Table_2_The Photorhabdus Virulence Cassettes RRSP-Like Effector Interacts With Cyclin-Dependent Kinase 1 and Causes Mitotic Defects in Mammalian Cells.xlsx

The “Photorhabdus virulence cassettes” (PVCs) secreted by Photorhabdus are defined as “extracellular contractile injection systems” (eCISs) and can deliver effectors to eukaryotic hosts for cytotoxicity. Previously, we demonstrated the cryogenic electron microscopy (cryo-EM) structure and assembly process of an intact PVC particle from Photorhabdus asymbiotica. In this work, we characterized the biological functions of a PVC effector, which is defined as a homologous protein of Ras/Rap1-specific endopeptidase domain (RRSP) in the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin from Vibrio vulnificus. In this work, we found that the RRSP homologous protein (RRSPPa) was associated with inhibition of cell proliferation and increased cell apoptosis and death of HeLa cells. Furthermore, we discovered that RRSPPa disturbed mitotic progression, including the induction of cell cycle alteration, retardation of cell abscission time, and regression of the cleavage furrow. In addition, we revealed that RRSPPa could target the cyclin-dependent kinase 1 (CDK1) protein and block activation of CDK1 through inhibition of Thr161 phosphorylation, which partially explained the crucial role of this effector in cell mitosis.

由发光杆菌（Photorhabdus）分泌的"光杆状菌毒力盒（Photorhabdus virulence cassettes，PVCs）"被定义为"细胞外收缩注射系统（extracellular contractile injection systems，eCISs）"，可将效应蛋白递送至真核宿主以发挥细胞毒性。此前，我们解析了不对称发光杆菌（Photorhabdus asymbiotica）来源的完整PVC颗粒的冷冻电子显微镜（cryo-EM）结构及其组装过程。本研究对一种PVC效应蛋白的生物学功能进行了表征，该蛋白为创伤弧菌（Vibrio vulnificus）来源的多功能自加工重复毒素（multifunctional autoprocessing repeats-in-toxin，MARTX毒素）中Ras/Rap1特异性内切酶结构域（Ras/Rap1-specific endopeptidase domain，RRSP）的同源蛋白。本研究发现，该RRSP同源蛋白（RRSPPa）可抑制HeLa细胞增殖，并促进细胞凋亡与死亡。进一步研究表明，RRSPPa可干扰细胞有丝分裂进程，具体表现为诱导细胞周期异常、延长胞质分裂时长以及引发分裂沟回缩。此外，本研究揭示RRSPPa可靶向结合细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1，CDK1），通过抑制其苏氨酸161（Thr161）位点的磷酸化阻断CDK1的激活，这一机制部分解释了该效应蛋白在细胞有丝分裂中发挥的关键作用。