Characterization of Erbb Receptors in Nanoparticles

描述（由申请人提供）：超过30%的蛋白质与细胞膜结合，这使得它们难以以可溶性和活性形式分离。我们建议实施最近开发的纳米脂蛋白颗粒（nlp），以允许分离活性形式的膜结合蛋白。nlp是一种盘状纳米颗粒，当载脂蛋白和一群磷脂在水环境中自组装成脂质双分子层时形成，这种双分子层可以溶解活性膜结合蛋白。特别是，我们将专注于酪氨酸激酶受体的表征，它介导许多癌症的扩散，是几种化疗药物的靶点。在细胞环境外研究这些受体的能力将使结构和机制研究成为可能，从而提高对癌变和其他疾病的理解。考虑到膜相关蛋白占大多数药物靶点，开发新技术来获得这类重要的蛋白质是很重要的。本研究的主要目标是建立普遍适用的方法来制备、分离和表征人类I型酪氨酸激酶受体ErbB1、ErbB2、ErbB3和ErbB4。已知表皮生长因子受体家族在细胞信号传导和癌变中起重要作用。由于这些蛋白质的不溶性和在水溶液中聚集的倾向，研究这些蛋白质具有挑战性。本提案的重点是开发一种新的生物技术应用，从而形成能够溶解这些和其他膜结合蛋白的nlp。与目前使用的模型膜相比，nlp在粒径、分散性和溶解度方面具有明显的优势。本研究将通过合成nlp中ErbB受体的同源和异源二聚体，对其结构和功能进行表征，并进行文库筛选，以确定新的高亲和力肽和小分子，这些小分子可以作为进一步开发药物的先导化合物。

DESCRIPTION (provided by applicant): Over 30% of proteins are bound to cell membranes, which makes them difficult to isolate in a soluble and active form. We propose to implement recently developed nanolipoprotein particles (NLPs) to allow the separation of membrane-bound proteins in an active form. NLPs are discoidal nanoparticles formed when an apolipoprotein and a population of phospholipids self-assemble into a lipid bilayer in an aqueous environment that can solubilize active membrane-bound proteins. In particular, we will focus on characterization of tyrosine kinase receptors, which mediate the spread of many cancers and are targets of several chemotherapeutics. The ability to study these receptors outside the cell environment will enable structural and mechanistic studies for improved understanding of carcinogenesis and other diseases. Given that membrane-associated proteins account for the majority of drug targets, it is important to develop novel technologies to gain access to this important class of proteins. This main goal is to develop generally applicable methods to produce, isolate and characterize the human type I tyrosine kinase receptors ErbB1, ErbB2, ErbB3 and ErbB4. The epidermal growth factor families of receptors are known to be important in cell signaling and carcinogenesis. These proteins are challenging to study because of their insolubility and tendency to aggregate in aqueous solutions. This proposal is focused on developing a new biotechnology application leading to formation of NLPs capable of solubilizing these and other membrane-bound proteins. The NLPs present a distinct advantage over currently used model membranes in terms of particle size monodispersity and solubility. This proposal will be carried out by synthesizing homo- and heterodimers of ErbB receptors in NLPs, characterizing them for structure and function and performing library screening in order to identify novel high-affinity peptides and small molecules that can serve as lead compounds for further development as drugs.