Genetically Encoded Sensors Shed Light on Zinc Homeostasis

描述(申请人提供)：正如我们所知，过渡金属离子对生命至关重要，并在广泛的基本过程中发挥重要作用。矛盾的是，这些必需金属也是有毒的，因此细胞必须严格控制金属的积累、分配和出口。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，包括神经退行性变、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媒介的条件，并阐明金属失衡是如何导致疾病和退化的。目前的建议集中在锌(锌)上，因为有新的证据表明，细胞内可以产生瞬时的锌信号，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式。锌是人体必需的微量营养素。它的缺乏会导致认知受损、免疫功能障碍、腹泻和死亡。在人类基因组中有近3000个基因含有锌指基序，这表明锌结合蛋白是细胞的基本成分。这确实是一个令人震惊的数字，占人类基因组编码蛋白质的近10%。我们的总体假设是，锌离子作为细胞功能的重要调节因子，协调许多细胞通路的活动，从而使锌离子状态的变化与疾病改变下游信号转导靶点，从而深刻地影响细胞生理。这一假说的基本前提是，锌离子是动态调节的，而游离锌离子的变化影响典型的信号通路，如钙离子，以及改变蛋白质组中金属离子的占有率，微调数百个，甚至数千个依赖于锌的蛋白质的活性。从历史上看，我们对细胞内锌离子稳态的理解一直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(即细胞内细胞器)的游离锌离子。在上一个资助周期中，我们开发了一套针对胞浆、细胞核、内质网、高尔基体和线粒体的荧光锌离子传感器，以满足这一需求。利用这些传感器，我们对锌离子的动力学、锌离子和钙离子之间的相互作用有了惊人的发现，并首次揭示了在疾病中游离锌离子的分布是如何改变的。在下一个周期中，我们将在这些发现的基础上进行扩展，以扩大细胞器靶向传感器的库，彻底了解正常细胞和疾病细胞中游离锌离子的分布，定义锌离子改变的机制(S)，并确定锌离子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)创造新的锌离子传感器，定量报告细胞器中的游离锌离子，以实现对自由锌离子的全面定量定位；(2)确定前列腺癌中游离锌离子的变化，并确定调控失调的机制；以及(3)确定锌离子调控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.