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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=652678
• 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 *我建议开展两个主要的研究项目。 **项目1：开发合成肽原用盒（PPB），以应用于膜 - 定位配体中，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，否定了这种策略的典型缺点，例如不良的水溶性和聚集。全球目标是设计和开发一种“弹头”，该弹头被原生蛋白酶认可，经历原始化并创建原位膜锚。我们已经创造了这个“弹头”一个肽前原化盒（PPB）。我们建议将此基序纳入各种小分子，蛋白质配体，以进一步了解我们对蛋白质 - 膜锚固涉及的基本科学的理解，并探索其功能效用和局限性。 在同一领域，我们将调查配位复合物配体在将磷酸化（激活的）蛋白锚定在脂质膜上的使用。*这项提出的工作代表了该分子识别分支的方向的重大转变，并为难以创造的挑战提供了一种更具创造性的方法。 **第二个项目需要开发转元，比率荧光传感器，选择性用于蛋白质靶标上近二磷酸化残基的选择性。我们的初步工作基于pyrene介导的准分子形成开发了第一个荧光测量传感器，该传感器有选择地检测到含有肽/蛋白质的PY上的PYPY。我们的目标是更好地理解这种方式，并优化基于凝胶和溶液的重要双磷酸化蛋白质的传感器。尽管已使用商业染料（Pro-Q Diamond）对单磷酸化位点（PY，PS和PT）进行非特异性检测来研究磷酸化 - 蛋白质，但它们并未选择性地检测蛋白质的子集，其激活需要PXPX基序磷酸化（其中x = y，x = y，x = y，x = y，t，t和s）。我们实验室中的初步研究基于准分子发射信号（480 nm）确定了创新的荧光传感器，该传感器在含Py的肽/蛋白质上有选择地检测到PYPY。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96孔应用以及SDS-PAGE GEL和NITROCELLULULOSE等固体支持，以赋予更高的选择性，效力和增强的灵敏度限制。**总结。 *我们将基于对合成配体的日益了解，以开发出优雅的PPB-蛋白质膜锚，以选择性地膜定位靶蛋白。这项拟议的工作代表了分子识别分支的方向的重大转变，并为艰难挑战提供了一种更具创造性的方法。我们的PXPX基因传感器对磷酸化蛋白质态产生了非常有希望的灵敏度和选择性曲线，我们希望通过较高的效力结合支架，以在SDS-PAGE凝胶测定中使用较高的效力结合支架和96-Well Plate Assay Format。