Controlling Protein Complexation and Cellular Localization using Novel Molecular Therapeutics

使用新型分子疗法控制蛋白质复合和细胞定位

• 批准号: RGPIN-2014-05767
• 负责人: Gunning, Patrick
• 金额: $ 6.12万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588605
• 关键词: Controlling; Protein; Complexation; Cellular; Localization

PROPOSAL SUMMARY I propose to conduct two major research projects. PROJECT 1: Develop Synthetic Peptide Prenylation Boxes (PPB’s) for application in Membrane-Localizing Ligands to Manipulate the Spatial and Temporal Location of Proteins in Cells. I will describe an entirely new and innovative molecular approach to facilitate protein-membrane anchorage, negating the typical shortcomings of this strategy such as poor water solubility and aggregation. The global objective is to design and develop a ‘warhead’ that is recognized by native prenylating enzymes, undergoes prenylation, and creates a membrane anchor in situ. We have coined this ‘warhead’ a Peptide Prenylation Box (PPB). We propose to incorporate this motif into a variety of small molecule, protein ligands to further our understanding of the fundamental science involved in protein-membrane anchorage and explore its functional utility and limitations. In the same field, we will investigate the use of coordination complex ligands in anchoring phosphorylated (activated) proteins to the lipid membrane. This proposed work represents a significant shift in the direction of this branch of molecular recognition and offers a more creative approach to a difficult challenge. PROJECT 2: Developing turn-on, ratiometric fluorescent sensors selective for di-phosphorylated protein motifs. The second project entails the development of a turn-on, ratiometric fluorescent sensor, selective for proximal diphosphorylated residues on a protein target. Our preliminary work has developed the first fluorometric sensor, based on pyrene-mediated excimer formation, that selectively detects pYpY over pY containing peptide/proteins. Our objective is to better understand this modality and optimize sensors for gel-based and solution-based quantification of important diphosphorylated proteins. While non-specific detection of mono-phosphorylated sites (pY, pS and pT) using commercial dyes (Pro-Q Diamond) has been employed to study the phospho-proteome, they do not selectively detect a subset of proteins, whose activation requires pXpX motif phosphorylation (where X = Y, T, and S). Initial studies in our lab have identified an innovative fluorescent sensor, based on excimer emission signal (480 nm), that selectively detects pYpY over pY-containing peptide/proteins. Our objective is to expand the application of these novel sensors to pXpX containing phosphoproteins, optimize structures to confer greater selectivity, potency, and enhanced sensitivity limits for application in 96-well and on solid supports such as SDS-PAGE gel and nitrocellulose membrane. Overall Summary. We will build on our growing knowledge of synthetic ligands to develop elegant PPB-protein-membrane anchors for selectively membrane localizing target proteins. This proposed work represents a significant shift in the direction of this branch of molecular recognition and offers a more creative approach to a difficult challenge. Our pXpX excimer-based sensors have yielded very promising sensitivity and selectivity profiles for phosphorylated protein states, which we expect to improve by an order of magnitude with higher potency binding scaffolds for ultimate application in SDS-PAGE gel assay and 96-well plate assay format.

提案摘要 我建议开展两个重大研究项目。 项目 1：开发合成肽异戊二烯化盒 (PPB)，用于膜定位配体，以操纵细胞中蛋白质的空间和时间位置。 我将描述一种全新的创新分子方法，以促进蛋白质膜锚定，消除该策略的典型缺点，例如水溶性差和聚集。全球目标是设计和开发一种“弹头”，它可以被天然异戊二烯化酶识别，进行异戊二烯化，并在原位形成膜锚。我们将这种“弹头”称为肽异戊二烯化盒（PPB）。我们建议将该基序整合到各种小分子、蛋白质配体中，以进一步了解蛋白质膜锚定所涉及的基础科学，并探索其功能用途和局限性。 在同一领域，我们将研究配位复合物配体在将磷酸化（活化）蛋白质锚定到脂质膜上的用途。 这项拟议的工作代表了分子识别这一分支方向的重大转变，并为应对艰巨的挑战提供了更具创造性的方法。 项目 2：开发选择性二磷酸化蛋白质基序的开启式比率荧光传感器。 第二个项目需要开发一种开启式比率荧光传感器，对蛋白质靶标上的近端二磷酸化残基具有选择性。我们的前期工作开发了第一个基于芘介导的准分子形成的荧光传感器，它选择性地检测 pYpY 而不是含有 pY 的肽/蛋白质。我们的目标是更好地了解这种模式并优化传感器，以对重要的二磷酸化蛋白质进行基于凝胶和基于溶液的定量。虽然使用商业染料 (Pro-Q Diamond) 对单磷酸化位点（pY、pS 和 pT）进行非特异性检测已用于研究磷酸化蛋白质组，但它们不能选择性地检测蛋白质子集，其激活需要 pXpX 基序磷酸化（其中 X = Y、T 和 S）。我们实验室的初步研究已经确定了一种基于准分子发射信号 (480 nm) 的创新荧光传感器，可选择性检测 pYpY 而不是含有 pY 的肽/蛋白质。我们的目标是将这些新型传感器的应用扩展到含有磷蛋白的 pXpX，优化结构以赋予更大的选择性、效力，并增强在 96 孔和固体支持物（如 SDS-PAGE 凝胶和硝酸纤维素膜）上的应用的灵敏度限制。 总体总结。 我们将基于对合成配体不断增长的了解，开发优雅的 PPB 蛋白膜锚，用于选择性膜定位靶蛋白。这项拟议的工作代表了分子识别这一分支方向的重大转变，并为应对艰巨的挑战提供了更具创造性的方法。我们的基于 pXpX 准分子的传感器对磷酸化蛋白质状态产生了非常有前途的灵敏度和选择性特征，我们预计通过更高效力的结合支架将其提高一个数量级，最终应用于 SDS-PAGE 凝胶测定和 96 孔板测定格式。