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

被引:17
作者
Mares, Rosa E. [1 ]
Melendez-Lopez, Samuel G. [1 ]
Ramos, Marco A. [1 ]
机构
[1] Univ Autonoma Baja California, Fac Ciencias Quim & Ingn, Tijuana 22390, Baja California, Mexico
关键词
green fluorescent protein; protein disulfide isomerase; folding; chaperone; ENDOPLASMIC-RETICULUM STRESS; GROUP-II CHAPERONIN; MOLECULAR CHARACTERIZATION; INHIBITORS; BINDING; GLYCOPROTEIN-120; CATALYST; REGION; BONDS;
D O I
10.3390/ijms12074625
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:4625 / 4636
页数:12
相关论文
共 39 条
  • [11] Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry
    Gallina, A
    Hanley, TM
    Mandel, R
    Trahey, M
    Broder, CC
    Viglianti, GA
    Ryser, HJP
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (52) : 50579 - 50588
  • [12] Substrate recognition by the protein disulfide isomerases
    Hatahet, Feras
    Ruddock, Lloyd W.
    [J]. FEBS JOURNAL, 2007, 274 (20) : 5223 - 5234
  • [13] WAVELENGTH MUTATIONS AND POSTTRANSLATIONAL AUTOXIDATION OF GREEN FLUORESCENT PROTEIN
    HEIM, R
    PRASHER, DC
    TSIEN, RY
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (26) : 12501 - 12504
  • [14] Studies on the function of yeast protein disulfide isomerase in renaturation of proteins
    Katiyar, S
    Till, EA
    Lennarz, WJ
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 2001, 1548 (01): : 47 - 56
  • [15] A structural overview of the PDI family of proteins
    Kozlov, Guennadi
    Maeaettaenen, Pekka
    Thomas, David Y.
    Gehring, Kalle
    [J]. FEBS JOURNAL, 2010, 277 (19) : 3924 - 3936
  • [16] Increasing melanoma cell death using inhibitors of protein disulfide isomerases to abrogate survival responses to endoplasmic reticulum stress
    Lovat, Penny E.
    Corazzari, Marco
    Armstrong, Jane L.
    Martin, Shaun
    Pagliarini, Vittoria
    Hill, David
    Brown, Anna M.
    Piacentini, Mauro
    Birch-Machin, Mark A.
    Redfern, Christopher P. F.
    [J]. CANCER RESEARCH, 2008, 68 (13) : 5363 - 5369
  • [17] Chaperonin-mediated folding of green fluorescent protein
    Makino, Y
    Amada, K
    Taguchi, H
    Yoshida, M
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (19) : 12468 - 12474
  • [18] INHIBITION OF A REDUCTIVE FUNCTION OF THE PLASMA-MEMBRANE BY BACITRACIN AND ANTIBODIES AGAINST PROTEIN DISULFIDE-ISOMERASE
    MANDEL, R
    RYSER, HJP
    GHANI, F
    WU, M
    PEAK, D
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (09) : 4112 - 4116
  • [19] Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes
    Morris, May C.
    [J]. CELL BIOCHEMISTRY AND BIOPHYSICS, 2010, 56 (01) : 19 - 37
  • [20] Thermal stability of chemically denatured green fluorescent protein (GFP) -: A preliminary study
    Nagy, A
    Málnási-Csizmadia, A
    Somogyi, B
    Lorinczy, D
    [J]. THERMOCHIMICA ACTA, 2004, 410 (1-2) : 161 - 163