Cells have developed strategies aiming to enhance both the

Cells have developed strategies aiming to enhance both the efficiency of proteins with chaperone activity as well as their overall antioxidant capacity to maintain proteostasis under oxidative stress conditions (Miyata et al., 2012). For instance, 2-Cys peroxiredoxin is an abundantly expressed, antioxidant enzyme that catalyzes the reduction of various peroxide substrates, thus controlling cellular peroxide levels. Under redox stress conditions, though, 2-Cys peroxiredoxin displays holdase-like chaperone activity, which is triggered by the oxidation of highly sensitive cysteine residues of the protein. Oxidation of these cysteines induces a conformational switch of 2-Cys peroxiredoxin which allows it to form high molecular weight complexes with misfolded proteins and thus prevent their aggregation (Jang et al., 2004; Kumsta and Jakob, 2009; Perkins et al., 2015). Similarly, alpha-crystallin (α-crystallin) is a small heat-shock protein responsible for the optical properties of the lens. Its abundant expression facilitates short-range organization in the cytoplasm of the lens fibers, thus diminishing the scattering of light (Jester, 2008), while its holdase chaperone activity prevents lens opacification, by inhibiting the aggregation of aged and/or denaturated proteins (Posner et al., 2012). Aldehyde dehydrogenase 3A1 (ALDH3A1) is a NAD(P)+-dependent enzyme that oxidizes medium chain aldehydes to their corresponding carboxylic acids (Pappa et al., 2003b; Pappa et al., 2001; Piatigorsky, 2000). It has recently been characterized as a corneal crystallin being constitutively expressed at high levels in the mammalian cornea (Jester et al., 1999; Pappa et al., 2005; Pappa et al., 2003a; Voulgaridou et al., 2016). ALDH3A1 appears to exert a variety of antioxidant and cytoprotective properties in corneal epithelial PF-04691502 mg and prevents the development of opacifications in the cornea under oxidative stress conditions thus suggesting a potential mechanism acting as a chaperone-like molecule, analogous to α-crystallin (Black et al., 2012; Lassen et al., 2007; Pappa et al., 2005; Pappa et al., 2003a; Pappa et al., 2003b; Stagos et al., 2010). Indeed, a number of findings like: (i) ALDH3A1 up-regulation after eyelids openings in mice, (ii) its down-regulation in rat corneal keratocytes cultured under dark conditions (Jester, 2008) and (iii) the increased formation of opacities in the cortex and corneal haze phenotype in Aldh3a1 (-/-) knock-out mice compared to wild-type mice (Lassen et al., 2007; Chen et al., 2016), support the notion that ALDH3A1 has a significant role in the maintenance of corneal optical properties. However, if such a role is accomplished through a chaperone-like activity remains elusive.
Material and methods
Results
Discussion ALDH3A1 is a cytoprotective corneal crystallin with multiple antioxidant modes of action and with an alleged role in the maintenance of corneal optical properties. To investigate the potential chaperone activity of ALDH3A1, we performed assays commonly used for studying proteins with holdase activity (Buchner et al., 1998; Buchner et al., 1991; Carver et al., 2002; Hess and FitzGerald, 1998; Horwitz, 1992; Kumar et al., 2005a; Kumar et al., 2005b; Olszewski and Wasserman, 1986; Raman et al., 1995; Reddy et al., 2002; Santhoshkumar and Sharma, 2001). Our results indicated that both MBP and 6xHis fusion ALDH3A1 recombinant proteins were able to maintain the enzymatic activity of SmaI as well as the solubility of CS under thermal stress conditions. On the contrary, only 6xHis-tagged ALDH3A1 prevented the heat-induced inactivation of CS. Additionally, dot heat shock and thermotolerance assays revealed that the expression of both wild-type and mutant 6xHis-tagged ALDH3A1 resulted in higher colony formation efficiency and enhanced tolerance to thermal stress in BL21(DE3) E. coli cells. The cytoprotective role of ALDH3A1 was also evident under oxidative stress conditions in the human corneal epithelial cell line HCE-2.