation analyses followed by Liquid chromatography-Mass Spectrometry (LC-MS/MS) revealed that melatonin may dissociate ribosomal proteins, such as RS19, RL23A, and nucleophosmin from Nedd4-1, while 40S ribosomal protein S7 and 60S ribosomal protein L35 came into contact with Nedd4-1 upon melatonin treatment. By using IPA analyses, we obtained further data indicated novel target molecules of melatonin in hypoxic conditions, including OTOF, SF3B2, IPO5, ST13, FGFR3, Mx1/Mx2, playing roles in RNA splicing and trafficking, growth factor and interferon signaling. Here, we described a new insight into the role of melatonin in UPS functioning by proposing a molecular mechanism through which melatonin regulates Nedd4-1.
褪黑素在结构上类似于非共价蛋白酶体抑制剂；然而，泛素蛋白酶体系统 (UPS) 在神经元存活中的作用以及褪黑素如何执行 UPS 抑制作用在很大程度上仍然未知。使用褪黑素处理的细胞，我们评估了 E3 连接酶 Nedd4-1 的表达，褪黑素如何调节其活性及其与神经元存活的关系。Nedd4-1 在对照和 Nedd4-1 过表达细胞的缺氧条件下均上调，褪黑素处理在常氧和缺氧条件下逆转其表达，这与细胞存活增加有关。褪黑素对 Nedd4-1 的 mRNA 水平表达没有影响。然而，当褪黑素与蛋白质合成抑制剂环己酰亚胺一起给药时，Nedd4-1 的蛋白水平进一步降低，表明褪黑素可能在其合成后下调 Nedd4-1。值得注意的是，随后进行的免疫沉淀分析和液相色谱-质谱联用 (LC-MS/MS) 表明，褪黑素可能使核糖体蛋白，如 RS19、RL23A 和核仁磷酸蛋白从 Nedd4-1 上解离，而 40S 核糖体蛋白 S7 和 60S 核糖体蛋白 L35 在褪黑素处理后与 Nedd4-1 接触。通过 IPA 分析，我们获得了更多数据，表明在缺氧条件下褪黑素的新靶分子，包括 OTOF、SF3B2、IPO5、ST13、FGFR3、Mx1/Mx2，在 RNA 剪接和运输、生长因子和干扰素信号转导中发挥作用。在这里，我们通过提出褪黑素调节 Nedd4-1 的分子机制，描述了褪黑素在 UPS 功能中的作用的新见解。
