Feng Bing, Liu Xujie, Chen Yuanyuan
Department of Ophthalmology, University of Pittsburgh.
Department of Ophthalmology, University of Pittsburgh; McGowan Institute for Regenerative Medicine, University of Pittsburgh;
J Vis Exp. 2019 Jan 16(143). doi: 10.3791/58703.
Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.
视紫红质错误折叠突变会导致视杆光感受器死亡，表现为常染色体显性遗传性视网膜色素变性（RP），这是一种缺乏有效治疗方法的进行性致盲疾病。我们假设，通过药理学方法稳定突变型视紫红质蛋白，可以减轻错误折叠的视紫红质突变体的细胞毒性。在其他II类视紫红质突变中，P23H突变编码一种结构不稳定的视紫红质突变蛋白，该蛋白在内质网（ER）中积累，而野生型视紫红质在哺乳动物细胞中被转运到质膜。我们之前进行了基于发光的高通量筛选（HTS），并鉴定出一组药理学伴侣，它们挽救了P23H视紫红质从内质网到质膜的转运。在这里，我们采用免疫染色方法，随后进行高内涵成像分析，定量了全细胞和质膜上突变型视紫红质蛋白的量。该方法对于从高通量筛选后的假阳性中识别真正的命中物是信息丰富且有效的。此外，高内涵图像分析使我们能够从单个实验中量化多个参数，以评估每种化合物的药理学特性。使用该检测方法，我们分析了11种不同化合物对6种与RP相关的视紫红质突变体的影响，获得了二维药理学图谱，以便对这些视紫红质突变体的结构稳定性以及不同化合物对这些突变体的疗效进行定量和定性的了解。
