Center for Pediatric Research

项目摘要 在桑福德研究所建立的儿科研究中心的目标是建立一个 多学科中心，支持具有广泛兴趣的研究人员进行的转化研究 在儿科疾病中。拟议补编将为一个新的合作项目提供支助 包括三个独立的IDEA机构研究者的实验室，包括一个以前的项目 我们COBRE的领导人（Kevin弗朗西斯博士）和两名研究人员目前由一名单独的COBRE提供支持 成立于南达科他州州立大学（娜塔莉·蒂克斯博士和布兰登斯科特博士）。项目本身 科学创新，扩展了最近由两个研究人员实验室撰写的出版物， 解决了儿科胆固醇合成障碍中内吞作用缺陷的新作用。工作 建议有可能为这些特征不明显的儿科疾病做出重大贡献。 此外，该项目通过将重叠的兴趣与 每个实验室都有独特的专业知识，同时利用COBRE支持的核心设施。虽然这三 研究人员在细胞功能的脂质调节方面有共同的兴趣，他们互补的专业知识， 培训将允许彻底解剖和理解甾醇生物化学对内吞作用的影响。 贩运与胆固醇合成障碍直接相关。 八种已知的疾病是由胆固醇合成酶的遗传破坏引起的， 胆固醇含量和胆固醇前体分子的异常表达。这些疾病的出现 在儿童时期，由于发育和 受影响儿童的功能缺陷。虽然这些疾病有着广泛的共同点， 发育迟缓，生化缺陷和组织畸形，疾病的发病机制差异很大 因此，对这些儿童的治疗发展受到限制。当疾病- 相关的缺陷可能是由于细胞信号传导、膜结构和细胞- 具体的过程，甾醇生化变化的确切影响和胆固醇的机制， 与疾病相关的固醇调节膜生物学仍然没有解决。因此，描绘如何 临床相关的甾醇生物化学影响基本的细胞功能，疾病相关性构成了 受影响儿童的科学需求未得到满足。 从固醇前体合成胆固醇是一种高度调节的途径，对细胞结构和生物活性至关重要。 在哺乳动物细胞和组织中传递信号。我们最近的工作详细说明了保守甾醇的关键作用 关键货物内化机制调控中的结构特征和表达水平 网格蛋白介导的内吞作用（CME）。由于分子的细胞内递送对于细胞的维持至关重要， 信号，功能和健康，充分定义疾病相关的甾醇生物化学对 膜生物学代表了一种新的生物学机制，它可以帮助解释细胞膜的差异。 胆固醇合成障碍的发病机制，并确定有针对性的功能途径，以改善细胞 健康对于这个建议，我们将利用疾病相关的细胞模型来测试假设， 膜需要高度特异性的甾醇生物化学来促进大分子的功能内化 和脂质排序。在目标1中，我们将首先定义临床相关固醇在功能内化中的作用 大分子，区分疾病特异性影响和定义细胞信号变化 特异于感兴趣的免疫细胞。在目标2中，我们将确定与疾病相关的 固醇生化变化通过内吞细胞超分辨率成像调节膜功能 过程和招募PtdIns物种到内吞位点。在目标3中，我们将利用基于CRISPR的整体 基因组筛选和后续验证，以描绘基因，信号通路和细胞过程， 有助于或补偿由胆固醇合成破坏引起的内吞缺陷。 总之，这些研究构成了一个新的建议，详细说明甾醇代谢如何调节哺乳动物 膜生物学与细胞功能和罕见儿科疾病直接相关。我们预计完成 的拟议工作将提供重要的洞察调节内吞作用的甾醇生物化学与 与这组致命的儿科疾病高度相关。此外，持续的支持将进一步促进这一点， 跨机构合作，并允许支持的研究人员以 多PI NIH申请完成后，建议的工作。

PROJECT SUMMARY The goal of the Center for Pediatric Research established at Sanford Research was to establish a multidisciplinary center to support translational research being performed by investigators with broad interests in pediatric disease. The proposed supplement will provide support for a new collaborative project encompassing the laboratories of three separate IDeA institution investigators, including one former project leader from our COBRE (Dr. Kevin Francis) and two investigators currently supported by a separate COBRE established at South Dakota State University (Dr. Natalie Thiex and Dr. Brandon Scott). The project itself is scientifically innovative, expanding upon a recent publication authored by two of the investigators’ laboratories, addressing a novel role for defects in endocytosis within pediatric disorders of cholesterol synthesis. The work proposed has the potential to provide significant contributions to these poorly characterized pediatric diseases. In addition, this project epitomizes a team science-based approach by combining overlapping interests with distinct expertise across each laboratory while utilizing COBRE-supported core facilities. While all three investigators have common interests in lipid regulation of cellular function, their complementary expertise and training will allow a thorough dissection and understanding of the impact of sterol biochemistry on endocytic trafficking with direct relevance to disorders of cholesterol synthesis. Eight known diseases are caused by genetic disruption of cholesterol synthesis enzymes, resulting in loss of cholesterol content and the aberrant expression of cholesterol precursor molecules. These disorders arise during childhood and create life-altering disabilities within affected individuals due to both developmental and functional deficits within affected children. Though these diseases share broad commonalities associated with developmental delay, biochemical deficits, and tissue malformations, disease pathogenesis varies greatly across individuals and thus therapeutic development for these children has been limited. While disease- associated deficits are likely due to combinatorial changes in cell signaling, membrane structure, and cell- specific processes, the precise effects of sterol biochemical changes and the mechanisms whereby cholesterol vs disease-associated sterols regulate membrane biology remain unresolved. Therefore, delineating how clinically relevant sterol biochemistry affects basic cellular functions with disease relevance constitutes an unmet scientific need for affected children. The synthesis of cholesterol from sterol precursors is a highly regulated pathway critical to cell structure and signaling across mammalian cells and tissues. Our recent work detailed a critical role for conserved sterol structural features and expression levels in the regulation of the critical cargo internalization mechanism clathrin-mediated endocytosis (CME). As the intracellular delivery of molecules is vital to maintenance of cell signaling, function, and health, fully defining the impact of disease-associated sterol biochemistry on membrane biology represents a novel biological mechanism which could help explain differences in the pathogenesis of cholesterol synthesis disorders and identify targetable, functional pathways to improve cell health. For this proposal, we will utilize disease-relevant cellular models to test the hypothesis that cellular membranes require highly specific sterol biochemistry to promote functional internalization of macromolecules and lipid ordering. In Aim 1, we will first define the role of clinically relevant sterols on functional internalization of macromolecules, differentiating between disease-specific impacts and defining cell signaling changes specific to immune cells of interest. In Aim 2, we will determine the mechanisms whereby disease-relevant sterol biochemical changes regulate membrane function through super resolution imaging of endocytic processes and recruitment of PtdIns species to endocytic sites. In Aim 3, we will utilize a CRISPR-based whole genome screen and follow-up validation to delineate genes, signaling pathways, and cellular processes that contribute to or compensate for endocytic defects resulting from cholesterol synthesis disruption. In summary, these studies constitute a novel proposal detailing how sterol metabolism regulates mammalian membrane biology with direct correlation to cell function and rare pediatric diseases. We anticipate completion of the proposed work will provide important insight into the regulation of endocytosis by sterol biochemistry with high relevance to this group of lethal pediatric disorders. Additionally, continued support will further foster this cross-institute collaboration and allow the supported investigators to extend these studies in the form of a multi-PI NIH application upon completion of the work proposed.