Department of Molecular Microbiology, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 3525433, Israel.
Cells. 2024 Nov 18;13(22):1900. doi: 10.3390/cells13221900.
causes amebiasis, a significant global health issue, with millions affected annually, especially in developing countries. EhDUF2419, an important protein involved in 's queuine salvage pathway and its interaction network, remains unclear. To explore this, we transfected trophozoites with a plasmid encoding Myc-tagged EhDUF2419 and achieved successful overexpression. Through immunoprecipitation with the Myc antibody followed by mass spectrometry, we identified 335 proteins interacting with Myc-tagged EhDUF2419, including over 100 ribosomal proteins, along with translation initiation and elongation factors, and aminoacyl-tRNA synthetases. Ribosome purification revealed the presence of EhDUF2419 in ribosomal protein-enriched fractions. Treatment with queuosine (Q) significantly reduced the EhDUF2419 protein levels and decreased the Q-modified tRNA in Myc-tagged EhDUF2419 overexpressing trophozoites. This effect, which was Q-dependent, was not observed in strains carrying an empty vector control or overexpressing a truncated form of EhDUF2419 lacking catalytic activity. The reduction in the EhDUF2419 protein levels was regulated by proteasome-mediated degradation, as evidenced by the reduced degradation in the presence of MG132, a proteasome inhibitor. Our study uncovers the novel interaction of EhDUF2419 with ribosomal proteins and its regulation by the proteasome machinery, providing new insights into its role in and potential therapeutic strategies.
导致阿米巴病，这是一个重大的全球健康问题，每年有数百万患者受到影响，尤其是在发展中国家。EhDUF2419 是参与“s 唾液酸补救途径及其相互作用网络”的重要蛋白质，其作用机制尚不清楚。为了探索这一点，我们用编码 Myc 标记的 EhDUF2419 的质粒转染滋养体，并成功实现了过表达。通过用 Myc 抗体进行免疫沉淀，然后进行质谱分析，我们鉴定出与 Myc 标记的 EhDUF2419 相互作用的 335 种蛋白质，其中包括 100 多种核糖体蛋白，以及翻译起始和延伸因子，以及氨酰-tRNA 合成酶。核糖体纯化显示 EhDUF2419 存在于富含核糖体蛋白的级分中。用 Queuosine (Q) 处理可显著降低 EhDUF2419 蛋白水平，并降低 Myc 标记的 EhDUF2419 过表达滋养体中的 Q 修饰 tRNA。这种效应是 Q 依赖性的，在携带空载体对照或过表达缺乏催化活性的 EhDUF2419 截断形式的菌株中未观察到。EhDUF2419 蛋白水平的降低受蛋白酶体介导的降解调节，这一点可以从蛋白酶体抑制剂 MG132 存在时降解减少得到证明。我们的研究揭示了 EhDUF2419 与核糖体蛋白的新相互作用及其受蛋白酶体机制的调节，为其在和潜在的治疗策略中的作用提供了新的见解。
