ALDH3A1: a corneal crystallin with diverse functions

Experimental Eye Research 84 (2007) 3e12 www.elsevier.com/locate/yexer

Review

ALDH3A1: a corneal crystallin with diverse functions Tia Estey a, Joram Piatigorsky b, Natalie Lassen c, Vasilis Vasiliou a,c,* a

Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA b Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA c Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 1 April 2006; accepted in revised form 19 April 2006 Available online 21 June 2006

Abstract Aldehyde dehydrogenase 3A1 (ALDH3A1) comprises a surprisingly high proportion (5e50% depending on species) of the water-soluble protein of the mammalian cornea, but is present little if at all in the cornea of other species. Mounting experimental evidence demonstrates that this abundant corneal protein plays an important role in the protection of ocular structures against oxidative damage. Corneal ALDH3A1 appears to protect against UV-induced oxidative stress through a variety of biological functions such as the metabolism of toxic aldehydes produced during the peroxidation of cellular lipids, the generation of the antioxidant NADPH, the direct absorption of UV-light, the scavenging of reactive oxygen species (ROS), and the possession of chaperone-like activity. With analogies to the abundant, multifunctional, and taxon-specific lens crystallins, mammalian ALDH3A1 has been considered a corneal crystallin, suggesting that it may contribute to the optical properties of the cornea as well. Recent studies have also revealed a novel role for ALDH3A1 in the regulation of the cell cycle. ALDH3A1-transfected HCE cells have increased population-doubling time, decreased plating efficiency, and reduced DNA synthesis, most likely due to a profound inhibition of cyclins and cyclin-dependent kinases. We have proposed that the ALDH3A1-induced reduction in cell growth may contribute to protection against oxidative stress by extending time for DNA and cell repair. Taken together, the multiple roles of ALDH3A1 against oxidative stress in addition to its contributions to the optical properties of the cornea are consistent with the idea that this specialized protein performs diverse biological functions as do the lens crystallins. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: ALDH3A1; corneal crystallin; oxidative stress; lipid peroxidation; cell growth

1. Introduction Located at the anterior surface of the eye, the mammalian cornea is an avascular tissue that serves as a protective barrier between the environment and the internal ocular structures. One of the primary sources of environmental stress for the cornea is solar radiation, specifically in the ultraviolet (UV)-range. The cornea absorbs UV-light differentially based * Corresponding author. Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA. Tel.: þ1 303 315 6153; fax: þ1 303 315 6281. E-mail address: [email protected] (V. Vasiliou). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.04.010

on the incident wavelength. Over 90% of both UVC- and UVB-light is absorbed by the cornea in addition to 60% of UVA radiation (Zigman, 1993), whereas the remaining UVA-light is absorbed by the lens so that only a minor amount (<1%) reaches the retina (Pitts, 1993) (Fig. 1). UV-radiation can damage ocular tissues. Long- and short-term UV-exposure to the eye may result in photokeratoconjunctivitis and play an active role in the etiology of cataracts, corneal and retinal degeneration, pterygium, uveal melanoma, and other neoplasms of the anterior portion of the eye (van Kuijk, 1991). On the level of corneal morphology, UV-exposure can cause a reduction in epithelial cell proliferation (Haaskjold et al., 1993), alterations in the thickness in the epithelial layer (Cullen et al., 1984; Koliopoulos and Margaritis, 1979; Riley et al., 1987),

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and loss in metabolic capacity (Lattimore, 1988, 1989). Additionally, a noticeable decrease in activity of the metabolic enzymes alcohol and aldehyde dehydrogenase (Downes et al., 1993; Manzer et al., 2003) as well as corneal antioxidant enzymes such as catalase and glutathione peroxidase, have been observed after UV-exposure (Cejkova et al., 2000). UVinduced lipid peroxidation, protein modification, and extensive DNA damage can singularly or collectively lead to a loss in the viability of epithelial cells and death by apoptotic and necrotic pathways (Hightower, 1995; Hightower et al., 1994; Shinohara et al., 2000). The combination of near continual exposure of the cornea to UV-radiation and molecular oxygen may lead to substantial oxidative stress and tissue damage. Oxidative stress is the result of an imbalance between reactive oxygen species (ROS) and antioxidants. When ROS production exceeds the capacity of the antioxidants and repair systems, oxidative stress occurs and cellular damage and death may follow. Commonly encountered ROS in biological systems include the hydroxyl radical (OH$), the superoxide anion (O$ 2 ), hydrogen peroxide (H2O2), and singlet oxygen (1O2). These species may not only be generated by environmental factors such as ultraviolet radiation (UVR) and exposure to exogenous toxins (i.e. nitric oxide, cigarette smoke), but are also the product of normal metabolic activity (Chance et al., 1979; Shacter, 2000; Stadtman and Levine, 2000). Since all biomolecules (DNA, proteins, lipids, and carbohydrates) are susceptible to oxidation, a variety of antioxidants have evolved to minimize the deleterious effects of ROS. Catalase (Chance et al., 1979) and superoxide dismutase (SOD) (Fridovich, 1995) catalytically remove ROS whereas ferritin (Harrison and Arosio, 1996) and transferrin (Bates and Schlabach, 1973) reduce the availability of metal ions, which are pro-oxidants that accelerate ROS formation. Reduced glutathione (GSH) (Chance et al., 1979; Dickinson and Forman, 2002), ascorbate (Machlin and Bendich, 1987), a-tocopherol (Machlin and Bendich, Cornea Lens

