McKeone Richard, Wikstrom Matthew, Kiel Christina, Rakoczy P Elizabeth
Department of Molecular Ophthalmology, Lions Eye Institute, Perth, Western Australia ; Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia.
Centre for Experimental Immunology, Lions Eye Institute, Perth, Western Australia ; Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia.
Mol Vis. 2014 Feb 7;20:183-99. eCollection 2014.
Following a previous study that demonstrated a correlation between rhodopsin stability and the severity of retinitis pigmentosa (RP), we investigated whether predictions of severity can be improved with a regional analysis of this correlation. The association between changes to the stability of the protein and the relative amount of rhodopsin reaching the plasma membrane was assessed.
Crystallography-based estimations of mutant rhodopsin stability were compared with descriptions in the scientific literature of the visual function of mutation carriers to determine the extent of associations between rhodopsin stability and clinical phenotype. To test the findings of this analysis, three residues of a green fluorescent protein (GFP) tagged rhodopsin plasmid were targeted with site-directed random mutagenesis to generate mutant variants with a range of stability changes. These plasmids were transfected into HEK-293 cells, and then flow cytometry was used to measure rhodopsin on the cells' plasma membrane. The GFP signal was used to measure the ratio between this membrane-bound rhodopsin and total cellular rhodopsin. FoldX stability predictions were then compared with the surface staining data and clinical data from the database to characterize the relationship between rhodopsin stability, the severity of RP, and the expression of rhodopsin at the cell surface.
There was a strong linear correlation between the scale of the destabilization of mutant variants and the severity of retinal disease. A correlation was also seen in vitro between stability and the amount of rhodopsin at the plasma membrane. Rhodopsin is drastically reduced on the surface of cells transfected with variants that differ in their inherent stability from the wild-type by more than 2 kcal/mol. Below this threshold, surface levels are closer to those of the wild-type.
There is a correlation between the stability of rhodopsin mutations and disease severity and levels of membrane-bound rhodopsin. Measuring membrane-bound rhodopsin with flow cytometry could improve prognoses for poorly characterized mutations and could provide a platform for measuring the effectiveness of treatments.
在先前一项证明视紫红质稳定性与色素性视网膜炎（RP）严重程度之间存在相关性的研究之后，我们调查了通过对这种相关性进行区域分析是否可以改善严重程度的预测。评估了蛋白质稳定性变化与到达质膜的视紫红质相对量之间的关联。
将基于晶体学的突变型视紫红质稳定性估计值与科学文献中对突变携带者视觉功能的描述进行比较，以确定视紫红质稳定性与临床表型之间的关联程度。为了检验该分析的结果，用定点随机诱变技术靶向绿色荧光蛋白（GFP）标记的视紫红质质粒的三个残基，以产生具有一系列稳定性变化的突变变体。将这些质粒转染到HEK-293细胞中，然后用流式细胞术测量细胞质膜上的视紫红质。GFP信号用于测量这种膜结合视紫红质与总细胞视紫红质之间的比率。然后将FoldX稳定性预测值与来自数据库的表面染色数据和临床数据进行比较，以表征视紫红质稳定性、RP严重程度和视紫红质在细胞表面表达之间的关系。
突变变体的去稳定化程度与视网膜疾病的严重程度之间存在很强的线性相关性。在体外也观察到稳定性与质膜上视紫红质的量之间存在相关性。与野生型相比，固有稳定性差异超过2千卡/摩尔（kcal/mol）的变体转染的细胞表面，视紫红质显著减少。低于此阈值时，表面水平更接近野生型。
视紫红质突变的稳定性与疾病严重程度以及膜结合视紫红质水平之间存在相关性。用流式细胞术测量膜结合视紫红质可以改善对特征不明确的突变的预后，并可为测量治疗效果提供一个平台。
