lluminating the biochemistry of zinc and RNA in live cells

阐明活细胞中锌和 RNA 的生物化学

• 批准号: 10308669
• 负责人: Amy E Palmer
• 金额: $ 52.52万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10308669
• 关键词: Address; Apoptosis; Benchmarking; Binding; Biochemistry; Biological Assay; Biology; Biophysics; Cell Cycle; Cell Proliferation; Cell physiology; Cells; Cellular biology; Chemicals; Complex; DNA Binding; DNA biosynthesis; Disease; Fluorescent Probes; Genetic Transcription; Genomics; Growth and Development function; Health; Human; Human Genome; Individual; Ions; Iron; Mammalian Cell; Mammals; Maps; Measurement; Mediating; Metals; Micronutrients; Organism; Pathway interactions; Performance; Physiological; Proteins; Proteome; Proteomics; RNA; Regulation; Research; Series; Signal Transduction; Suggestion; Transcription Factor 3; Transition Elements; Work; Zinc; base; fluorophore; human disease; immune function; improved; live cell imaging; mRNA tagging; programs; tool; tool development

Project Summary Zinc (Zn2+) is the second most abundant transition metal in mammals after iron. There are over two thousand proteins encoded by the human genome that contain zinc binding motifs, where zinc binding is predicted to be essential for function. At the cellular level zinc is important for DNA synthesis, cell proliferation, differentiation, and apoptosis, while at the organism level zinc is required for growth, development and immune function. Given the importance of Zn2+ in cell biology and human health, it is astounding that we still don’t understand the mechanisms of how Zn2+ levels and dynamics impact basic cellular functions and give rise to disease. Although the conventional view of Zn2+ in biology is that it is constitutively and stably bound to the proteins that comprise the zinc proteome, there is growing evidence that Zn2+ in cells is dynamic. Further, our lab has shown that Zn2+ dynamics profoundly influence fundamental cellular processes such as transcription, secretory pathway function, and the cell cycle, firmly establishing that Zn2+ is a signaling ion. However, the proteins and pathways that sense Zn2+ dynamics to effect cellular change remain a mystery. My research program is poised to tackle this question by exploring the hypothesis that Zn2+ dynamics titrate occupancy and hence activity of the Zn2+ proteome. Thus, changes in Zn2+ – during physiological signaling, environmental perturbation, or as a consequence of disease – could fine-tune the activity of thousands of zinc-dependent proteins, establishing Zn2+ as a major regulator of cellular function. We are addressing this hypothesis by tackling 4 overarching questions: (1) Which proteins across the zinc proteome sense dynamic changes in Zn2+ status? (2) Does zinc regulate transcription by titrating function and DNA-binding of transcription factors? (3) What are the pathways and proteins that mediate Zn2+ regulation of the mammalian cell cycle? And (4) how does Zn2+ deficiency influence the regulation of other essential metals (Fe, Cu and Mn). To tackle these questions, we will use a combination of genomics, chemical proteomics, live cell imaging, and biochemistry approaches. Recently, my lab exploited our expertise in tool development, biophysical and photophysical characterization of fluorescent probes, and analytical approaches to live cell measurements, to develop a new platform for tagging mRNA and ncRNA with fluorophores to track them in live cells. This platform fills an important technological need, as there are tantalizing suggestions of connections between RNA localization, dynamics and function, but there are major limitations in the existing repertoire of tools. Thus, there is a pressing need for robust, complementary, and minimally perturbing tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA biology. Therefore, the final component of my research program is to (5) Meet the technological need for improved tools to tag and track RNA in live cells. Specifically, we will develop a suite of riboswitch-based RNA tags that bind modular chemical probes. We will also develop a series of robust assays for benchmarking the performance of RNA tagging tools in live cells.

项目概要 锌 (Zn2+) 是哺乳动物体内含量第二丰富的过渡金属，仅次于铁。有两千多 由人类基因组编码的含有锌结合基序的蛋白质，其中锌结合预计是 功能所必需的。在细胞水平上，锌对于 DNA 合成、细胞增殖、分化、 和细胞凋亡，而在有机体水平上，锌是生长、发育和免疫功能所必需的。 鉴于 Zn2+ 在细胞生物学和人类健康中的重要性，令人惊讶的是我们仍然不了解 Zn2+ 水平和动态如何影响基本细胞功能并引起疾病的机制。 尽管生物学上 Zn2+ 的传统观点是它与蛋白质组成型且稳定地结合， Zn2+ 包含锌蛋白质组，越来越多的证据表明细胞中的 Zn2+ 是动态的。此外，我们实验室还有 研究表明 Zn2+ 动力学深刻影响基本的细胞过程，例如转录、分泌 途径功能和细胞周期，牢固地确立了 Zn2+ 是一种信号离子。然而，蛋白质和 感知 Zn2+ 动态以影响细胞变化的途径仍然是一个谜。我的研究计划是 准备通过探索 Zn2+ 动力学滴定占有率并因此 Zn2+蛋白质组的活性。因此，Zn2+ 的变化 – 在生理信号传导、环境信号传导过程中 扰动或疾病的结果——可以微调数千种锌依赖性细胞的活性 蛋白质，将 Zn2+ 确立为细胞功能的主要调节剂。我们正在通过以下方式解决这个假设 解决 4 个首要问题：(1) 锌蛋白质组中的哪些蛋白质感知 Zn2+ 的动态变化 地位？ (2) 锌是否通过转录因子的滴定功能和 DNA 结合来调节转录？ (3) 介导哺乳动物细胞周期 Zn2+ 调节的途径和蛋白质有哪些？以及（4）如何 Zn2+ 缺乏是否会影响其他必需金属（Fe、Cu 和 Mn）的调节？为了解决这些 问题，我们将结合基因组学、化学蛋白质组学、活细胞成像和生物化学 接近。最近，我的实验室利用了我们在工具开发、生物物理和光物理方面的专业知识 荧光探针的表征以及活细胞测量的分析方法，以开发新的 用荧光团标记 mRNA 和 ncRNA 的平台，以在活细胞中追踪它们。该平台填补了 重要的技术需求，因为有关于 RNA 定位之间联系的诱人建议， 动力学和功能，但现有的工具库存在重大限制。因此，有一个 迫切需要强大、互补且干扰最小的工具来可视化单个 RNA 分子 在活细胞中绘制 RNA 生物学复杂且不断演变的图景。因此，最终的组成部分 我的研究计划是 (5) 满足对标记和跟踪活细胞中 RNA 的改进工具的技术需求。 具体来说，我们将开发一套基于核糖开关的 RNA 标签，用于结合模块化化学探针。我们将 还开发了一系列强大的检测方法，用于对活细胞中 RNA 标记工具的性能进行基准测试。