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Corneal ALDH3A1 and UV Protection

Final Activity Report Summary - CORNEAL ALDH3A1 (Corneal ALDH3A1 and UV Protection)

Corneal epithelium is a self-renewing stratified epithelial tissue that constitutes the first line of defence functioning as a barrier against environmental assaults. It is characterised by unique features such as optical transparency, light refraction, and resistance to oxidative stress and neoplasia. Because the corneal epithelium is constantly exposed to sunlight, it has evolved defence mechanisms to prevent the eye from cellular oxidative damage. Among these defence mechanisms is the abundant expression of aldehyde dehydrogenase 3A1 (ALDH3A1), an NAD(P)-dependent metabolic enzyme that represents nearly half of the total soluble protein in the corneal epithelium whose functions are yet to be determined.

Previous studies have supported a protective role in corneal epithelium against apoptosis caused by UV radiation and other oxidative agents. The aim of this project was to further characterize the role(s) and the function(s) of the enzyme in the corneal epithelium and study the molecular mechanism(s) through which ALDH3A1 influences the apoptotic and cell surveillance pathways.

The project was organised under three specific aims.
- Specific aim 1 involved the characterisation of the functional domain(s) of ALDH3A1 protein responsible for the cytoprotective actions of ALDH3A1 in the corneal epithelium.
- Specific aim 2 involved the elucidation of the molecular mechanism(s) by which ALDH3A1 exerts its cytoprotective actions in response to UV-induced cellular damage.
- Specific aim 3 involved the characterisation of the effects of ALDH3A1 on the apoptotic and DNA repair signalling cascades in response to UV-induced cellular damage.

By using stably transfected corneal epithelial cell lines expressing the wild type and the mutant from for the active site of ALDH3A1 we demonstrated that ALD3A1 protects corneal epithelial cells from oxidative damage as significant lower levels of reactive oxygen species, reduced glutathione, lipid peroxidic aldehydes and oxidative DNA damage were observed in ALDH3A1-expressing cells. The metabolic activity of ALDH3A1 protein is dispensable for the cellular protection against oxidative damage although its metabolic active state potentiates its cytoprotective effects. Expression of ALDH3A1 provides a cell survival advantage by causing retardation effect in the cell cycle and by altering the expression profile of proapoptotic / antiapoptotic proteins and reducing the basal levels of p53 in corneal epithelial cells.

Preliminary towards evaluating the DNA repair efficiency in the ALDH3A1-expressing and -non expressing cells lines demonstrated that expression of ALDH3A1 improves the DNA repair efficiency in corneal epithelial cells. Experiments using the recombinant protein showed that ALDH3A1 protects the UV-induced inactivation of glucose-6-phosphate dehydrogenase in vitro and suppressed the thermal aggregation of other model proteins supporting a potential chaperone-like role in the corneal epithelium.

Based on these results, a new picture is arisen for ALDH3A1 as a multifunctional protein that plays an important role in corneal homeostasis. Elucidating the role of corneal ALD3A1 provides invaluable information about the proteins that are recruited in corneal epithelium as crystallins and shed light into their physiological function(s) and their role into the pathophysiology of corneal related diseases.