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: 10808798
• 负责人: Amy E Palmer
• 金额: $ 0.83万
• 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/10808798
• 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; Development; Disease; Fluorescent Probes; Genetic Transcription; Genomics; Growth; 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; Technology; Titrations; Transition Elements; Visualization; Work; Zinc; fluorophore; human disease; immune function; improved; live cell imaging; mRNA tagging; programs; tool; tool development; transcription factor

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.

项目摘要 锌（Zn 2+）是哺乳动物体内第二丰富的过渡金属，仅次于铁。有两千多个 由人类基因组编码的含有锌结合基序的蛋白质，其中锌结合被预测为 功能必不可少。在细胞水平上，锌对DNA合成、细胞增殖、分化 而在机体水平，锌是生长、发育和免疫功能所必需的。 考虑到Zn 2+在细胞生物学和人类健康中的重要性，令人震惊的是，我们仍然不了解 Zn 2+水平和动态如何影响基本细胞功能并引起疾病的机制。 虽然生物学中对Zn 2+的传统观点是它组成性地和稳定地结合于蛋白质， 包括锌蛋白质组，越来越多的证据表明细胞中的Zn 2+是动态的。此外，我们的实验室 显示Zn 2+动力学深刻地影响基本的细胞过程，如转录、分泌 途径功能和细胞周期，坚定地确立了Zn 2+是信号传导离子。然而，蛋白质和 感应Zn 2+动力学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Zn 2+动态滴定占有率的假设来解决这个问题， Zn 2+蛋白质组的活性。因此，Zn 2 + -在生理信号传导过程中的变化，环境 干扰，或作为疾病的结果-可以微调成千上万的锌依赖的活动， 蛋白质，确立Zn 2+作为细胞功能的主要调节剂。我们正在解决这个假设， 解决4个首要问题：（1）锌蛋白质组中的哪些蛋白质感知Zn 2+的动态变化 状态如何？(2)锌是否通过滴定功能和转录因子的DNA结合来调节转录？（三） 哪些途径和蛋白质介导Zn 2+对哺乳动物细胞周期的调节？（4）如何 Zn 2+缺乏是否影响其他必需金属（Fe、Cu和Mn）的调节。解决这些 问题，我们将使用基因组学，化学蛋白质组学，活细胞成像和生物化学的组合 接近。最近，我的实验室利用我们在工具开发、生物物理和生物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，以开发一种新的 用荧光团标记mRNA和ncRNA以在活细胞中跟踪它们的平台。该平台填补了 重要的技术需求，因为有诱人的建议之间的联系RNA定位， 动态和功能，但现有工具库存在重大局限性。因此，有一个 迫切需要强大的，互补的和最小干扰的工具来可视化单个RNA分子 在活细胞中绘制RNA生物学的复杂和不断发展的景观。因此，最后一部分 我的研究计划是（5）满足对改进工具的技术需求，以标记和跟踪活细胞中的RNA。 具体来说，我们将开发一套基于核糖开关的RNA标签，结合模块化的化学探针。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。