Controlling Protein Complexation and Cellular Localization using Novel Molecular Therapeutics

我建议进行两项主要研究计划。项目1：开发用于膜定位配体的合成肽异戊烯化盒（PPB），以操纵细胞中蛋白质的空间和时间位置。 我将描述一个全新的和创新的分子方法，以促进蛋白质膜锚定，否定这种策略的典型缺点，如水溶性差和聚集。全球目标是设计和开发一种“弹头”，它能被天然异戊二烯化酶识别，经历异戊二烯化，并在原位产生膜锚。我们已经创造了这个'弹头'肽Prenylation盒（PPB）。我们建议将这个基序纳入各种小分子，蛋白质配体，以进一步了解蛋白质膜锚定的基础科学，并探索其功能实用性和局限性。在同一领域，我们将研究使用的配位络合物配体锚定磷酸化（活化）蛋白质的脂质膜。 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，并为应对困难的挑战提供了更具创造性的方法。项目2：开发对二磷酸化蛋白基序有选择性的开启型比率荧光传感器。 第二个项目需要开发一种开启的比率荧光传感器，选择性地用于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，基于芘介导的激基缔合物的形成，选择性地检测pYpY超过pY含有肽/蛋白质。我们的目标是更好地了解这种模式，并优化传感器的凝胶和溶液为基础的重要的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化蛋白质组，但它们不能选择性地检测其活化需要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，该传感器选择性地检测pYpY而不是含pY的肽/蛋白。我们的目标是将这些新型传感器的应用扩展到含pXpX的磷蛋白，优化结构以赋予更大的选择性，效力和增强的灵敏度限制，用于96孔和固体支持物，如SDS-PAGE凝胶和硝酸纤维素膜。总体总结。 我们将建立在我们不断增长的合成配体的知识，以开发优雅的PPB-蛋白质-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，并为应对困难的挑战提供了更具创造性的方法。我们的基于pXpX准分子的传感器已经产生了非常有前途的磷酸化蛋白质状态的灵敏度和选择性概况，我们期望通过更高效力的结合支架将其提高一个数量级，以最终应用于SDS-PAGE凝胶测定和96孔板测定形式。

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.