Mares Rosa E, Meléndez-López Samuel G, Ramos Marco A
Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional, Tijuana, Baja California 22390, México; E-Mails:
Int J Mol Sci. 2011;12(7):4625-36. doi: 10.3390/ijms12074625. Epub 2011 Jul 18.
Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.
绿色荧光蛋白（GFP）因其在体外和体内都具有显著的稳定性，已被广泛应用于多种分子和细胞生物学领域。有趣的是，天然GFP对最常见的化学变性剂具有抗性；然而，在酸诱导变性后观察到荧光信号较低。此外，这种酸变性的GFP已被用作某些细菌伴侣蛋白和其他类似伴侣分子折叠活性研究的底物。蛋白质二硫键异构酶是一类真核氧化还原酶，可催化新生多肽中二硫键的氧化和异构化，在蛋白质折叠中起关键作用，并且可能表现出伴侣活性。然而，使用不同蛋白质作为模型底物时报道了相互矛盾的结果。在这里，我们报告了GFP作为模型底物在研究蛋白质二硫键异构酶（PDI）伴侣活性方面的进一步应用。由于酸变性GFP的复性可以轻松直接地监测，因此我们使用一种简单的微量测定法来研究PDI辅助蛋白质复性过程中分子参与者的作用。此外，还分析了一种著名的PDI伴侣活性抑制剂的作用。由于其功能活性的多样性，PDI酶可能是潜在的有趣药物靶点。由于PDI可能参与保护细胞免受内质网应激，包括癌细胞，PDI抑制剂可能能够增强癌症化疗的疗效；此外，已经证明阻断与细胞表面相关蛋白质二硫键的还原裂解可显著降低人类免疫缺陷病毒的感染性。尽管已经描述了几种用于测试PDI还原酶活性的高通量筛选（HTS）测定法，但我们在此报告一种新颖且简单的微量测定法来测试PDI酶的伴侣活性，该方法适用于PDI抑制剂的高通量筛选。
