Genetically Encoded Sensors Shed Light on Zinc Homeostasis

描述（由申请人提供）：众所周知，过渡金属离子对生命至关重要，在一系列基本过程中发挥着重要作用。矛盾的是，这些必需的金属也是有毒的，因此细胞必须严格控制金属的积累、分布和出口。毫不奇怪，金属失衡对人类健康有着深远的影响，并与许多病理生理学相关，包括神经变性、糖尿病、癌症和免疫功能障碍。我们研究的长期目标是确定细胞平衡金属离子的机制，确定细胞使用金属作为信号剂的条件，并阐明金属失衡如何导致疾病和变性。目前的研究重点是锌（Zn2+），因为有新的证据表明，瞬态Zn2+信号可以在细胞内产生，这代表了金属离子如何影响细胞功能的一个令人兴奋的新范例。锌离子是人类生命所必需的微量元素。它的缺乏会导致认知受损、免疫功能紊乱、腹泻和死亡。人类基因组中有近3000个基因含有Zn2+指基序，这表明Zn2+结合蛋白是必不可少的细胞成分。这是一个非常惊人的数字，它代表了人类基因组编码蛋白质的近10%。我们的总体假设是，Zn2+作为细胞功能的重要调节因子，协调多种细胞通路的活性，因此疾病时Zn2+状态的改变改变了下游信号靶点，深刻影响细胞生理。该假设的基本前提是，Zn2+是动态调节的，游离Zn2+的变化会影响典型的信号通路，如Ca2+，以及改变蛋白质组的金属离子占用，微调数百个（如果不是数千个）Zn2+依赖蛋白的活性。从历史上看，我们对细胞Zn2+稳态的理解受到缺乏工具的限制，无法以高空间和时间分辨率可视化和量化活细胞中特定位置（即胞内细胞器）的游离Zn2+。在上一个资助周期中，我们通过开发一套针对细胞质、细胞核、内质网、高尔基体和线粒体的荧光Zn2+传感器来解决这一需求。有了这些传感器，我们对Zn2+动力学，Zn2+和Ca2+之间的相互作用有了显著的发现，并提供了游离Zn2+的分布如何在疾病中改变的第一眼。在下一个周期中，我们将以这些发现为基础，扩展它们，以扩大细胞器靶向传感器的范围，全面分析正常细胞与患病细胞中游离Zn2+的分布，定义Zn2+改变的机制，并确定Zn2+失调对细胞功能的影响。我们提出的工作有三个具体目标：(1)创建新的Zn2+传感器，定量报告细胞器中的游离Zn2+，从而实现游离Zn2+的全面定量映射；(2)明确游离Zn2+在前列腺癌中的变化，明确其失调机制；(3)确定Zn2+失调是否在影响下游靶标中起致病作用。

DESCRIPTION (provided by applicant): Transition metal ions are critical to life as we know it and play essential roles in a wide swath of fundamental processes. Paradoxically, these essential metals are also toxic and therefore cells must tightly regulate metal accumulation, distribution and export. Not surprisingly, metal imbalance has profound implications for human health and is correlated with a host of pathophysiologies, including neurodegeneration, diabetes, cancer, and immune dysfunction. The long term goals of our research are to identify the mechanisms by which cells balance metal ions, define conditions under which cells use metals as signaling agents, and elucidate how metal imbalance leads to disease and degeneration. The current proposal focuses on zinc (Zn2+) as there is emerging evidence that transient Zn2+ signals can be generated within the cell, representing an exciting new paradigm for how metal ions influence cellular function. Zn2+ is an essential micronutrient required for human life. Its deficiency leads to impaired cognition, immune dysfunction, diarrhea, and death. Close to 3,000 genes in the human genome contain Zn2+ finger motifs, indicating that Zn2+ binding proteins are essential cell constituents. This is a truly staggering number, and represents close to 10% of the proteins encoded by the human genome. Our overall hypothesis is that Zn2+ serves as an important regulator of cell function, coordinating the activity of numerous cellular pathways, such that changes in Zn2+ status with disease alter downstream signaling targets, profoundly influencing cellular physiology. The basic premise of this hypothesis is that Zn2+ is dynamically regulated, and that changes in free Zn2+ influence canonical signaling pathways such as Ca2+, as well as alter the metal ion occupancy of the proteome, fine tuning the activity of hundreds, if not thousands of Zn2+-dependent proteins. Historically, our understanding of cellular Zn2+ homeostasis has been limited by the lack of tools to visualize and quantify free Zn2+ in specific locations (i.e. intracellular organelles) in living cells with high spatial and temporal resolution. In the last grant cycle, we addressed this need by developing a suite of fluorescent Zn2+ sensors genetically targeted to the cytosol, nucleus, ER, Golgi, and mitochondria. With these sensors we made remarkable discoveries about Zn2+ dynamics, interplay between Zn2+ and Ca2+, and provided the first glimpse of how the distribution of free Zn2+ may be altered in disease. In the next cycle, we will build on these discoveries and extend them to expand the repertoire of organelle-targeted sensors, thoroughly profile free Zn2+ distribution in normal versus diseased cells, define the mechanism(s) by which Zn2+ is altered, and identify the consequences of Zn2+ dysregulation for cellular function. Our proposed work has 3 specific aims: (1) Create new Zn2+ sensors that quantitatively report on free Zn2+ in organelles to enable comprehensive quantitative mapping of free Zn2+; (2) Define the changes in free Zn2+ in prostate cancer and identify the mechanism of dysregulation; and (3) Identify whether Zn2+ dysregulation plays a causative role in influencing downstream targets.