ABSTRACT
A typical 2-cysteine peroxiredoxin (2-Cys Prx) PaPrx can act alternatively as thioredoxin (Trx)-dependent peroxidase and molecular chaperone in Pseudomonas aeruginosa PAO1. In addition, the functional switch of PaPrx is regulated by its structural change which is dependently induced by stress conditions. In the present study, we examined the effect of gamma ray on structural modification related to chaperone activity of PaPrx. The structural change of PaPrx occupied with gamma ray irradiation (2 kGy) based on polyacrylamide gel electrophoresis (PAGE) analysis and the functional change also began. The enhanced chaperone activity was increased about 3-4 folds at 30 kGy gamma irradiation compared with nonirradiated PaPrx, while the peroxidase activity was significantly decreased. We also investigated the influence of the gamma ray on protein hydrophobicity as related to chaperone function. The exposure of hydrophobic domains reached a peak at 30 kGy gamma ray and then decreased dependently with increasing gamma irradiation. Our results suggest that highly enhanced chaperone activity could be adapted for use in bio-engineering systems and industrial applications such as enzyme stabilization during industrial process (inactivation protection), improvement of useful protein productivity (refolding and secretion) and industrial animal cell cultivation (stress protection).
Subject(s)
Gamma Rays , Peroxiredoxins/metabolism , Peroxiredoxins/radiation effects , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/radiation effects , Hydrophobic and Hydrophilic Interactions/radiation effects , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Chaperones/radiation effects , Peroxiredoxins/chemistry , Peroxiredoxins/genetics , Protein Engineering , Protein Structure, Secondary/radiation effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/radiation effects , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/radiation effectsABSTRACT
We study the effect of microwaves at 2,450 MHz on protein unfolding using surface plasmon resonance sensing. Our experimental method makes use of the fact that unfolding proteins tend to bind to chaperones on their unfolding pathway and this attachment is readily monitored by surface plasmon resonance. We use the protein citrate synthase (CS) for this study as it shows strong binding to the chaperone alpha crystallin when stressed by exposure to excess temperature. The results of microwave heating are compared with the effect of ambient heating and a combination of ambient and microwave heating to the same final temperature. We study the temperature distributions during the heating process. We show that microwaves cause a significantly higher degree of unfolding than conventional thermal stress for protein solutions heated to the same maximum temperature.
Subject(s)
Citrate (si)-Synthase/chemistry , Citrate (si)-Synthase/radiation effects , Microwaves , Molecular Chaperones/chemistry , Molecular Chaperones/radiation effects , alpha-Crystallins/chemistry , alpha-Crystallins/radiation effects , Dose-Response Relationship, Radiation , Hot Temperature , Protein Binding/radiation effects , Radiation DosageABSTRACT
A range of environmental conditions can lead to oxidative stress; thus, a prompt and effective response to oxidative stress is crucial for the survival of plants. Microarray and northern-blot analyses were performed toward the identification of the factors and signaling pathways that enable plants to limit oxidative damage caused by exposure to high light (HL). Arabidopsis plants grown under moderate light (100 micromol m(-2) s(-1)) were exposed to HL (1,000 micromol m(-2) s(-1)) for 1 h. The microarray analyses revealed that exposure of Arabidopsis to HL caused an increase in known antioxidant genes, as well as several unknown genes. Some of these unknown genes had homologies to possible regulatory genes and metabolic enzymes. Furthermore, it was found that a range of chaperones were up-regulated in the HL treatment and that this induction was specifically due to the HL stress. The temporal expression under HL and different oxidative stress conditions of a subset of HL-responsive genes was confirmed via northern-blot analysis. Results from the arrays were also compared with publicly available microarray data sets from a range of different stress conditions at the Arabidopsis Functional Genomics Consortium. This cross comparison enabled the identification of genes that may be induced by changes in redox poise. Finally, to determine if the genes that were differentially expressed by HL stress were under similar transcriptional control, we analyzed the promoter sequences for the presence of common motifs.
Subject(s)
Arabidopsis/radiation effects , Oxidative Stress/radiation effects , Antioxidants/metabolism , Antioxidants/radiation effects , Arabidopsis/genetics , Arabidopsis/metabolism , Blotting, Northern , Environment , Gene Expression Regulation, Plant/radiation effects , Heat-Shock Proteins/genetics , Heat-Shock Proteins/radiation effects , Light , Molecular Chaperones/genetics , Molecular Chaperones/radiation effects , Oligonucleotide Array Sequence Analysis , Oxidative Stress/genetics , Promoter Regions, Genetic/genetics , Signal Transduction/genetics , Signal Transduction/radiation effectsABSTRACT
Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.
