Investigating ubiquitination-regulated cell cycle events underpinning malaria transmission

疟疾是由单细胞病原体疟原虫引起的，其威胁全球约4亿人，每年导致超过50万人死亡，因此仍然是一个主要的公共卫生问题，迫切需要新的治疗方法。疟疾寄生虫复杂的生命周期的完成需要一个哺乳动物宿主，在那里它引起疾病，以及蚊子媒介负责疾病的传播。因此，要有效消除疟疾，就需要采取治疗和阻断传播的战略。与其他真核生物相似，疟原虫复制其基因组以分裂，增殖和传播。然而，寄生虫基因组复制的几个不寻常的特征可以用来对抗寄生虫，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生殖的雄性和雌性配子母细胞启动的。在经历蚊子的环境后，雄性配子体以10分钟的惊人速度经历了三轮基因组复制（称为有丝分裂），形成与雌性细胞融合的精子样配子。受精后的受精卵进一步进行基因组复制（称为减数分裂），发育成一个能动的动合子，负责感染蚊子。那么配子母细胞的有丝分裂和合子的减数分裂是如何被调控的呢？我们发现配子母细胞和合子中的许多蛋白质被小分子蛋白泛素动态修饰。泛素可逆地附着在蛋白质上以调节它们的命运，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在寄生虫基因组复制过程中可能发挥关键作用。重要的是，作为泛素标记的橡皮擦，疟原虫USP 7（泛素特异性蛋白酶7）对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（伯氏疟原虫）来研究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如何准备寄生虫在传播阶段启动DNA复制。使用最先进的蛋白质组学和显微镜技术，我们将确定USP7的合作伙伴和响应者，并确定寄生虫USP7的酶活性和结构如何与其宿主的等效物不同。我们的研究结果将在建立筛选药理学USP7抑制剂的平台方面产生影响。此外，确定USP7如何协调有丝分裂和减数分裂将有助于开发改进的治疗策略，靶向和阻断寄生虫传播的多个步骤。

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.