Retina Optic Nerve

1987), and NAD(P)H (Forni and Willson, 1986a; Kirsch and De Groot, 2001) are low molecular weight compounds that function as ROS scavengers. In addition to compounds and proteins that reduce ROS concentrations in the cell, repair systems also exist to help reverse oxidative damage to DNA and proteins. Oxidized DNA, which may lead to genetic mutation and cell death if left unchecked, can be repaired by base excision (Sancar, 1996). The majority of damaged protein molecules are typically ubiquitinated and then degraded by the proteasome (Hilt and Wolf, 1996), though protein oxidation reactions such as non-native disulfide cross-linking and the formation of methionine sulfoxide may be reversed (Stadtman and Levine, 2003). ALDH3A1 contributes to the corneal defenses by playing a critical and multifunctional role in the protection of the cornea, and perhaps the entire eye, against UV-induced oxidative stress. ALDH3A1 is one of the most abundantly expressed proteins in the corneal epithelium, accounting for 5 to 50% of the total water-soluble protein fraction in mammalian species (Abedinia et al., 1990; Nees et al., 2002; Pappa et al., 2001; Piatigorsky, 2001). ALDH3A1 is a member of the ALDH superfamily of proteins that catalyze the NAD(P)þ-dependent oxidation of a wide range of endogenous and exogenous aldehydes (Vasiliou et al., 2004; Vasiliou and Nebert, 2005). An early observation that SWR/J mice are susceptible to corneal clouding after exposure to UVR (Downes et al., 1994) led to the finding that these mice are ‘‘natural knockouts’’ due to structural mutations in the gene coding sequences that result in the near absence of ALDH3A1 expression (Pappa et al., 2001; Shiao et al., 1999). The central roles and mechanisms by which ALDH3A1 protects against UV-induced corneal damage are thought to include (i) metabolism of the toxic aldehydes produced during lipid peroxidation (King and Holmes, 1993), (ii) direct absorption of UV-light (Abedinia et al., 1990; Mitchell and Cenedella, 1995), (iii) antioxidant function either directly through the scavenging of free radicals (Uma et al., 1996b) or indirectly through the production of NADPH (Atherton et al., 1999), (iv) maintaining corneal refractive and transparent properties as a corneal crystallin (Piatigorsky, 1998a), and (v) chaperone-like activity (Manzer et al., 2003; Uma et al., 1996a) (Fig. 2).

SOLAR RADIATION

UVC

(190-290 nm)

2. The role of ALDH3A1 in detoxification

UVB

(290-320 nm)

UVA

(320-400 nm)

Visible

(400-700 nm)

Fig. 1. Schematic representation of the absorption of solar radiation by the eye. UVC-light (190e290 nm) is nearly entirely absorbed by the cornea. Though the majority of both UVB (290e320 nm) and UVA (320e400 nm) is also absorbed by the cornea, a small amount of UVA-light penetrates the cornea and is later absorbed by the lens. This ensures that only a trivial amount of UVAlight (<1%) reaches the retina. On the contrary, visible light is transmitted through both the lens and cornea to the retina to provide the basis of vision.

