沙眼衣原体pORF5质粒蛋白通过激活Drp1诱导细胞线粒体自噬与线粒体分裂

Chlamydia trachomatis plasmid protein pORF5 induces mitophagy and mitochondrial fission by activating Drp1

摘要:

目的 探讨沙眼衣原体(Chlamydia trachomatis)质粒蛋白pORF5对细胞线粒体自噬及线粒体分裂的影响，并阐明其机制是否与Drp1激活相关。方法 采用慢病毒转染技术构建稳定表达pORF5基因的HeLa细胞株及对照细胞株，经血清饥饿处理后，采用Western blotting检测自噬相关蛋白微管相关蛋白1轻链3 (microtubule-associated protein light chain 3, LC3)、Beclin-1和泛素结合蛋白p62的表达水平，利用间接免疫荧光检测LC3与线粒体外膜转位酶20 (translocase of outer mitochondrial membrane 20, TOMM20)共定位水平；采用MitoTracker Red CMXRos对线粒体进行染色，利用激光共聚焦显微镜观察线粒体形态并分析相关形态参数；通过Western blotting和间接免疫荧光检测动力相关蛋白1 (dynamin-related protein 1, Drp1)的磷酸化水平及其在线粒体上的转位情况。为探究Drp1在细胞自噬和线粒体分裂中的作用，采用激光共聚焦及间接免疫荧光评估Drp1特异性线粒体分裂抑制剂1 (mitochondrial division inhibitor 1, Mdivi-1)预处理后细胞线粒体形态变化及Drp1转位情况，采用Western blotting检测自噬相关蛋白的表达水平，并运用间接免疫荧光法分析LC3的荧光强度及其与TOMM20的共定位情况。结果 与对照组相比，pORF5可显著上调LC3-Ⅱ和Beclin-1蛋白表达，下调p62表达，并增强LC3与TOMM20的共定位；pORF5表达导致线粒体网络结构碎片化；pORF5能促进Drp1 Ser616位点磷酸化，并增强Drp1在线粒体上的转位；使用Mdivi-1抑制Drp1后可减弱Drp1磷酸化与转位，使线粒体形态趋于延长；Mdivi-1抑制剂处理组LC3-Ⅱ、Beclin-1表达下调，p62表达上调，且LC3与TOMM20共定位减弱。结论 沙眼衣原体pORF5蛋白通过促进Drp1磷酸化及线粒体转位，进而诱导细胞线粒体自噬与线粒体分裂。

Abstract:

Objective To investigate the effects of Chlamydia trachomatis plasmid protein pORF5 on cellular mitophagy and mitochondrial fission and to elucidate whether its mechanism is related to Drp1 activation.Methods HeLa cells stably expressing pORF5 and control cells were constructed by lentiviral transfection. After serum starvation treatment, the expression levels of autophagy-related proteins—microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and p62—were determined by Western blotting. Co-localization of LC3 and translocase of outer mitochondrial membrane 20 (TOMM20) was assessed by indirect immunofluorescence. Mitochondria were stained with MitoTracker Red CMXRos, and mitochondrial morphology was observed and analyzed through confocal laser scanning microscopy. Dynamin-related protein 1 (Drp1) phosphorylation and its translocation to mitochondria were examined by Western blotting and indirect immunofluorescence. To explore the role of Drp1 in autophagy and mitochondrial fission, we pretreated cells with the Drp1-specific mitochondrial division inhibitor 1 (Mdivi-1). Changes in mitochondrial morphology and Drp1 translocation were evaluated by confocal microscopy and indirect immunofluorescence. Then, Western blotting was employed to determine the expression levels of autophagy-related proteins, and indirect immunofluorescence assay to analyze LC3 fluorescence intensity and its co-localization with TOMM20.Results Compared with the control group, pORF5 significantly upregulated the expression of LC3-Ⅱ and Beclin-1, downregulated the expression of p62, and enhanced the co-localization of LC3 and TOMM20. pORF5 expression led to the fragmentation of the mitochondrial network structure. It promoted Drp1 phosphorylation at Ser616 and enhanced Drp1 translocation to mitochondria. Inhibition of Drp1 with Mdivi-1 attenuated Drp1 phosphorylation and translocation, resulting in elongated mitochondrial morphology. In addition, the Mdivi-1 inhibitor group showed downregulated expression of LC3-Ⅱ and Beclin-1, upregulated the expression of p62, and attenuated co-localization of LC3 and TOMM20.Conclusion The C. trachomatis plasmid protein pORF5 may induce mitophagy and mitochondrial fission by promoting Drp1 phosphorylation and its mitochondrial translocation.