Subject(s)
Crystallins/chemistry , Crystallins/radiation effects , Molecular Chaperones/chemistry , Molecular Chaperones/radiation effects , Animals , Aspartic Acid/chemistry , Aspartic Acid/radiation effects , Cattle , Gamma Rays/adverse effects , In Vitro Techniques , Isomerism , Oxidation-Reduction , Photochemistry , Protein Structure, Secondary/radiation effects , Protein Structure, Tertiary/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , StereoisomerismABSTRACT
alpha-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of alpha-crystallin towards photo-induced aggregation of gamma-crystallin, aggregation of insulin and on the refolding induced aggregation of beta- and gamma-crystallins. We observed that alpha-crystallin could prevent photo-aggregation of gamma-crystallin and this chaperone-like activity of alpha-crystallin is enhanced several fold at temperatures above 30 degrees C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or re-organized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to alpha-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.
Subject(s)
Crystallins/chemistry , Crystallins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Animals , Crystallins/radiation effects , Dithiothreitol , In Vitro Techniques , Insulin/chemistry , Insulin/metabolism , Macromolecular Substances , Molecular Chaperones/radiation effects , Photochemistry , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Tertiary , TemperatureABSTRACT
Previous reports on the chaperone activity of alpha-crystallin to prevent protein denaturation and thermal aggregation have suggested that partially denatured proteins can bind alpha-crystallin in its central region. Likewise, beta- and gamma-crystallin can also be localized to the central cavity of alpha-crystallin particle in vivo, which provides indirect evidence that alpha-crystallin can function as a chaperone in the intact lens. In this study, we have further demonstrated that the binding of the substrate proteins to alpha-crystallin by short-term preincubation may mimic the in vivo conditions of crystallin association. Preheating of alpha-crystallin with its substrate proteins at 60 degrees C for 20 min resulted in the formation of stable complexes between alpha-crystallin and its substrates (8.0% of insulin or 5.3% of gamma-crystallin was involved in complex formation). Under such conditions, the chaperone activity of alpha-crystallin to inhibit dithiothreitol-, ultraviolet-, or oxidation-induced protein aggregation can be greatly enhanced. Since UV-irradiation and oxidative stress are common insults to eye lenses under normal physiological conditions, the presence of alpha/gamma and alpha/beta complex in vivo may play an important role to maintain the lens in a transparent state.
Subject(s)
Crystallins/chemistry , Crystallins/physiology , Molecular Chaperones/chemistry , Molecular Chaperones/physiology , Animals , Cattle , Crystallins/metabolism , Crystallins/radiation effects , In Vitro Techniques , Insulin/chemistry , Insulin/metabolism , Macromolecular Substances , Molecular Chaperones/radiation effects , Oxidative Stress , Protein Binding , Protein Denaturation , Ultraviolet Rays/adverse effectsABSTRACT
alpha-Crystallin, a major protein of the eye lens, is known to have chaperone activity in preventing heat-induced aggregation of enzymes and other crystallins. In this study, we investigate the ability of alpha-crystallin to inhibit UV-light-induced aggregation of other lens proteins and the effect of exposure of alpha-crystallin to UV irradiation on its chaperone activity. The chaperone activities of alpha-crystallin preincubated at different temperatures were found to be different and could be correlated with its change in quaternary structure as determined by the fluorescence probe ANS (8-anilo-1-naphthalene sulfonate). alpha-Crystallin can inhibit the aggregation of gamma-crystallin from UV irradiation at room temperature, and the preheated alpha-crystallins provide more protection than the native one. Upon irradiation by UV light, alpha-crystallin gradually lost its ability to protect beta-crystallin against thermal aggregation. The loss of the chaperone efficacy of alpha-crystallin to protect other lens proteins may shed light on human cataract formation induced by long-term exposure to UV irradiation.
Subject(s)
Crystallins/metabolism , Molecular Chaperones/metabolism , Ultraviolet Rays/adverse effects , Anilino Naphthalenesulfonates/analysis , Animals , Crystallins/drug effects , Crystallins/pharmacology , Crystallins/radiation effects , Fluorescent Dyes , Heating/adverse effects , Humans , Molecular Chaperones/pharmacology , Molecular Chaperones/radiation effects , Protein Binding , Protein Conformation/drug effects , Protein Denaturation/drug effects , Protein Denaturation/radiation effects , SwineABSTRACT
Buffer solutions of the lens protein gamma-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63 degrees C or to UV radiation at 308 nm. When alpha-crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of alpha-crystallin. Hence, normal alpha-crystallin functioned as a "molecular chaperone," providing protection against both UV and heat-induced protein aggregation. When alpha-crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis-a-vis both UV and heat-induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of alpha-crystallin was to cause interpeptide crosslinking among the alpha A2 and alpha B2 subunits of the alpha-crystallin macromolecule. In our experiments alpha-crystallin was exposed to UV doses, which resulted in 0.50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated alpha-crystallin. The results are consistent with the notion that UV irradiation of alpha-crystallin results in loss of chaperone binding sites.
