Upagupta Chandak, Carlisle Rachel E, Dickhout Jeffrey G
Department of Biochemistry, McMaster University, 1280 Main St W, Hamilton, ON L8S 4L8, Canada.
Department of Medicine, Division of Nephrology, McMaster University, St. Joseph's Healthcare Hamilton, 50 Charlton Ave E, Hamilton, ON L8N 4A6, Canada.
Biochem Biophys Res Commun. 2017 Apr 22;486(1):163-170. doi: 10.1016/j.bbrc.2017.03.019. Epub 2017 Mar 8.
Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.
新翻译的蛋白质必须经过适当折叠以确保其功能。为了进入低能状态，错误折叠的蛋白质会形成聚集体，这与许多退行性疾病有关，如亨廷顿舞蹈症和慢性肾病（CKD）。最近的研究表明，使用低分子量化学伴侣是减少各种细胞类型中蛋白质聚集的有效方法。本研究展示了一种新颖的无偏差检测方法，用于评估这些化合物在预防蛋白质错误折叠和/或聚集方面的分子功效。该检测方法利用硫黄素T荧光染色来定性和定量测量细胞内蛋白质的错误折叠情况。此方法的功能首先在经各种内质网（ER）应激诱导剂处理的肾近端小管上皮细胞中进行评估。一旦在肾脏模型系统中确立，我们分析了一些已知化学伴侣减轻ER应激的能力。总共选择了五种不同的化合物：4-苯丁酸盐（4-PBA）、二十二碳六烯酸（DHA）、牛磺熊去氧胆酸、海藻糖和甘油。然后分析了这些化合物在减轻毒胡萝卜素诱导的ER应激方面的剂量依赖性效应，并用于确定它们的半数有效浓度（EC）值。在这些伴侣中，4-PBA和DHA在相对较低的浓度下就能最大程度地减轻ER应激。在分析这些化合物的效率及其相应结构后，确定具有局部亲水极性末端且随后接有长疏水链的伴侣，如4-PBA和DHA，在减轻ER应激方面最有效。本研究为低分子量化学伴侣的使用提供了一些见解，并可能作为开发更高效新伴侣的第一步，从而为蛋白质聚集引起的疾病提供潜在治疗方法。
