PDF(Pubmed)
Abstract：
Smoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO in their native state is highly beneficial for the development of effective pharmaceutical drugs, as their structures and functions are retained to the highest extent in this state. Therefore, although SMO protein is conventionally solubilized in detergent micelles, incorporating the protein in a lipid-based membrane mimic is still required. In this study, we used styrene maleic acid (SMA) copolymer that directly extracted membrane protein and surrounding lipids as well as formed the so-called polymer nanodiscs, to solubilize and purify the SMO transmembrane domain encapsulated by SMA-nanodiscs. The obtained SMA-nanodiscs showed high homogeneity and maintained the physiological activity of SMO protein, thereby enabling the measurement of the dissociation constant (Kd) for SMO ligands SMO-ligands Shh Signaling Antagonist V (SANT-1) and Smoothened Agonist (SAG) using ligand-based solution nuclear magnetic resonance spectroscopy. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment.
摘要：
平滑（SMO）蛋白是G蛋白偶联受体（GPCR）家族的成员，参与Hedgehog（Hh）信号通路。它是治疗各种癌症的假定靶点，包括髓母细胞瘤和基底细胞癌（BCC）。表征膜蛋白，如SMO在其天然状态是非常有益的开发有效的药物，因为它们的结构和功能在这种状态下被最大程度地保留。因此,虽然SMO蛋白通常溶解在洗涤剂胶束中，仍然需要将蛋白质掺入基于脂质的膜模拟物中。在这项研究中,我们使用苯乙烯马来酸（SMA）共聚物直接提取膜蛋白和周围的脂质以及形成所谓的聚合物纳米盘，以溶解和纯化由SMA-纳米盘封装的SMO跨膜结构域。获得的SMA-nanodisks显示出较高的均匀性，并保持了SMO蛋白的生理活性，从而能够使用基于配体的溶液核磁共振波谱来测量SMO配体SMO-配体Shh信号拮抗剂V(SANT-1)和平滑激动剂(SAG)的解离常数(Kd)。这项工作为研究结构铺平了道路，函数,以及在天然类脂质环境中SMO蛋白的药物开发。
