Controlling Protein Complexation and Cellular Localization using Novel Molecular Therapeutics

建议书摘要 我建议进行两个主要的研究项目。 项目1：开发用于膜定位配体的合成肽预链化盒(PPB‘s)，以操纵蛋白质在细胞中的空间和时间位置。 我将描述一种全新的、创新的分子方法，以促进蛋白质膜的锚定，消除这种策略的典型缺点，如较差的水溶解性和聚集性。全球目标是设计和开发一种可被天然预烯基化酶识别、经历预烯基化并在原位产生膜锚的“弹头”。我们把这种“弹头”命名为多肽预聚合盒(PPB)。我们建议将这个基序结合到各种小分子、蛋白质配体中，以加深我们对蛋白质膜锚定所涉及的基础科学的理解，并探索其功能用途和局限性。在同一领域，我们将研究配位络合物配体在将磷酸化(活化)蛋白锚定到脂膜上的使用。 这项拟议的工作代表了这一分子识别分支方向的重大转变，并提供了一种更具创造性的方法来应对这一困难的挑战。 项目2：开发选择性用于二磷酸化蛋白质模体的可开启的比率荧光传感器。 第二个项目需要开发一种开启的比率荧光传感器，选择性地检测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出第一个荧光传感器，基于Py介导的准分子形成，它选择性地检测含有Py的多肽/蛋白质上的PyPy。我们的目标是更好地了解这种模式，并优化传感器，用于基于凝胶和基于溶液的重要二磷酸化蛋白质的定量。虽然使用商业染料(Pro-Q Diamond)对单磷酸化位点(Py、PS和PT)的非特异性检测已被用于研究磷酸蛋白质组，但他们并没有选择性地检测到需要pXpX基序磷酸化(其中X=Y、T和S)的蛋白质子集。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，它可以选择性地检测含Py的多肽/蛋白质上的PyPy。我们的目标是扩大这些新型传感器在含有磷蛋白的pXpX中的应用，优化结构以提供更高的选择性、有效性和更高的灵敏度限制，用于96孔和固体载体，如SDS-PAGE凝胶和硝酸纤维素膜。 总体摘要。 我们将建立在我们日益增长的合成配体知识的基础上，开发出优雅的PPB-蛋白质-膜锚，用于选择性地定位目标蛋白质。这项拟议的工作代表了这一分子识别分支方向的重大转变，并提供了一种更具创造性的方法来应对这一困难的挑战。我们基于pXpX准分子的传感器已经为磷酸化蛋白质状态提供了非常有前景的灵敏度和选择性曲线，我们希望通过更高效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.