Lipid peroxidation is causally implicated in a wide variety of disorders such as atherogenesis (Steinberg, 1999; Witting et al., 1999), cataractogenesis (Ansari et al., 1996; Awasthi et al., 1996; Srivastava et al., 1995), neurogenerative diseases (Butterfield et al., 2001; Sayre et al., 2001), and retinopathy (Kumar et al., 2001; Totan et al., 2001). UVR induces lipid peroxidation in many cell types including human corneal epithelial (HCE) cells (Marks-Hull et al., 1997). UVR leads to the production of ROS, which initiate lipid peroxidation by attacking the polyunsaturated fatty acids in cell membrane phospholipids and eventually cause the accumulation of toxic and reactive aldehydes. Unlike ROS, aldehydes are generally long-lived compounds that can diffuse through the cell and

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react with biological targets distant from the site of origin. More than 200 aldehyde species are associated with the peroxidation of cellular lipids including 4-hydroxy-2-nonenal (4HNE) (Esterbauer et al., 1991). 4-HNE is one of the most toxic aldehydes generated by lipid peroxidation, and the cytotoxic and genotoxic effects of 4-HNE have been reviewed (Esterbauer et al., 1991; Esterbauer, 1993). The biological impact of 4-HNE depends upon its concentration and involves cell signaling, protein and DNA damage, and apoptosis (Herbst et al., 1999; Li et al., 1996; Malecki, 2000). Like many other a,b-unsaturated aldehydes, 4-HNE is a potent electrophile that can react with nucleophilic sites including those of DNA and proteins (Esterbauer et al., 1991). The electrophilic nature of 4-HNE arises due to the conjugated double bond of the aldehyde moiety, and is further enhanced by the nearby electron-withdrawing alcohol group. This produces a strong electrophilic center on the C-3 carbon that is particularly reactive toward the Cys residues of proteins (Doorn and Petersen, 2002; Esterbauer et al., 1991). 4-HNE can also attack His (Doorn and Petersen, 2002; Isom et al., 2004; Uchida and Stadtman, 1992), Lys (Doorn and Petersen, 2002; Isom et al., 2004; Szweda et al., 1993), and Arg residues (Isom et al., 2004). The covalent binding of 4-HNE to protein molecules, which can create intermolecular cross-links (Petersen and Doorn, 2004; Uchida and Stadtman, 1993), often results in a loss in protein and cellular functions (Carini et al., 2004; Crabb et al., 2002). For example, inactivation of glutathione reductase by 4-HNE enhances the potential for cellular oxidative damage (Vander Jagt et al., 1997) and Cell membrane (1)

ALDH3A1

UVR

(2) ROS Lipid peroxidation Adducts to corneal proteins (5)

Aldehydes NADP+

ALDH3A1

Aggregation

GSSG

AL

DH

(3)

3A

1

Accumulation of damaged proteins Cellular toxicity

Changes in refraction and/or transparency

NADPH (4) GSH Carboxylic acids

Excretion

Fig. 2. Proposed roles of ALDH3A1 in the protection of the cornea against UV-induced oxidative stress. ALDH3A1 may be involved in various protective functions including: (1) direct absorption of ultraviolet radiation (UVR); (2) scavenging reactive oxygen species (ROS); (3) metabolism of toxic aldehydes produced through UV-induced lipid peroxidation; and (4) production of the antioxidant NADPH, which directly absorbs UVR and is required for the regeneration of reduced glutathione (GSH). ALDH3A1 has also been proposed to protect against non-native protein aggregation through chaperone-like activity (5). Additionally, ALDH3A1 may serve as a structural element (i.e. corneal crystallin) to maintain the optical properties of the cornea.

