The role of lysine methylation in regulating the motility of Toxoplasma gondii

描述（由申请人提供）：弓形虫病是由寄生虫弓形虫引起的。T.当免疫系统受损（如艾滋病患者）或发育不全（如胎儿）时，弓形虫可导致严重的组织损伤。如不及时治疗，T.弓形虫的增殖（即急性弓形虫病）将产生毁灭性的后果，包括弓形虫脑炎的发展。目前用于治疗弓形虫病的药物阻止了寄生虫的生长，但不能杀死寄生虫;因此必须长期服用，在此期间通常会产生严重的副作用。对于耐药寄生虫株感染，治疗选择要么非常有限，要么不存在。因此，迫切需要更特异性地针对寄生虫的新药。为了引起疾病，T.弓形虫必须通过宿主细胞侵入、复制和寄生虫排出来重复其裂解周期。 成功完成这个循环需要寄生虫感知环境条件的变化，并相应地在非运动状态和运动状态之间切换。尽管它在寄生虫生理学中的重要性，但调节这种开关的信号继电器却知之甚少。最近，我们发现了一个以前未知的调节T细胞运动的机制。弓形虫，介导的一种新的蛋白质赖氨酸甲基转移酶，AKMT（顶端复合赖氨酸（K）甲基转移酶）。当AKMT不存在时，寄生虫保持不动。侵袭和排出，以及因此整个溶解循环，都被抑制。如果我们了解这种抑制的详细性质，那么它可以被用来开发新的寄生虫特异性药物。为了实现这一目标，需要回答三个主要问题。1)在没有AKMT的情况下，发动机本身是否受损？2)无论运动是否受损，运动器官的其他重要组成部分是否依赖于甲基化？3)为什么，在功能方面，甲基化是每个敏感组分所必需的（即，是否需要正确组装器械？ATP水解或其他催化活性？正确的亚细胞定位？）为了回答这些关键问题，我们设计了以下三个具体目标：Aim1-确定AKMT是否直接调节肌球蛋白运动复合体的活性; Aim2-确定AKMT靶点; Aim3-确定AKMT靶点的功能和时空分布。我们将使用多方面的方法来实现我们的目标，结合蛋白质组学，最先进的成像技术，生物物理测定和靶向基因破坏。

DESCRIPTION (provided by applicant): Toxoplasmosis is caused by the parasite Toxoplasma gondii. The infection of T. gondii can cause severe tissue damages when the immune system is compromised (such as in AIDS patients) or underdeveloped (such as in fetuses). If not treated in time, uncontrolled T. gondii proliferation (i.e. acute toxoplasmosis) will have devastating consequences, including the development of toxoplasmic encephalitis. Current medications for treating toxoplasmosis drugs block growth but do not kill the parasite; thus they must be taken for long periods, during which severe side-effects routinely develop. For infection with drug resistant parasite strains, the treatment options either are very limited or do not exist. New drug that target parasites more specifically are therefore desperately needed. To cause disease, T. gondii must reiterate its lytic cycle through host cell invasion, replication, and parasite egress. The successful completion of this cycle requires that the parasite sense changes in environmental conditions and switch between non-motile and motile states, accordingly. Despite its importance in parasite physiology, the signal relay that regulates this switch is poorly understood. Recently we discovered a previously unknown mechanism of regulating cell motility in T. gondii, mediated by a novel protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). When AKMT is absent, the parasite remains immotile. Both invasion and egress, and thus the complete lytic cycle, are inhibited. If we understood the detailed nature of this inhibition, then it could be exploited to develop new parasite specific drugs. Towards that goal, three major questions need to be answered. 1) Is the motor itself crippled in the absence of AKMT? 2) Whether or not the motor is crippled, are other essential components of the motility apparatus dependent on methylation? 3) Why, in functional terms, is methylation required for each sensitive component (i.e., required for proper assembly of the apparatus?; for ATP hydrolysis or other catalytic activity?; for correct subcellular localization?) To answer these critical questions, we have designed the following three specific aims: Aim1-Determine if AKMT directly regulates the activity of the myosin motor complex; Aim2- Identify AKMT targets; and Aim3- Determine the function and spatial-temporal distribution of the AKMT targets. We will use a multi-faceted approach to pursue our aims, combining proteomics, state of the art imaging technologies, biophysical assays and targeted gene disruption.