弓形虫TgAtg8-TgAtg3蛋白-蛋白相互作用在顶质体内稳态中的作用机制研究

Toxoplasma gondii autophagy-related protein 8 (TgAtg8) is usually recognized as the most important element in T. gondii autophagy pathway. However, recent data obtained in Toxoplasma have shed light on a very important function for TgAtg8 in the process of apicoplast inheritance. Nevertheless, very little is known how does TgAtg8 arrive at apicoplast membrane. We have previously determined that TgAtg8 can interact directly with TgAtg3 in a fixed ratio of 1 molecule of TgAtg8 bound to 1 molecule of TgAtg3. Furthermore, prevention of the protein-protein interaction of full length TgAtg8 with TgAtg3 suppressed the apicoplast DNA duplication and parasite proliferation. Therefore, we hypothesize there is possibility for parasite to regulate the localization of TgAtg8 on apicoplast by utilizing TgAtg3. To confirm this hypothesis, in this project, we are going to make positive and negative regulation of the TgAtg8-TgAtg3 protein-protein interaction by both gene regulation and positive and negative small molecular regulators strategies. Based on these strategies, the density of the protein-protein interaction, dynamic change of apicoplast and the proliferation of parasite would be observed in real time in order to confirm that the function of TgAtg8-TgAtg3 interaction is associated with the localization of TgAtg8, apicoplast homeostasis and division and proliferation of parasite and clarify further the relevant regulation mechanism. We forecast that the increase of expression level of TgAtg3 at the initial stage of parasite division will promote a bulk of free TgAtg8 to localize on the apicoplast membrane, which will be able to maintain the apicoplast inheritance and guarantee the normal division of parasite. In conjunction with our earlier work, our work will expand the biological function of TgAtg8-TgAtg3 protein-protein interaction, , which presents an attractive strategy for the development of drugs for treatment of infections caused by Toxoplasma gondii.

弓形虫自噬相关蛋白8（TgAtg8）已证实是自噬通路中关键蛋白。然而，近期发现TgAtg8还有促进顶质体遗传的功能，但TgAtg8如何到达顶质体发挥功能尚不清楚。我们前期证实TgAtg8-TgAtg3相互作用在顶质体DNA复制、虫体增殖中发挥作用。推测：虫体可能通过TgAtg3调控TgAtg8定位顶质体而发挥功能。本项目拟通过基因调控与特异性小分子化合物两种策略分别对该相互作用进行正、负调控，实时观察分裂过程中蛋白相互作用强度、顶质体动态变化与虫体增殖情况，明确该蛋白相互作用对TgAtg8定位、顶质体内稳态、虫体分裂增殖的作用，并揭示其作用机制。我们预测：虫体分裂初期TgAtg3表达量增加，通过蛋白相互作用促进大量游离TgAtg8定位顶质体，从而维持顶质体稳定遗传、保证虫体正常分裂。本研究将拓展TgAtg8-TgAtg3蛋白相互作用的生物学功能，也为研发高效、低毒抗弓形虫药物提供理论依据。

弓形虫自噬相关蛋白（Toxoplasma gondii Autophagy-related protein, TgAtg）8可调控速殖子的顶质体内稳态，但调控机制尚不清楚。基于Atg3是介导Atg8与膜结合的关键分子，我们推测TgAtg3参与调控TgAtg8介导的顶质体内稳态。.本研究首先构建了TgAtg3条件性敲减株，证实TgAtg3通过调控TgAtg8脂化从而维持顶质体内稳态及虫体分裂增殖。随后，利用点突变与pulldown实验明确了TgAtg8中第27位精氨酸是介导与TgAtg3相互作用的关键位点。同时构建了TgAtg8条件性敲减株，并以此为母本株，分别构建了TgAtg8野生型与R27E、G124A点突变补回株。以它们为研究对象，我们发现敲减TgAtg8可显著抑制虫体胞内增殖，但不影响入侵与出胞；当补回野生型TgAtg8后，虫体增殖恢复正常，但在补回点突变型TgAtg8后，虫体增殖无法恢复，充分证实TgAtg8全长蛋白（野生型补回株）、TgAtg8-TgAtg3相互作用（R27E补回株）及TgAtg8定位于膜表面的能力（G124A点突变补回株）对虫体增殖有重要调控作用。并且通过观察虫体分裂过程中顶质体丢失情况，证实TgAtg8调控虫体增殖主要与维持顶质体内稳态有关，而这一功能依赖于TgAtg8-TgAtg3的相互作用。最后，我们发现无论是敲减TgAtg8还是补回点突变蛋白，虫体胞内增殖能力被显著抑制、虫株毒力显著降低；当补回野生型TgAtg8后，虫体可正常增殖。表明阻断TgAtg8-TgAtg3相互作用可显著抑制虫体胞内增殖、降低虫株毒力。.至此，本课题首次阐明了TgAtg8蛋白中介导TgAtg8-TgAtg3相互作用的关键氨基酸位点，证实两者相互作用可通过调控TgAtg8参与的顶质体内稳态功能，从而维持虫体的胞内增殖与虫株毒力。此研究结果将为研发抗弓形虫药物提供重要的理论依据。