Functional analysis of novel testis-expressed secreted and transmembrane proteins

项目摘要 我们团队的发现不是在提案中研究一个基因功能的传统方法，而是- 的方法使我们能够同时定义许多新基因的功能。我们 小鼠生殖道富集新基因的生物信息学发现及其功能的确定 模型提供了强有力的手段，将这些基因产物置于独特的生物途径中， 哺乳动物精子发生的分子过程。在R 01赠款的支持下，我们创建了 使用CRISPR/Cas9介导的基因组工程敲除超过150个新基因。我们优先考虑27个 编码分泌的、糖基磷脂酰肌醇（GPI）锚定的和跨膜蛋白的基因， 是潜在的避孕靶点和/或在精子发生或精子成熟或功能中起作用;敲除 这27个基因中有17个导致了生育缺陷。选择这些基因是因为它们 与男性不育症的潜在相关性及其作为男性避孕目标的可能性;约70%的FDA- 批准的药物靶向膜结合或分泌的蛋白质，因此这些蛋白质可以是靶点 用于抗体或小分子避孕药。到目前为止，这些R 01研究已经产生了38篇论文， 包括9篇发表在《科学》、《美国国家科学院院刊》和《公共科学图书馆遗传学》上的论文。在R 01补助金更新提案中，我们将继续 利用我们在信息学、生物化学、分子生物学、化学生物学和 操纵小鼠基因组。这一补助金更新的总体目标是机械地界定在 六种新的男性生殖道必需蛋白的体内功能，这些蛋白是精子通过生殖道所必需的。 子宫输卵管连接处（UTJ），并使用DNA编码化学技术（DEC-Tec）来寻找化学物质 可以作为非激素避孕药的实体。我们的假设是这些新的生殖道- 特定的蛋白质在精子功能所需的进化上保守的途径中起作用， 易受避孕目标战略的影响。使用DEC-Tec，我们已经筛选了数十亿 与避孕靶蛋白的复合文库，并确定了具有优异的 结构-富集关系（预测的特异性和泛抑制剂分子）。我们的假设是 在以下具体目的测试：1）定义五个睾丸特异性的分子作用机制 精子通过UTJ的蛋白质; 2）确定附睾的功能和功能域- 精子附睾成熟和通过UTJ转运的特定基因; 3）使用小分子 通过DEC-Tec筛选鉴定，以阻止精子通过UTJ。我们的原理验证研究 人类生殖遗传学和避孕发展的重要翻译意义：这些 精子UTJ转运蛋白和相互作用途径蛋白在不育男性中可能发生突变 并且是小分子避孕的有希望的靶点，这是NICHD科学愿景的优先领域。

PROJECT SUMMARY Rather than the traditional approach of studying the function of one gene in a proposal, our team’s discovery- based approaches have allowed us to define the functions of many novel genes simultaneously. Our bioinformatic discovery of novel reproductive tract-enriched genes and definition of their functions in mouse models provide powerful means to place these gene products into unique biological pathways and gain insight into the molecular processes of mammalian spermatogenesis. With the support of this R01 grant, we created knockouts of over 150 novel genes using CRISPR/Cas9-mediated genome engineering. We prioritized 27 genes that encode secreted, glycosylphosphatidylinositol (GPI)-anchored, and transmembrane proteins that are potential contraceptive targets and/or act in spermiogenesis or for sperm maturation or function; knockouts of 17 of these 27 genes resulted in fertility defects. These classes of genes were chosen because of their potential relevance to infertility in men and their likelihood as targets for male contraception; ~70% of FDA- approved drugs target either membrane-bound or secreted proteins, and thus these proteins could be targets for antibody-based or small molecule contraceptives. To date, these R01 studies have resulted in 38 papers, including 9 papers in Science, PNAS, and PLoS Genetics. In this R01 grant renewal proposal, we will continue to utilize our combined expertise in informatics, biochemistry, molecular biology, chemical biology, and manipulation of the mouse genome. The overall goals of this grant renewal are to mechanistically define the in vivo functions of six novel male reproductive tract-essential proteins that are required for sperm transit through the uterotubal junction (UTJ) and to use DNA-Encoded Chemistry Technology (DEC-Tec) to find chemical entities that can serve as non-hormonal contraceptives. Our hypothesis is that these novel reproductive tract- specific proteins act in evolutionarily conserved pathways required for sperm function, processes that are vulnerable to targeting strategies for contraception. Using DEC-Tec, we have already screened multi-billion compound libraries with contraceptive target proteins in this proposal and identified hits with excellent structure-enrichment relationships (both predicted specific and pan-inhibitor molecules). Our hypothesis will be tested in the following Specific Aims: 1) Define the molecular mechanisms of action of five testis-specific proteins in sperm transit through the UTJ; 2) Determine the functions and functional domains of an epididymis- specific gene in sperm epididymal maturation and transit through the UTJ; and 3) Use small molecules identified through DEC-Tec screens to block sperm transit through the UTJ. Our proof-of-principle studies have important translational implications for human reproductive genetics and contraceptive development: these essential sperm UTJ transit proteins and interacting pathway proteins are potentially mutated in infertile men and are promising targets for small molecule contraception, priority areas of the NICHD scientific vision.