Quantitative Cu-Homeostasis in Live Mammalian Cells at the Single-Molecule Level

单分子水平上活哺乳动物细胞的定量铜稳态

• 批准号: 10405568
• 负责人: Tai-Yen Chen
• 金额: $ 38.06万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10405568
• 关键词: Achievement; Affect; Bacteria; Behavior; Biochemical; Biological Assay; Biological Process; Cells; Collaborations; Complex; Copper; DNA-Protein Interaction; Diffuse; Disease; Environment; Equilibrium; Family; Fluorescence; Foundations; Genetic Transcription; Goals; Hepatocyte; Hepatolenticular Degeneration; Homeostasis; Human; Imaging Techniques; In Vitro; Individual; Knowledge; Mammalian Cell; Measurement; Mediating; Membrane; Metalloproteins; Metals; Microscopy; Mission; Mutation; National Institute of General Medical Sciences; Pathogenesis; Pathway interactions; Prevention; Proteins; Public Health; Research; Resolution; Roentgen Rays; Role; Spatial Distribution; Synchrotrons; Technology; Therapeutic; Wilson disease protein; efflux pump; experimental study; fluorescence imaging; imaging approach; induced pluripotent stem cell; innovation; insight; knowledge base; live cell imaging; novel; programs; reconstitution; response; secretion process; single molecule; single-molecule FRET; spatiotemporal; stem cells; uptake

PROJECT SUMMARY/ABSTRACT Understanding responsive mechanisms of metalloproteins is key to elucidate biological functions of copper (Cu) and to identify the causes of diseases resulting from abnormal metal homeostasis. The cellular Cu uptake and secretion require relevant metalloproteins to adjust in a spatiotemporally coordinated manner to assure proper cellular Cu level. However, in the Cu field, little is known about how metalloproteins are individually regulated nor systematically cooperate with each other in their native environment, i.e., in cells. Our research goal is to understand the responsive mechanisms of Cu-uptake and secretory metalloproteins in live mammalian cells, with specific focuses on how metalloproteins adjust their behaviors such as spatial distributions, oligomeric states, inter-protein and inter-domain interactions for proper Cu balance in a spatiotemporally defined manner. Previous achievements of the PI include discoveries of novel mechanisms of MerR-family metalloregulators in regulating transcription and Cu-responsive dynamic assembly of efflux pumps by examining the protein-DNA interaction and protein diffusive behaviors in live bacteria using single-molecule super-resolution microscopy. Leveraging the power of these technologies, in combined with the recently developed live-cell single-molecule fluorescence- resonance-energy-transfer assay, we will elucidate the responsive mechanisms of metalloproteins in the uptake and secretory pathways in live mammalian cells. Using CTR1 and ATOX1-ATP7A/B as the initial examples of uptake and secretory metalloproteins, the proposed experiments will (1) quantify Cu-dependent oligomeric state distribution and identify the Cu-responsive moiety of CTR1; (2) define the preferential interaction of ATOX1 to ATP7A and ATP7B and how mutations in ATP7B affect Cu homeostasis in cellular Cu defending using induced pluripotent stem cells derived hepatocytes. In addition to primary approaches of single-molecule super-resolution fluorescence imaging techniques, complementary bulk spectroscopic and biochemical measurements will be compared. The research program is further enhanced by collaborations with the experts in Cu homeostasis and stem cell fields. The research is significant because it will provide mechanistic insights into metalloprotein- mediated Cu-uptake and secretion processes as well as complementary information for synchrotron X-ray fluorescence studies on intracellular Cu-redistribution. The comparison between human induced pluripotent stem cell (hiPSC)-derived healthy and diseased hepatocytes will inform how disease mutations disrupt cellular Cu balance, providing the knowledge base to devise therapeutic strategies for Wilson's diseases. The research is innovative because it represents a substantive departure from the status quo by shifting focus to define response mechanisms of metalloproteins using advanced approaches including single-molecule super-resolution microscopy and hiPSC-derived hepatocytes. The live-cell imaging approach also circumvents the general challenge in studying membrane complexes, whose in vitro reconstitution is technically demanding. The hiPSC- derived diseased hepatocytes provide an ideal platform to study the pathogenesis of Wilson's disease.

项目摘要/摘要 了解金属蛋白的反应性机制是阐明铜（CU）生物学功能的关键 并确定因异常金属稳态引起的疾病原因。细胞Cu摄取和 分泌需要相关金属蛋白以时空协调的方式进行调整以确保正确 细胞铜水平。但是，在CU领域，对于金属蛋白如何单独调节的知识知之甚少 在本机环境（即细胞）中，也没有系统地合作。我们的研究目标是 了解活哺乳动物细胞中Cu摄取和分泌金属蛋白的反应性机制， 特定的重点是金属蛋白如何调整其行为，例如空间分布，寡聚状态， 蛋白间和域间相互作用以时空定义的方式进行适当的CU平衡。以前的 PI的成就包括在调节中的新型机制的发现 通过检查蛋白-DNA相互作用 使用单分子超分辨率显微镜在活细菌中蛋白质扩散行为。利用 这些技术的功能，结合最近开发的活细胞单分子荧光 - 共振 - 能量转移测定法，我们将阐明摄取中金属蛋白的响应机制 和活哺乳动物细胞中的分泌途径。使用CTR1和ATOX1-ATP7A/B作为初始示例 摄取和分泌金属蛋白，提出的实验将（1）量化Cu依赖性寡聚状态 分布并确定CTR1的CU响应部分； （2）定义ATOX1与 ATP7A和ATP7B以及ATP7B中的突变如何影响CU使用诱导的细胞Cu防御 多能干细胞衍生出肝细胞。除了单分子超分辨率的主要方法 荧光成像技术，互补的散装光谱和生化测量将为 比较的。与CU稳态和专家的合作进一步增强了研究计划 干细胞场。这项研究很重要，因为它将为金属蛋白 - 介导的Cu摄取和分泌过程以及同步加速器X射线的互补信息 荧光研究对细胞内CU重新分配。人类诱导的多能茎之间的比较 细胞（HIPSC）衍生的健康和患病的肝细胞将告知疾病突变如何破坏细胞Cu 平衡，为威尔逊疾病制定治疗策略提供知识库。研究是 创新性是因为它通过转移重点来定义响应来代表与现状的实质性不同 使用包括单分子超分辨率在内的高级方法的金属蛋白机制 显微镜和HIPSC衍生的肝细胞。活电池成像方法还规避了一般 研究膜复合物的挑战，其体外重构在技术上要求。 hipsc- 衍生的患病肝细胞为研究威尔逊氏病发病机理提供了理想的平台。