Design of Ca2+ Sensors to Monitor Ca2+ Signaling in ER

用于监测 ER 中 Ca2 信号传导的 Ca2 传感器的设计

• 批准号: 6759006
• 负责人: Jenny J. Yang
• 金额: $ 18.19万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6759006
• 关键词: binding sites; biological signal transduction; biosensor device; biotherapeutic agent; calcium flux; calcium indicator; calcium transporting ATPase; calreticulin; cell line; chemical kinetics; chemical stability; chromophore; confocal scanning microscopy; cytoplasm; drug design /synthesis /production; endoplasmic reticulum; green fluorescent proteins; histamine; histochemistry /cytochemistry; immunocytochemistry; intracellular transport; ionophores; ligands; protein binding; tissue /cell culture

DESCRIPTION (provided by applicant): The goal of this project is to develop a novel class of Ca(ll) sensor proteins that will have wide applicability in studies of human diseases including various cardiomyopathies, Alzheimer diseases, cancer, and lens cataract formation that are known to be associated with altered Ca(ll) signaling. A major barrier to the understanding of specific spatio-temporal patterns of intracellular Ca(ll) signaling is the lack of targeted sensors that monitor concentration from submicromolar to greater than 5 mM. We have developed a novel approach for developing Ca(ll) sensors by grafting Ca(ll) binding motifs into Green Fluorescence Protein (GFP) that exhibit Ca(ll) dependent fluorescence changes. In addition, we can design de novo Ca(ll) binding sites in GFP such that they exhibit different Ca(ll) binding affinities and strong selectivity. This proposal has two objectives. First, we will develop GFP-based Ca(ll) sensors for the ER with a broad-range of Ca(ll) affinities (10 micromolar < Kd < 5mM) and optimized optical properties by grafting different Ca(ll) binding motifs. We will examine their optical properties, metal binding affinity and selectivity, stability and kinetic properties. The cellular locations of ER-directed Ca(ll) sensors will be verified cytochemically and immunocytochemically. We will monitor changes in ER Ca(ll) signaling in real time in response to agonists such as ATP and histamine. Second, we will develop high affinity and selective Ca(ll) sensors for the cytosol (0.1 <Kd < 10 microM) by designing de novo Ca(ll) binding sites within GFP. We will enhance metal binding affinities and selectivity of Ca(ll) sensors with different types of Ca(il) ligand residues and number of charged residues. In addition to optimizing optical properties and kinetic properties of the Ca(ll) sensors, we will evaluate our developed Ca(ll) sensors in vivo by varying the intracellular cytosolic Ca(ll) concentration using Ca(ll) ionophores and appropriate Ca(ll) pump inhibitors, and comparing the estimated Ca(ll) concentrations with that derived using small synthetic dyes and currently available CaM-based sensors. Calibration method for in vivo detection of [Ca(ll)] will be developed using established HeLa cell lines and primary lens cell cultures. The proposed work will have a significant impact on the field because it will not only significantly expand the repertoire of tools for studying cell signaling and related diseases, but also it will result in technological advances toward designing Ca(ll) switches that are capable of controlling protein function.

描述（由申请人提供）：该项目的目标是开发一类新型 Ca(II) 传感器蛋白，该蛋白将在人类疾病的研究中具有广泛的适用性，包括各种心肌病、阿尔茨海默病、癌症和晶状体白内障形成，这些疾病已知与改变的 Ca(II) 信号传导有关。了解细胞内 Ca(II) 信号传导的特定时空模式的一个主要障碍是缺乏可监测从亚微摩尔到大于 5 mM 浓度的靶向传感器。我们开发了一种开发 Ca(II) 传感器的新方法，通过将 Ca(II) 结合基序移植到绿色荧光蛋白 (GFP) 中，表现出 Ca(II) 依赖性荧光变化。此外，我们可以在GFP中从头设计Ca(II)结合位点，使其表现出不同的Ca(II)结合亲和力和强选择性。该提案有两个目标。首先，我们将为 ER 开发基于 GFP 的 Ca(II) 传感器，具有广泛的 Ca(II) 亲和力（10 微摩尔 < Kd < 5mM），并通过嫁接不同的 Ca(II) 结合基序来优化光学特性。我们将检查它们的光学特性、金属结合亲和力和选择性、稳定性和动力学特性。 ER 导向的 Ca(II) 传感器的细胞位置将通过细胞化学和免疫细胞化学进行验证。我们将实时监测 ER Ca(II) 信号传导对 ATP 和组胺等激动剂的反应变化。其次，我们将通过设计 GFP 内的从头 Ca(II) 结合位点，开发用于细胞溶胶 (0.1 <Kd < 10 microM) 的高亲和力和选择性 Ca(II) 传感器。我们将通过不同类型的 Ca(II) 配体残基和带电残基数量来增强 Ca(II) 传感器的金属结合亲和力和选择性。除了优化 Ca(ll) 传感器的光学特性和动力学特性外，我们还将通过使用 Ca(ll) 离子载体和适当的 Ca(ll) 改变细胞内胞质 Ca(ll) 浓度来评估我们开发的体内 Ca(ll) 传感器。 ) 泵抑制剂，并将估计的 Ca(II) 浓度与使用小型合成染料和当前可用的基于 CaM 的传感器得出的浓度进行比较。将使用已建立的 HeLa 细胞系和原代晶状体细胞培养物开发体内检测 [Ca(II)] 的校准方法。拟议的工作将对该领域产生重大影响，因为它不仅将显着扩展用于研究细胞信号传导和相关疾病的工具库，而且还将导致设计能够控制 Ca(II) 开关的技术进步蛋白质功能。