Abstract
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.
Highlights
Green fluorescent protein (GFP) is an autofluorescent protein that was first identified and isolated from the jellyfish, Aequorea victoria [1]
Several high-throughput screening (HTS) assays to test protein disulfide isomerase (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
Eukaryotic PDI enzymes have been shown to be up-regulated during ER stress, suggesting that they might be involved in cellular protection, including protection against induced apoptosis [30,31]
Summary
Green fluorescent protein (GFP) is an autofluorescent protein that was first identified and isolated from the jellyfish, Aequorea victoria [1]. PDI enzymes play a key role in the folding of proteins delivered to the secretory pathway; they are multifunctional proteins that display chaperone activity [16,17] Their function as oxidoreductase or chaperone is substrate-dependent [18,19,20]. Since refolding of acid-denatured GFP can be and directly monitored by real-time fluorescence, a simple micro-assay was used to study the effect of the chaperone or substrate concentration on protein folding kinetics. This micro-assay format was used to evaluate the effect of a well-known inhibitor of PDI enzymes
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