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4-HNE-inactivation of glutathione peroxidase is associated with a decrease in cellular glutathione levels (Kinter and Roberts, 1996). High levels of 4-HNE-modified proteins may play a causative role in the pathophysiology of degenerative disease and cellular aging (Lucas and Szweda, 1998; Sayre et al., 1997; Yoritaka et al., 1996), and 4-HNE accumulation has been observed in both cataracts (Ansari et al., 1996; Bhuyan et al., 1992) and pathologic corneas (Buddi et al., 2002). The deleterious effects of 4-HNE, including protein adduct formation and apoptosis, are substantially reduced by the expression of ALDH3A1 in at least two cell lines: RAW 264.7 murine macrophages (Haynes et al., 2000) and HCE cells (Pappa et al., 2003a). 4-HNE-induced cytotoxicity in HCE cells (TD50 ¼ 11 mM) was less pronounced in the ALDH3A1transfected cells (TD50 ¼ 48 mM) (Pappa et al., 2003a). Additionally, HCE cells expressing ALDH3A1 at high levels were less sensitive to UV-light and showed a lower rate of cell death after UVB- and UVC-exposure when compared to the mocktransfected HCE control cells (Pappa et al., 2003a). In both experiments DNA laddering, caspase-3 activation, and 4HNEeprotein adducts were reduced in the presence of ALDH3A1. The protective effect provided by ALDH3A1 can be attributed to detoxification of 4-HNE. The apparent affinity constant (Km) for 4-HNE in the cytosolic extracts of the ALDH3A1-transfected HCE cells is approximately 56 mM (Pappa et al., 2003a) and is consistent with ALDH3A1 being able to metabolize 4-HNE in vivo. Additionally, purified recombinant human ALDH3A1 metabolizes 4-HNE with an apparent Km of approximately 45 mM, which indicates high specificity for the aldehyde (Pappa et al., 2003b). Purified ALDH3A1 can also enzymatically oxidize other a, b-unsaturated aldehydes produced by the peroxidation of cellular lipids including trans-2-hexenal (Km w 155 mM), trans-2octenal (Km w 35 mM), and trans-2-nonenal (Km w 12 mM) (Pappa et al., 2003b). Collectively, these data indicate that ALDH3A1 can contribute to the detoxification of various aldehydes produced by the peroxidation of cellular lipids in response to oxidative stress. The cytotoxicity of aldehydes associated with lipid peroxidation may arise in part due to direct and indirect inhibition of the proteasome. In eukaryotic organisms, the primary route of intracellular protein degradation is the proteasome (Davies, 2001), a multi-catalytic enzyme complex found in the cytosol and nucleus of the cell (Gaczynska et al., 2001). Proteasome degradation encompasses both normal protein turnover as well as the removal of damaged and/or misfolded proteins (Orlowski and Wilk, 2000). Oxidized proteins are also selectively degraded by the proteasome (Grune et al., 1997; Shaeffer, 1988), consistent with it being a fundamental element of the cellular defense against oxidative stress (Davies, 2001). Extensively oxidized proteins, however, may actually inhibit proteasome function (Bulteau et al., 2002b). Proteins modified by either prolonged exposure to oxidative conditions or to 4-HNE have been shown to reduce the activity of the proteasome (Davies, 2001; Friguet and Szweda, 1997). 4-HNE-modified glucose-6-phosphate dehydrogenase (G6PD) is resistant to proteasome degradation and, in some cases,

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4-HNE-modified proteins were actually found to be noncompetitive inhibitors of the proteasome (Bulteau et al., 2002b; Friguet and Szweda, 1997; Friguet et al., 1994). Additionally, direct exposure of cells to either UVR or 4HNE can have a deleterious impact on the function of the proteasome. UVA- and UVB-exposure to human keratinocytes resulted in a loss in proteolytic degradation (Bulteau et al., 2002a), and 4-HNE can directly inhibit the trypsin and peptidylglutamyl peptide hydrolase activities of the proteasome (Okada et al., 1999). Loss of proteasome function can lead to the accumulation of damaged proteins and consequent disruption of cellular function. Decreased activity of the proteasome has been implicated in a variety of diseases and may contribute to ageing (Carrard et al., 2002; Stadtman, 2001). In the lens, the accumulation and eventual precipitation of damaged crystallin proteins are thought to contribute to cataract formation (Taylor and Davies, 1987) and may result in part from a loss in proteasome function (David and Shearer, 1989; Garland et al., 1988; Zetterberg et al., 2003). Recent studies in our laboratory provide evidence for the role of ALDH3A1 in protecting against proteasome inhibition in cultured rabbit stromal fibroblasts (TRK43 cells) and in Aldh3a1(/) mice (Lassen, N. et al., in preparation). TRK43 cells lose proteasome activity after exposure to UVR and 4-HNE. This effect was markedly reduced in TRK43 cells stably-transfected to express human ALDH3A1. In addition, Aldh3a1(/) mice have reduced proteasome activity in the cornea and lens when compared to the age-matched control animals. We propose that the detoxification of aldehydes, specifically 4-HNE, by ALDH3A1 preserves the normal function of the proteasome. ALDH3A1 can prevent both direct (i.e. inactivation by 4-HNE) and indirect (i.e. non-competitive inhibition by extensively oxidized proteins) pathways of proteasome inhibition by efficiently reducing the cellular concentration of 4-HNE (Fig. 3). Normal function of the proteasome would help to remove damaged and oxidized proteins as well as prevent non-native protein aggregation, both of which may be important in maintaining corneal transparency.

3. Direct absorption of UVR and ‘‘suicide’’ response It was proposed early on that bovine ALDH3A1, first identified as BCP54 (Alexander et al., 1981; Silverman et al., 1981), serves an important UV absorptive role in the cornea in addition to metabolizing aldehyde by-products of UV-induced lipid peroxidation (Abedinia et al., 1990). The role of this protein in the direct absorption of UVR was supported by the finding that the water-soluble protein fraction of bovine corneas accounts for only 17% of the total protein but for nearly 50% of the total absorption of UVB-light (290e300 nm), leading to the suggestion that the watersoluble proteins in the cornea be termed absorbins (Mitchell and Cenedella, 1995). The UV absorbing power of ALDH3A1 is enhanced by its ability to bind NADPH (Atherton et al., 1999). It is thus likely that ALDH3A1 substantially contributes to the UV-absorption properties of the cornea.

