Controlling Protein Complexation and Cellular Localization using Novel Molecular Therapeutics

*我建议进行两个主要的研究项目。**项目1：开发合成肽前置酰化盒（PPB’s），用于膜定位配体，以操纵细胞中蛋白质的时空定位。**我将描述一种全新的、创新的分子方法来促进蛋白质-膜锚定，消除这种策略的典型缺点，如水溶性差和聚集性差。总体目标是设计和开发一种“弹头”，它可以被天然的戊烯酰化酶识别，进行戊烯酰化，并在原位产生膜锚。我们创造了这种“弹头”——肽戊酰化盒（PPB）。我们建议将该基序整合到各种小分子，蛋白质配体中，以进一步了解涉及蛋白质-膜锚定的基础科学，并探索其功能用途和局限性。在同一领域，我们将研究配合物配体在将磷酸化（活化）蛋白锚定到脂质膜上的应用。*这项工作代表了分子识别分支方向的重大转变，并为解决困难挑战提供了更具创造性的方法。**项目2：开发可选择二磷酸化蛋白基序的开启比例荧光传感器。**第二个项目需要开发一种开启的比例荧光传感器，可选择蛋白质靶标上的近端二磷酸化残基。我们的初步工作已经开发了第一个荧光传感器，基于芘介导的准分子形成，选择性地检测pYpY超过含有pY的肽/蛋白质。我们的目标是更好地理解这种模式，并优化基于凝胶和基于溶液的重要二磷酸化蛋白定量传感器。虽然使用商业染料（Pro-Q Diamond）非特异性检测单磷酸化位点（pY， pS和pT）已被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创新的荧光传感器，基于准分子发射信号（480 nm），可以选择性地检测pYpY和含有pYpY的肽/蛋白质。我们的目标是将这些新型传感器的应用范围扩大到含有磷蛋白的pXpX，优化结构以获得更高的选择性，效力和灵敏度限制，适用于96孔和固体载体（如SDS-PAGE凝胶和硝化纤维素膜）。* *整体总结。*我们将利用我们在合成配体方面不断增长的知识，开发优雅的ppb蛋白-膜锚，用于选择性地对靶蛋白进行膜定位。这项提议的工作代表了分子识别分支方向的重大转变，并为解决困难挑战提供了更具创造性的方法。我们基于pXpX样品的传感器已经对磷酸化蛋白状态产生了非常有希望的灵敏度和选择性，我们希望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凝胶分析和96孔板分析格式。

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.