弓形虫ROP18激酶磷酸化宿主RTN1-C促进神经细胞凋亡的分子机制研究

Toxoplasma gondii is a neurotropic parasite, and the central nervous system (CNS) is the most easily damaged of all invaded organs. The ROP18 kinase has been identified as a key virulence molecule conferring a high mortality phenotype characteristic of type I T.gondii strains. Our preliminary data indicated that ROP18 significantly induced the apoptosis of neural cells, and the activity of ROP18 kinase is required for the apoptosis of neural cells. To identify the host targets of ROP18, we employed a yeast two-hybrid assay to screen for ROP18-interacting proteins. To our surprise, we found that RTN1-C, an endoplasmic reticulum (ER) protein preferentially expressing in the CNS, directly interacted with ROP18. Previous study showed that RTN1-C acts as a molecular switch to induce ER stress and mitochondria mediated apoptosis in human neural cells. Therefore, our central hypothesis is that the interaction between ROP18 and the host RTN1-C may be responsible for neuropathogenic action of Toxoplasmosis. Strikingly, our preliminary results also found that ROP18 kinase directly phosphorylated RTN1-C at Ser/Thr sites. According to these results, the present study will analyze the apoptosis of neural cells infected with expressing ROR18 wild type, kinase death and ROR18 knockout transgenic strains. We will uncover the molecular mechanism of ER stress and mitochondria-mediated apoptosis pathway induced by ROP18/RTN1-C interaction. We propose to define the interaction domains of ROP18/RTN1-C and determine the special phosphorylated sites. We will define the related key molecules targeted by the interaction of ROP18/RTN1-C using siRNA, mutation and inhibitors of signal pathway. The systematic studies proposed in this application will be relevant in interpreting reports of neuropathogenesis during T. gondii infection and provide experiment basis of targeted therapy for blocking the progress of Toxoplasmic encephalitis.

对神经系统的偏嗜性是弓形虫感染的显著特征，然而原因尚不清楚。我们前期发现中枢神经组织特异性表达蛋白RTN1-C是弓形虫毒力因子ROP18激酶的直接作用的宿主蛋白；ROP18的激酶活性是神经细胞凋亡的重要因素；鉴于RTN1-C自身可通过线粒体和内质网应激(ERS)相关通路引起细胞凋亡，我们推测弓形虫ROP18激酶磷酸化RTN1-C进而激活线粒体和ERS相关的凋亡通路引起神经损伤，可能是高致病性弓形虫感染致神经损伤的重要原因。本课题拟采用ROP18野生型和激酶失活型转基因虫株感染神经细胞，分析ROP18/RTN1-C靶向的凋亡通路关键蛋白；探究两者之间相互作用机制并鉴定作用功能域和具体磷酸化位点；通过siRNA、点突变和信号通路阻断技术寻找和验证ROP18/RTN1-C对凋亡通路中关键环节的影响。该研究不仅能解释弓形虫对中枢神经系统偏嗜性的原因，也为阻断疾病进程和针对性治疗提供研究基础。

本项目研究揭示了急性期弓形虫病引起弓形虫脑炎致神经损伤的分子机制。ROP18是由虫体棒状体分泌的Ser/Thr 蛋白激酶，与虫体的毒力密切相关。研究发现：中枢神经组织特异性表达的内质网蛋白RTN1-C是弓形虫毒力因子ROP18 激酶的直接作用的宿主蛋白；ROP18磷酸化RTN1-C的Ser7/134，Thr4/8/118 位点，磷酸化修饰的RTN1-C可使组蛋白去乙酰化酶HDAC的活性下降，进而引起定位于内质网的伴侣蛋白GRP78发生乙酰化，诱发内质网应激以及下游的内质网应激相关的凋亡通路活化，这是弓形虫脑炎病理损伤的重要机制之一；同时，鉴于RTN1-C是特异性表达于中枢神经系统的蛋白，ROP18-RTN1-C 之间的特异性相互作用，可能是弓形虫对神经系统偏嗜性的一个重要原因。