UVR ROS Lipid peroxidation 4-HNE

Oxidized proteins

Proteasome degradation

ALDH3A1

Amino acids

Proteasomeresistant proteins Protein aggregation

Loss in corneal transparency

Fig. 3. The role of ALDH3A1 in the preservation of proteasome activity through 4-HNE metabolism. The inhibition of the proteasome may occur through both direct and indirect mechanisms; 4-HNE can directly bind and inactivate the proteasome whereas 4-HNE-modified proteins are known inhibitors of proteasome degradation. In either case, loss of proteasome activity can lead to the accumulation of damaged proteins that may affect the transparency of the cornea. ALDH3A1 efficiently catalyzes 4-HNE and thus blocks both direct and indirect pathways of 4-HNE-induced proteasome inhibition. Ultraviolet radiation (UVR) and reactive oxygen species (ROS) can also contribute to the pool of oxidized proteins in the cell.

The direct absorption of UV-light by ALDH3A1 may be a critical mechanism for protecting proteins and other cellular elements because UVR is a major source of oxidative stress for the cornea. UV-induced oxidative stress arises from the direct absorption of the UV energy by the constituents of the cells, including protein molecules themselves. Proteins absorb UV-light primarily through electronic transitions of the peptide bond (190e220 nm) and of the aromatic moieties of Trp and Tyr (ca. 250e300 nm). Phe, His, and disulfide bonds also contribute to near UV absorption of proteins but to a significantly lesser extent than Trp or Tyr. Some protein-bound chromophores (i.e. NADH/NADPH) also absorb in the near UV range. In addition, nucleic acids have high absorbance at 250e260 nm. Absorption of UVR can lead to the oxidation of the absorbing species in the presence of molecular oxygen through photo-ionization, which is often referred to as direct (or Type I) photo-oxidation. In addition, indirect (Type II) photo-oxidation reactions can occur and lead to the production of singlet oxygen and other ROS that can in turn damage proteins, lipids, or DNA (Davies and Truscott, 2001). Consequences of UV-induced modifications to proteins include enzyme inactivation, partial unfolding, and non-native aggregation (Dean et al., 1997; Stadtman and Levine, 2003). Such modifications may be partly responsible for the accumulation of aggregated proteins in the lens and cataract formation (Bloemendal et al., 2004; Derham and Harding, 1999). Experimental evidence supports the notion that direct absorption of UV light by ALDH3A1 protects other corneal proteins by virtue of its abundance in that tissue. In C57BL/6J inbred mice, UV-light (302 nm) caused a reduction in ALDH3A1 activity by 85% whereas other metabolic enzyme

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activities remained intact (Downes et al., 1993). Similarly, a large excess of ALDH3A1 in vitro reduces the UV-induced inactivation of G6PD (Estey, T. et al., in preparation). That UV absorption by ALDH3A1 may be a major protective mechanism in the cornea is paradoxical since it is especially sensitive to destruction by UV radiation. UVR inactivates this enzyme by what is known as a ‘‘suicide’’ response (Downes et al., 1993; Manzer et al., 2003; Piatigorsky, 1998b). UVR dosedependent inactivation of ALDH3A1 occurred in HCE cells stably-transfected with ALDH3A1 cDNA, and purified recombinant ALDH3A1 was inactivated and covalently cross-linked by direct UV-exposure (Manzer et al., 2003). However, the high concentration of ALDH3A1 in the cornea should permit a sufficient amount of active molecules to remain for its metabolic functions even under conditions of UV-induced inactivation. 4. Direct and indirect antioxidant properties of ALDH3A1 In view of its direct exposure to the environment, it is not surprising that the cornea possesses a myriad of antioxidant defenses to counteract the deleterious effects of oxidative damage from UV irradiation. For example, ascorbate is found at high levels in the corneal epithelium where it serves as a low molecular weight scavenger and may additionally function as a UV-filter (Ringvold et al., 2000). Various proteins also participate in the antioxidant defense of the cornea. The metabolic elimination of H2O2 in the corneal epithelium is attributed to both catalase (Atalla et al., 1987; Bhuyan and Bhuyan, 1970) and glutathione (GSH) peroxidase (Atalla et al., 1988; Bhuyan and Bhuyan, 1977). Superoxide dismutase (SOD) is found in the human corneal epithelium and endothelium (Redmond et al., 1984) and includes expression of both intracellular copper/zinc SOD as well as extracellular SOD (Behndig et al., 1998). Interestingly, ferritin, typically a cytosolic protein, is localized in the nucleus of avian (chicken) corneal epithelial cells (Cai et al., 1998). Free iron can induce DNA strand breaks through Fenton reactions, and ferritin sequesters the free iron atoms, thus reducing the availability of the pro-oxidants for such reactions. The expression of nuclear ferritin greatly reduces DNA damage after exposure to UVR in cultured cells, a finding consistent with the role of this protein as an antioxidant involved in the protection of DNA against UV-induced damage in the corneal epithelium (Cai et al., 1998). The evolutionary application of ferritin for UV protection in the corneas of birds is noteworthy since the accumulation of ALDH3A1 in the cornea does not occur outside of mammals (Cuthbertson et al., 1992). ALDH3A1 may also contribute to the antioxidant arsenal of the cornea by scavenging ROS (Uma et al., 1996a). Bovine ALDH3 protected RNAse A from modifications induced by hydroxyl radical, probably due in part to the conserved, free Cys residue in the active site of the enzyme (Uma et al., 1996b). Other abundant corneal proteins, including isocitrate dehydrogenase and serum albumin, possess antioxidant properties as well. Isocitrate dehydrogenase is an NADPþ-dependent bovine

