Investigating ubiquitination-regulated cell cycle events underpinning malaria transmission

疟疾是由单细胞病原体疟疾引起的，这种病原体威胁着全球约4亿人，每年导致50多万人死亡，因此仍然是一个重大的公共卫生问题，迫切需要新的治疗方法。要完成疟疾寄生虫复杂的生命周期，需要有导致疾病的哺乳动物宿主，以及负责传播疾病的蚊子媒介。因此，有效消除疟疾将需要治疗和传播阻断战略。与其他真核生物类似，疟原虫复制自己的基因组进行分裂、增殖和传播。然而，寄生虫基因组复制的几个不同寻常的特征可以被用来对抗寄生虫，特别是在寄生虫传播的关键阶段。寄生虫对蚊子的传播是由有性雄配子体和雌性配子体启动的。在经历了蚊子的环境后，雄配子体经历了三轮基因组复制(称为有丝分裂)，以令人难以置信的10分钟的速度形成与雌性细胞融合的精子样配子。然后受精的受精卵进行进一步的基因组复制(称为减数分裂)，发育成可移动的卵母细胞，负责感染蚊子。那么配子细胞的有丝分裂和受精卵的减数分裂是如何调节的呢？我们发现，配子体和受精卵中的许多蛋白质都被泛素这种小蛋白动态修饰。泛素可逆地附着在蛋白质上，调节它们的命运，包括它们的稳定性、细胞定位和活性水平，这表明这些可逆标记可能在寄生虫基因组复制过程中发挥关键作用。重要的是，作为泛素标记的擦除器，疟原虫USP7(泛素特异性蛋白酶7)对这种寄生虫完成有丝分裂和减数分裂至关重要。由于在实验上不可能研究人类疟疾寄生虫(恶性疟原虫)的减数分裂，我们将利用高度保守的啮齿动物疟疾模型(P.berghei)，研究USP7如何在有丝分裂和减数分裂期间调节基因组复制。在这项提案中，我们将揭示USP7如何在传播阶段为启动DNA复制准备寄生虫。利用最先进的蛋白质组学和显微技术，我们将确定USP7的伙伴和响应者，并确定寄生虫USP7的酶活性和结构如何与其宿主的等价物不同。我们的发现将对建立筛选药物性USP7抑制剂的平台产生影响。此外，确定USP7如何协调有丝分裂和减数分裂，将有助于开发针对和阻止寄生虫传播的多个步骤的改进治疗策略。

Malaria is caused by the unicellular pathogen Plasmodium that threatens around 400 million people globally and results in over 0.5 million deaths annually, thereby continuing to be a major public health problem, and urgently requiring new therapeutics. Completion of the malaria parasite's complex lifecycle requires a mammalian host where it causes disease, as well as a mosquito vector responsible for spread of the disease. Consequently, effective elimination of malaria will require both curative and transmission blocking strategies. Similar to other eukaryotes, Plasmodium replicates it's genome to divide, proliferate and spread. However, several unusual characteristics of parasite genome replication can be exploited to combat the parasite, especially in stages crucial for parasite transmission. Parasite transmission to the mosquito is initiated by sexual male and female gametocytes. Upon experiencing the mosquito environment, the male gametocyte undergoes three rounds of genome replication (called mitosis) with an incredible speed of 10 minutes to form sperm-like gametes that fuse with the female cell. The fertilised zygote then undergoes further genome replication (called meiosis) to develop into a motile ookinete that is responsible for infecting mosquitoes. So how is mitosis in gametocytes and meiosis in zygotes regulated? We discovered that many proteins in gametocytes and zygotes are dynamically modified by the small protein, ubiquitin. Ubiquitin is reversibly attached to proteins to modulate their fate, including their stability, cellular localisation and level of activity, suggesting these reversible marks could play a key role during parasite genome replication. Importantly, an eraser of ubiquitin marks, Plasmodium USP7 (ubiquitin specific protease 7) is crucial for the parasite to complete both mitosis and meiosis. Since it is not experimentally possible to study meiosis in the human malaria parasite (P. falciparum), we will exploit the highly conserved rodent malaria model (P. berghei), to examine how USP7 regulates genome replication both during mitosis and meiosis. In this proposal we will uncover how USP7 prepares the parasite for initiation of DNA replication during transmission stages. Using state-of-the-art proteomics and microscopy techniques, we will identify partners and responders of USP7 and also determine how the enzyme activity and structure of parasite USP7 is divergent from its host's equivalent. Our findings will be influential in establishing platforms to screen for pharmacological USP7 inhibitors. Moreover, identifying how USP7 orchestrates both mitosis and meiosis will be useful in the development of improved therapeutic strategies that target and block multiple steps in parasite transmission.