Regulating the trans-regulators: Investigating the PRMT7 molecular pathway as an epigenetic regulator of Leishmania spp. virulence

利什曼原虫威胁着全球四大洲的3.5亿人。利什曼病是第二致命的寄生虫病，巴西出现了这种多种疾病的所有病状。世界卫生组织估计，目前全球有1200万人感染，每年新增病例超过100万。目前没有疫苗，现有的利什曼病治疗受到日益增长的耐药性的威胁，而且往往无法应对发生和严重程度日益增加的急性流行病。迫切需要新的治疗方法和疫苗，英国和巴西政府承诺支持世界卫生组织最近的呼吁，进一步支持被忽视的热带病研究。单细胞利什曼原虫在其生命周期中会转变成许多不同的形式，以适应不同的宿主；从哺乳动物传染给白蛉，再通过白蛉叮咬传染给哺乳动物。只有某些生命周期阶段形式的利什曼原虫才能感染并在人类中存活。利什曼原虫的外观、代谢和毒力在这些转变过程中发生了重大变化，使它们能够生存。利什曼原虫的基因表达几乎完全依赖mRNA调控。为了应对环境的变化，特定的寄生虫蛋白结合mrna并将其定位为蛋白质生产，以指导和促进适应。控制这些寄生虫适应能力的蛋白质使它们能够在人类体内存活并感染人类。这些蛋白质对利什曼原虫感染的毒力和传播至关重要。我们最近分离出一种主要的控制板“调节器”蛋白PRMT7，它控制哺乳动物感染中的利什曼原虫毒力。目前已鉴定的利什曼原虫调节蛋白很少，这一发现代表了分离和检查这一调节途径并阻断寄生虫毒力的重大飞跃。为了研究PRMT7的调控途径并确定该蛋白的功能方式，我们组建了一个利什曼原虫PRMT蛋白、RNA调节因子和蛋白相互作用方面的专家团队。我们相信，对这一途径的深入了解将使我们能够阻止寄生虫建立人类感染。我们已经确定了PRMT7的一些下游靶蛋白，现在正试图确定它们是否受PRMT7的调控，以及它们是否参与利什曼原虫的毒力。利什曼原虫蛋白与人类蛋白不同；因此，我们可以利用这些差异来针对利什曼原虫特异性毒力因子，阻断它们的功能并阻止利什曼病的发展。重大发现将为利什曼病以及由相关寄生虫、恰加斯病和非洲锥虫病引起的疾病的研究提供见解。我们建议利用PRMT7毒力途径寻找更多利什曼原虫毒力的调节因子。我们将确定这些调节因子是如何起作用的，并测试它们是否对寄生虫的生存至关重要。基本调节因子可以作为潜在的药物靶点来阻断利什曼原虫感染。我们项目的新颖性和重要性有四个方面：1。PRMT7是迄今为止唯一在利什曼原虫中发现的蛋白精氨酸甲基转移酶，它似乎可以阻断毒性（Ferreira等人，2014）。2. 甲基化作为蛋白质修饰在利什曼原虫属寄生虫中是没有特征的。3. 调控RNA结合蛋白（rbp）在寄生虫生命周期分化、人类传染性和毒力中起重要作用，但在利什曼原虫中仍未发现。在所有寄生虫中，rbp和mRNA：蛋白复合物的三维分子结构在很大程度上是未知的，在利什曼原虫中则不存在。

Species of Leishmania threaten 350 million people worldwide on four continents. The Leishmaniases are the second deadliest parasitic disease and all pathologies of this diverse disease are present in Brazil. The World Health Organisation estimates 12 million people globally are currently infected and over 1 million new cases occur annually. No vaccine currently exists and available Leishmaniasis treatments are threatened by growing resistances and often overwhelmed by acute epidemics that are increasing in occurrence and severity. New treatments and vaccines are desperately needed and the UK and Brazilian governments are committed to the World Health Organisation's recent call to further support Neglected Tropical Disease research.The single-cell Leishmania parasite transforms into many different forms during its lifecycle to adapt to very different hosts; moving from mammals to sandflies and back to mammals by sandfly bites. Only Leishmania parasites of certain lifecycle stage forms can infect and survive in humans. Major changes to the Leishmania parasite's appearance, metabolism and virulence occur during these transitions that enable them to survive. Gene expression in Leishmania relies almost exclusively upon mRNA regulation. In response to changes in the environment, specific parasite proteins bind mRNAs and target them for protein production to guide and promote adaptation. Proteins that control the adaptation of these parasites enable them to survive in and infect humans. Such proteins are essential for the virulence and spread of the Leishmania parasite infection. We have recently isolated a major control panel "Regulator" protein, PRMT7, which controls Leishmania parasite virulence in mammalian infections. Very few Leishmania regulator proteins have yet been identified and this finding represents a major leap forward to isolate and examine this regulatory pathway and block parasite virulence. To study the PRMT7 regulation pathway and identify the way this protein functions, we have assembled a team of experts in Leishmania parasite PRMT proteins, RNA regulators and protein interactions. We believe insight into this pathway will enable us to block the parasite from establishing human infections. We have identified some downstream target proteins of PRMT7 and now seek to determine if they are regulated by PRMT7 and whether they participate in Leishmania parasite virulence. Leishmania proteins are different from human proteins; therefore we can use these differences to target Leishmania- specific virulence factors, block their function and block Leishmaniasis from developing. Significant findings will provide insight to Leishmaniasis research as well as diseases caused by related parasites, Chagas Disease and African Trypanosomiasis.We propose to find more regulators of Leishmania spp. virulence using the PRMT7 virulence pathway. We will identify how these regulators function, and test whether any are essential for parasite survival. Essential Regulators could serve as potential drug targets to block Leishmania parasite infection.The novelty and importance of our project is four fold:1. PRMT7 is the only Protein aRginine Methyl Transferase enzyme that has been characterised in Leishmania spp. parasites thus far and it appears to block virulence (Ferreira et al., 2014). 2. Methylation as a protein modification is uncharacterised in Leishmania spp. parasites. 3. Regulatory RNA binding proteins (RBPs) that are important in parasite lifecycle differentiation, human infectivity and virulence are largely unknown in Leishmania spp.4. The 3-dimensional molecular structures of RBPs and mRNA:protein complexes are largely unknown in all parasites and are absent in Leishmania spp.