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corneal crystallin (Sun et al., 1999) that protects against UVinduced oxidative stress by preventing lipid peroxidation, protein and DNA oxidation, and intracellular peroxide generation (Jo et al., 2002; Lee et al., 2002). Albumin, one of the most abundant corneal proteins in the extracellular stroma (Nees et al., 2003), scavenges H2O2 in the rat cornea (Zhu and Crouch, 1992). The antioxidant properties of albumin were reduced by approximately 20% after thiol alkylation, implicating Cys residues in the ROS scavenging potential of albumin (Soriani et al., 1994). ALDH3A1 may contribute to the antioxidant capacity of the cornea indirectly by producing NADPH during metabolism. The function of NADPH as an antioxidant in the cornea is multifaceted. It is required for the regeneration cycle of reduced glutathione (GSH), necessary to maintain the normal reduced state of the cell, from oxidized glutathione (GSSG) by the glutathione reductase/peroxidase system (Dickinson and Forman, 2002) (Fig. 2). NAD(P)H may also function as a direct antioxidant by reducing glutathyl and tyrosyl radicals generated during oxidative stress (Forni and Willson, 1986b; Kirsch and De Groot, 2001). NADPH directly absorbs light in the UV-range (Atherton et al., 1999) and may therefore contribute to UV filtration by the cornea (Davies and Truscott, 2001). Furthermore, antioxidant properties of NADPH can arise through binding to macromolecules to protect against oxidative damage. For example, bound NADPH offsets H2O2induced inactivation of catalase (Kirkman and Gaetani, 1984; Kirkman et al., 1999). Therefore, the oxidation of aldehydes by ALDH3A1 may simultaneously increase the intercellular concentration of NADPH and consequently increase the antioxidant defense of the cell under conditions of oxidative stress. 5. ALDH3A1 as a ‘‘corneal crystallin’’ The relative abundance of corneal BCP54 (bovine corneal protein of 54 kDa) (Alexander et al., 1981; Holt and Kinoshita, 1973; Silverman et al., 1981), subsequently identified as ALDH3A1 (Abedinia et al., 1990; Cooper et al., 1991; Cuthbertson et al., 1992; Verhagen et al., 1991), prompted the idea of corneal crystallins (Piatigorsky, 1998b, 2001). Consequently, BCP54/ALDH3A1 was called transparentin at the time of its discovery (Rabaey and Segers, 1981). More recently the notion that the abundant intracellular corneal proteins are crystallins has been strengthened by analogy with lens crystallins: (i) both accumulate in transparent, refractive tissue, (ii) both comprise diverse, water-soluble, cytoplasmic proteins with metabolic functions when expressed at lower levels in other tissues, (iii) both are taxon-specific, and (iv) both are present in great excess over that which might be expected for a strictly metabolic role. The taxon-specific accumulation of ALDH3A1 in the cornea is confounding (see Piatigorsky, 2001 for further discussion and references). For example, if the major or only roles of this enzyme in the cornea were catalytic detoxification of aldehydes and absorption of UV light, why does it accumulate in such high proportions in nocturnal rodents (50% of the

