Aging Institute, University of Pittsburgh/UPMC, Pittsburgh, Pennsylvania, USA; Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Aging Institute, University of Pittsburgh/UPMC, Pittsburgh, Pennsylvania, USA.
J Biol Chem. 2023 Dec;299(12):105388. doi: 10.1016/j.jbc.2023.105388. Epub 2023 Oct 27.
The main protease of severe acute respiratory syndrome coronavirus 2, Mpro, is a key viral protein essential for viral infection and replication. Mpro has been the target of many pharmacological efforts; however, the host-specific regulation of Mpro protein remains unclear. Here, we report the ubiquitin-proteasome-dependent degradation of Mpro protein in human cells, facilitated by the human E3 ubiquitin ligase ZBTB25. We demonstrate that Mpro has a short half-life that is prolonged via proteasomal inhibition, with its Lys-100 residue serving as a potential ubiquitin acceptor. Using in vitro binding assays, we observed ZBTB25 and Mpro bind to each other in vitro, and using progressive deletional mapping, we further uncovered the required domains for this interaction. Finally, we used an orthologous beta-coronavirus infection model and observed that genetic ablation of ZBTB25 resulted in a more highly infective virus, an effect lost upon reconstitution of ZBTB25 to deleted cells. In conclusion, these data suggest a new mechanism of Mpro protein regulation as well as identify ZBTB25 as an anticoronaviral E3 ubiquitin ligase.
严重急性呼吸综合征冠状病毒 2 的主要蛋白酶（Mpro）是一种关键的病毒蛋白，对病毒感染和复制至关重要。Mpro 一直是许多药物研发努力的目标；然而，Mpro 蛋白的宿主特异性调节仍不清楚。在这里，我们报告了人类细胞中 Mpro 蛋白的泛素-蛋白酶体依赖性降解，这是由人类 E3 泛素连接酶 ZBTB25 介导的。我们证明 Mpro 具有短的半衰期，通过蛋白酶体抑制可以延长其半衰期，其 Lys-100 残基可能是一个潜在的泛素受体。通过体外结合实验，我们观察到 ZBTB25 和 Mpro 在体外相互结合，通过逐步缺失作图，我们进一步揭示了这种相互作用所需的结构域。最后，我们使用了一个同源的β-冠状病毒感染模型，观察到 ZBTB25 的基因缺失导致病毒的感染性更高，而在缺失的细胞中重新构建 ZBTB25 则丧失了这种作用。总之，这些数据表明了 Mpro 蛋白调节的一种新机制，并确定了 ZBTB25 作为一种抗病毒 E3 泛素连接酶。
