Mechanisms of mitochondrial DNA replication licensing in trypanosomes

描述(由申请人提供)：维持基因组稳定性需要准确的DNA复制。真核细胞通过将许可因子招募到复制起始点来启动复制，然后通过26S蛋白酶体介导的许可参与者的降解来阻止DNA的再次复制，从而对复制起始点进行许可。真核生物还必须维持核外基因组线粒体DNA(MtDNA)的多个拷贝，但维持mtDNA拷贝数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复制的重要调节因子的基础上。布氏锥虫是一种原生动物寄生虫，含有一个不寻常的mtDNA网络，称为动泡DNA(KDNA)。该酶是已知的第一个调节kDNA复制的酶，也是第一个在真核生物中发现的细菌样HslVU酶，但它如何发挥其功能尚不清楚，主要是因为它的调节蛋白尚未鉴定。通过串联亲和纯化，我们在体内鉴定了两个与TbHslVU相关的新的线粒体蛋白，并在这一应用中探讨了它们在调节TbHslVU中的潜在作用以及它们可能参与的kDNA复制。我们推测，TbHslVU伙伴1和2的两个新蛋白VUP1和VUP2对TbHslVU具有调节作用。它们可以激活或抑制TbHslVU的活性，或调节TbHslVU复合体的组装，或介导底物识别。TbHslVU调节蛋白的鉴定表明，在锥体中对kDNA的复制和kDNA复制调控机制的复杂性具有额外的控制水平。最重要的是，由于在人类中没有发现TbHslVU及其合作伙伴VUP1和VUP2的同源物，它们是抗锥虫病化疗的潜在药物靶点。 公共卫生相关性：非洲人类锥虫病，又称昏睡病，是一种通过媒介传播的寄生虫病。目前世界卫生组织(WHO)估计，撒哈拉以南非洲36个国家约有6000万人面临感染风险，每年有30万至50万人感染。由于治愈这种疾病的药物很少，而且往往对人类有害，因此迫切需要进一步了解这种寄生虫和药物开发。锥虫具有一种独特的线粒体DNA复合体--动泡体DNA(KDNA)，其复制受到严格控制，对锥虫的生存至关重要。本应用中拟议的研究将通过鉴定和表征两个新的蛋白质VUP1和VUP2来探索kDNA复制许可的机制，这两个蛋白质都是新的线粒体蛋白水解酶TbHslVU的调节因子。TbHslVU是真核26S蛋白酶体复合体的结构等价物，在人类中没有同源基因。因此，TbHslVU及其调控蛋白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.