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: 7005233
• 负责人: Jenny J. Yang
• 金额: $ 0.92万
• 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/7005233
• 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）：该项目的目的是开发一种新型的CA（LL）传感器蛋白，该蛋白将在包括各种心肌病，阿尔茨海默氏症，癌症和癌细胞型形成在内的人类疾病研究中具有广泛的适用性，这些形成与改变的CA（LL）信号有关。理解细胞内CA（LL）信号传导特定时空模式的主要障碍是缺乏靶向传感器，该传感器监测从亚旋风到大于5 mm的浓度。我们通过将CA（LL）结合拟合到绿色荧光蛋白（GFP）中，开发了一种新的CA（LL）传感器的方法，这些方法表现出CA（LL）依赖性荧光变化。此外，我们可以在GFP中设计新的CA（LL）结合位点，以表明它们具有不同的CA（LL）结合亲和力和强烈的选择性。该提议有两个目标。首先，我们将通过将不同的CA（LL）亲和力（10微摩尔<kd <5mm）和通过接种不同的CA（LL）结合基序来开发基于GFP的CA（LL）传感器。我们将检查它们的光学特性，金属结合亲和力以及选择性，稳定性和动力学特性。 ER导向CA（LL）传感器的细胞位置将通过细胞化学和免疫细胞化学验证。我们将根据ATP和组胺等激动剂，实时监测ER CA（LL）信号的变化。其次，我们将通过在GFP内设计DE NOVO CA（LL）结合位点来开发细胞质（0.1 <kd <10 microM）的高亲和力（LL）传感器。我们将增强具有不同类型的Ca（IL）配体残基和带电残基数的CA（LL）传感器的金属结合亲密关系和选择性。除了优化CA（LL）传感器的光学性能和动力学特性外，我们还将通过使用CA（LL）离子载体（LL）离子载体和适当的CA（LL）泵基抑制剂的CA（LL）浓度来评估我们在体内开发的CA（LL）传感器，并使用估计的（LL）浓度进行比较。使用已建立的HELA细胞系和原代晶状体细胞培养物开发用于[CA（LL）]体内检测的校准方法。拟议的工作将对该领域产生重大影响，因为它不仅会显着扩展研究细胞信号传导和相关疾病的工具曲目，而且还将导致能够控制蛋白质功能的CA（LL）开关的技术进步。

项目成果

Designing caspase-3 sensors for imaging of apoptosis in living cells.

• DOI: 10.1002/chem.200901439
• 发表时间: 2009-09-21
• 期刊: CHEMISTRY-A EUROPEAN JOURNAL
• 影响因子: 4.3
• 作者: Chen, Ning;Huang, Yun;Yang, Lily;Liu, Rihe;Yang, Jenny J.
• 通讯作者: Yang, Jenny J.