Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

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.

Heterotypic seeding of Tau fibrillization by pre-aggregated Abeta provides potent seeds for prion-like seeding and propagation of Tau-pathology in vivo

“…DAPI staining to label nuclei was performed in a PBS washing step. The GFP bleaching experiments were performed as previously described with minor modifications [ 32 ]. Briefly, a 5 µM acid-denatured GFP solution (pH 1.5) was prepared by mixing equal amounts of solution A (50 mM Tris–HCl pH 7.5) with solution B (125 mM HCl) and incubated with cells 1 min at RT.…”

Protein polarization driven by nucleoid exclusion of DnaK(HSP70)–substrate complexes

“…CiΔU-His folding was measured using GFP fluorescence detection 61 . CiΔU-His at a final concentration of 20 μM was acid-denatured with 33 mM HCl for 10 min at 21 °C.…”

Novel roles for protein disulphide isomerase in disease states: a double edged sword?

“…Several methods are used to measure the chaperone activity of PDI in vitro . The rate of protein aggregation is assessed using protein substrates that do not possess cysteine residues, including GAPDH (Cai et al, 1994 ), rhodanese (Song and Wang, 1995 ), citrate synthase, alcohol dehydrogenase (Primm et al, 1996 ), or GFP, which on interaction with PDI causes increased fluorescent intensity as it folds into its native conformation (Mares et al, 2011 ).…”