Mechanisms of mitochondrial DNA replication licensing in trypanosomes

描述（由申请人提供）：基因组稳定性的维持需要精确的DNA复制。真核细胞通过向起始点招募许可因子来启动复制，随后通过26S蛋白酶体介导的许可因子降解来阻止DNA的再复制，从而获得复制起始点的许可。真核生物也必须维持核外基因组线粒体DNA （mtDNA）的多个拷贝，但mtDNA拷贝数维持的潜在机制仍然是一个谜。目前的建议旨在了解mtDNA复制许可的分子机制，并建立在我们最近发现的HslVU蛋白酶作为布鲁氏锥虫mtDNA复制的重要调节剂的基础上，布鲁氏锥虫是一种原生动物寄生虫，含有一个不寻常的mtDNA网络，称为着丝体DNA （kDNA）。该蛋白酶是已知的第一个kDNA复制调节剂，也是真核生物中发现的第一个细菌样HslVU蛋白酶，但人们对其如何发挥功能知之甚少，主要是因为其调节蛋白尚未确定。通过串联亲和纯化，我们在体内鉴定了两种与TbHslVU相关的新型线粒体蛋白，我们建议在本应用程序中研究它们在调节TbHslVU以及参与kDNA复制中的潜在作用。我们假设这两个新蛋白，分别被命名为VUP1和VUP2，分别代表TbHslVU伙伴1和2，作为TbHslVU的调节因子。它们可以激活或抑制TbHslVU的活性，或调节TbHslVU复合物的组装或介导底物识别。TbHslVU调控蛋白的鉴定表明，在锥虫中，对kDNA复制的控制和kDNA复制调控方案的复杂性存在额外的水平。最重要的是，由于在人类中没有发现TbHslVU及其伴侣VUP1和VUP2的同源物，因此它们是抗锥虫病化疗的潜在药物靶点。

DESCRIPTION (provided by applicant): Maintenance of genomic stability requires accurate DNA replication. Eukaryotic cells achieve this by licensing the replication origins through recruiting the licensing factors to the origins to initiate replication and subsequently by 26S proteasome-mediated degradation of the licensing actors to prevent DNA re-replication. Eukaryotes must also maintain multiple copies of an extranuclear genome, the mitochondrial DNA (mtDNA), but the underlying mechanism for maintenance of mtDNA copy number remains a mystery. The current proposal is aimed at understanding the molecular mechanism of mtDNA replication licensing and is built on our recent discovery of HslVU protease as an essential regulator of mtDNA replication in Trypanosoma brucei, a protozoan parasite that contains an unusual mtDNA network, known as the kinetoplast DNA (kDNA). This protease is the first known regulator of kDNA replication and first bacterial-like HslVU protease identified in a eukaryote, but how it exerts its function is poorly understood, mainly because its regulatory proteins are not identified. Through tandem affinity purification, we identified two novel mitochondrial proteins that associate with TbHslVU in vivo, and we propose in this application to examine their potential roles in regulating TbHslVU as well as their potential involvement in kDNA replication. We hypothesize that the two novel proteins, named VUP1 and VUP2 for TbHslVU Partner 1 and 2, function as regulators of TbHslVU. They could either activate or inhibit the activity of TbHslVU or regulate the assembly of TbHslVU complex or mediate substrate recognition. The identification of regulatory proteins of TbHslVU suggests an additional level of control over the replication of kDNA and the complexity of the regulatory scheme of kDNA replication in trypanosomes. Most importantly, since no homologs of TbHslVU and its partners, VUP1 and VUP2, are found in humans, they are potential drug targets for anti-trypanosomiasis chemotherapy. PUBLIC HEALTH RELEVANCE: Human African Trypanosomiasis, also known as sleeping sickness, is a vector-borne parasitic disease. Current World Health Organization (WHO) estimates that around sixty million people in thirty-six sub-Saharan African countries are at risk of infection and 300,000 to 500,000 people are infected each year. Since drugs to cure the disease are few and often toxic to humans, further understanding of the parasite and drug development are urgently needed. Trypanosomes possess a unique mitochondrial DNA complex, kinetoplast DNA (kDNA), whose replication is under tight control and is essential for trypanosome survival. The proposed studies in this application will explore the mechanism of kDNA replication licensing by identifying and characterizing two novel proteins, VUP1 and VUP2, both of which are regulators of a novel mitochondrial protease, TbHslVU. TbHslVU is a structural equivalent of the eukaryotic 26S proteasome complex and does not have a homolog in humans. Therefore TbHslVU and its regulatory proteins are potential drug targets for anti-trypanosomiasis chemotherapy.