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water-soluble protein), at lower concentrations in diurnal primates (5e10%) and negligibly, if at all in birds, which are more exposed to UV irradiation than the land-locked terrestrial mammals? While the answers remain uncertain, it is reasonable to speculate that their resolution will be advanced by appreciating the multiple functions carried out by ALDH3A1 in the cornea. Surprisingly, the corneas lacking ALDH3A1 in mice are clear and appear grossly normal (Nees et al., 2002). Thus, it does not appear as if the abundant ALDH3A1 is required to reduce relatively large discontinuities in refractive index to achieve cellular transparency (Benedek, 1983; Delaye and Tardieu, 1983). However, the use of ALDH3A1 and other abundant intracellular corneal proteins to reduce small spatial fluctuations in refractive index that minimize light scattering cannot be excluded and is beginning to receive experimental support at the single cell level (Jester et al., 2005). In addition, it is possible that the nuclear content of ALDH3A1 indicates a novel role in transcription (Pappa et al., 2005). A transcriptional role for ALDH3A1 is consistent with significant changes in gene expression that we noted between wild type and ALDH3A1 null mouse corneas (Vasiliou, V. et al., in preparation). Adhesive differences also exist between the wild type and ALDH3A1 null corneas. The direct or indirect role of ALDH3A1 in these phenotypes requires further investigation. 6. Chaperone-like activity of ALDH3A1 It has been suggested that ALDH3A1 reduces protein aggregation under various stresses by exhibiting a chaperonelike activity (Manzer et al., 2003; Uma et al., 1996a). This would be analogous to the chaperone activity of the small heat shock/a-crystallins of the lens (Horwitz, 1992). a-Crystallin undergoes a structural transition at approximately 30  C, which leads to partial unfolding of the molecule and may result in exposure of previously-buried hydrophobic regions of the protein (Raman et al., 1997). Such hydrophobic patches on the surface of a-crystallins might provide the basis for its chaperone-like activity towards other partially denatured proteins. However, our recent investigations have not supported the idea that ALDH3A1 acts as a chaperone (Estey, T. et al., in preparation). Although recombinant human ALDH3A1 undergoes a partial loss in native tertiary structure at temperatures greater than 30  C in vitro, this structural transition exacerbates the aggregation of the target protein L-lactic dehydrogenase under thermal stress (37  C). By contrast, a-crystallin suppresses LDH aggregation under similar conditions (Horwitz, 1992). Moreover, while ALDH3A1 can protect G6PD from inactivation by both 4-HNE and MDA, this can be attributed mostly to a metabolic effect of NADPþ rather than a chaperone-like activity of ALDH3A1. Finally, a 50-fold excess of ALDH3A1 was no more effective than bovine serum albumin, which has no known chaperone-like activity, in decreasing UV-induced inactivation of G6PD. Collectively, our studies demonstrate that ALDH3A1 does not prevent protein aggregation and enzyme inactivation through chaperone-like activity. However,

ALDH3A1 can substantially protect target proteins from inactivation by lipid peroxidation aldehydes, especially 4-HNE, through metabolism and can also reduce UV-induced inactivation of target proteins by directly absorbing UVR. These two mechanisms of protection may reduce protein aggregation and inactivation under oxidative stress and, therefore, help maintain corneal transparency (Fig. 2). Thus, the protection of other proteins by ALDH3A1 may indirectly contribute to the optical quality of the cornea. 7. Cell cycle regulation: a novel role for ALDH3A1? HCE cells transfected with human ALDH3A1 cDNA revealed phenotypic differences in cell proliferation. HCE cells expressing ALDH3A1 at levels comparable to those found in vivo in the human cornea had a longer doubling time, an increased cell cycle length, and a decreased efficiency of colony formation when compared to parental HCE cells and mocktransfected cells. Similar effects were also observed with an ALDH3A1-transfected human skin keratinocyte (NCTC 2455) cell line, indicating the relationship between ALDH3A1 levels and proliferative phenotype is not confined to HCE cells. BrdU incorporation assays revealed that DNA synthesis was substantially suppressed in both HCE and NCTC 2455 cell lines. Consequently, a novel role in cell cycle regulation was proposed for ALDH3A1 (Pappa et al., 2005). Cell cycle regulation encompasses a complex system of biological signals involving an array of proteins, including cyclins and cylin-dependent kinases. Cyclin A is responsible for the progression through S-phase of the cell cycle, cyclin B is required for entry into M phase, and the expression of cyclin E is the highest in late G1 phase (Zieske et al., 2004). To further investigate the relationship between ALDH3A1 and cell cycle regulation, the expression and kinase activity of the cyclins were monitored in the ALDH3A1-transfected HCE and NCTC 2455 cell lines (Pappa et al., 2005). Histone H1 kinase assays showed inhibition of kinase activity of all three cyclins in the cells expressing ALDH3A1. Cyclin A was significantly down-regulated in the ALDH3A1-transfected cell lines when compared to the control cell line, whereas cyclin B and E expression became undetectable. A decrease in retinoblastoma protein (pRb) phosphorylation and down-regulation of transcription factor E2F1 and cellregulatory protein p21 were also observed in the ALDH3A1transfected cell lines (Pappa et al., 2005). The ALDH3A1-induced reduction in DNA synthesis and cell proliferation rates may be an additional mechanism used by this versatile protein to protect corneal cells from oxidative damage. DNA is one of the major targets of UV-induced oxidative stress and modified DNA, if left unchecked, may result in genetic mutations or cell death (Cadet et al., 2003; Klaunig et al., 1998). Primary cancers of the cornea are rare and confined to the limbal region containing the stem cells (Miller et al., 2005), suggesting that the cornea has effective mechanisms for minimizing damage to DNA. The localization of ferritin in the nucleus of avian corneal epithelial cells is an example of an imaginative species-specific antioxidant

