title: 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.
authors.affiliation: Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional, Tijuana, Baja California 22390, México; E-Mails: rmares@uabc.edu.mx (R.E.M.); samuelmelendez@uabc.edu.mx (S.G.M.-L.).
authors.forename: Rosa E
authors.initials: RE
authors.lastname: Mares
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Meléndez-López
authors.forename: Marco A
authors.initials: MA
authors.lastname: Ramos
mesh_terms: D000143:Acids / Q000737:chemistry*; D001414:Bacitracin / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D000143:Acids; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D001414:Bacitracin; D049452:Green Fluorescent Proteins; D019704:Protein Disulfide-Isomerases