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Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

ABSTRACT: As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys.

SUBMITTER: Estey T

PROVIDER: S-EPMC3006428 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

Estey Tia T Chen Ying Y Carpenter John F JF Vasiliou Vasilis V

PloS one 20101221 12

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3 ...[more]

PMID: 21203538

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Project description:Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1(-/-)/3a1(-/-) double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.

Project description:?-Crystallin is the major protein of the eye lens and a member of the family of small heat-shock proteins. Its concentration in the human eye lens is extremely high (about 450 mg/mL). Three-dimensional structure of native ?-crystallin is unknown. First of all, this is the result of the highly heterogeneous nature of ?-crystallin, which hampers obtaining it in a crystalline form. The modeling based on the electron microscopy (EM) analysis of ?-crystallin preparations shows that the main population of the ?-crystallin polydisperse complex is represented by oligomeric particles of rounded, slightly ellipsoidal shape with the diameter of about 13.5 nm. These complexes have molecular mass of about 700 kDa. In our opinion, the heterogeneity of the ?-crystallin complex makes it impossible to obtain a reliable 3D model. In the literature, there is evidence of an enhanced chaperone function of ?-crystallin during its dissociation into smaller components. This may indirectly indicate that the formation of heterogeneous complexes is probably necessary to preserve ?-crystallin in a state inactive before stressful conditions. Then, not only the heterogeneity of the ?-crystallin complex is an evolutionary adaptation that protects ?-crystallin from crystallization but also the enhancement of the function of ?-crystallin during its dissociation is also an evolutionary acquisition. An analysis of the literature on the study of ?-crystallin in vitro led us to the assumption that, of the two ?-crystallin isoforms (?A- and ?B-crystallins), it is ?A-crystallin that plays the role of a special chaperone for ?B-crystallin. In addition, our data on X-ray diffraction analysis of ?-crystallin at the sample concentration of about 170-190 mg/mL allowed us to assume that, at a high concentration, the eye lens ?-crystallin can be in a gel-like stage. Finally, we conclude that, since all the accumulated data on structural-functional studies of ?-crystallin were carried out under conditions far from native, they cannot adequately reflect the features of the functioning of ?-crystallin in vivo.

Dataset Information

Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

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Structural and functional modifications of corneal crystallin ALDH3A1 by UVB light.

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