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defense in the cornea for protection against UV-induced DNA damage (Cai et al., 1998). We propose that another protective mechanism may involve a decrease in cell proliferation associated with high levels of ALDH3A1 expression. ALDH3A1 protection against DNA damage and apoptosis induced by mitomycin C and Vp-16 support a role for this enzyme in the control of cell proliferation (Pappa et al., 2005; Vasiliou et al., 2005). Mock-transfected HCE cells that were exposed to mitomycin C and Vp-16 displayed substantial DNA laddering, an indication of DNA damage and a marker of apoptosis. In contrast, cell lines expressing ALDH3A1 showed considerably less DNA laddering after the same treatment. ALDH3A1 regulation of the cell cycle may arise through two possible mechanisms (Pappa et al., 2005). First, ALDH3A1 may catalytically reduce the concentration of 4HNE, which can modulate cell proliferation and differentiation at low micromolar concentrations (Dianzani, 2003; Poli and Schaur, 2000). It is also possible that the effect of ALDH3A1 on the cell cycle may be accomplished non-catalytically such as, for example, by involvement in mitotic signaling. Other ALDH proteins are known to have non-enzymatic in addition to metabolic functions (Vasiliou and Nebert, 2005; Vasiliou et al., 2000). One example is the hormone binding role of ALDH1A1 (Yamauchi et al., 1999). Indeed, protein multifunctionality of the corneal crystallins is directly analogous to the multifunctionality of the lens crystallins, which have both structural and metabolic roles (Piatigorsky, 1998a). A thorough examination of the literature indicates that protein multifunctionality is more the rule than an exception (Piatigorsky, in press). Cell cycle control by corneal ALDH3A1 resonates with the anti-apoptotic (Kamradt et al., 2001, 2002; Mao et al., 2004) and cell growth regulating (Andley et al., 1998, 2001) roles of the lens a-crystallins. Taken together, then, we suggest that the suppression of cell growth by ALDH3A1 in the cornea may facilitate DNA and cellular repair after oxidative stress and prolong survival by reducing apoptosis (Pappa et al., 2005; Vasiliou et al., 2005). 8. Conclusions ALDH3A1 is a corneal crystallin in mammals that plays multifaceted roles in protecting the cornea (as well as other ocular and non-ocular tissues) during UV-induced oxidative stress. ALDH3A1 metabolizes damaging aldehydes produced from the peroxidation of cellular lipids, including 4-HNE, which substantiates the role of the enzyme in corneal detoxification. This was demonstrated directly by reducing the inactivation of G6PD via enzymatic detoxification of 4-HNE in vitro by added ALDH3A1. ALDH3A1 also protects the cornea and no doubt the underlying lens by directly absorbing UV light. Other protective mechanisms involving ALDH3A1 include a direct antioxidant role through ROS scavenging and an indirect role by producing NADPH. We have also found a novel role for ALDH3A1 in the modulation of the cell cycle, which may be essential in reducing UV-induced apoptosis. The unexplained abundance of intracellular ALDH3A1 in some mammalian corneas far exceeds its enzymatic needs,

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consistent with it having additional, possibly structural roles. While such a structural role may be confined to UV absorption, the taxon specificity of the abundant corneal proteins, including ALDH3A1, suggests additional functions as well. Other possible functions currently under investigation consider the role of ALDH3A1 in gene expression, cell adhesion, and light refraction. Whatever the total number of different molecular functions, ALDH3A1 conceptually joins the ranks of lens crystallins in being (1) expressed highly in the cornea and to a lesser extent in other tissues, (2) selectively expressed as an abundant corneal protein in some species and not others, and (3) almost certainly having a number of metabolic and structural roles.

Acknowledgements This work was supported by NEI R01 EY11490 as well as an AFPE Pre-doctoral Fellowship (T.E.).

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