Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • Our results add to previously published

    2024-04-23

    Our results add to previously published data on the contribution of ALDH3A1 to the optical properties of the cornea. Specifically, the study by Nees et al. (2002) was among the first to examine whether ALDH3A1 serves as a structural component in the cornea and similarly to lens crystallins. Their experiments established that ALDH3A1 is not mandatory for the obtainment of corneal transparency, as Aldh3a1 (-/-) knockout mice exhibited structurally physiological and indiscriminate from wild-type corneas (Nees et al., 2002). Nevertheless, a number of studies provide compelling evidences of the importance of ALDH3A1 for the maintenance of corneal clarity, especially under UVR exposure conditions. For example, Downes et al. (1994) reported that the ALDH3A1 null mouse strain SWR/J was susceptible to extensive corneal hazing after UVB exposure (Downes et al., 1994). Accordingly, a study by Lassen et al. (2007) evaluating the ocular phenotype of an Aldh3a1 (-/-) knock-out mouse strain showed that ALDH3A1 deficient mice developed marked opacities in their cortex, along with enhanced cataract formation by one month of age. Additionally, after UVB exposure, 1–3 months old Aldh3a1 (-/-) mice also exhibited increased anterior lens subcapsular opacities compared to wild-type mice. Interestingly, biochemical analysis revealed that the ocular opacities of the Aldh3a1 (-/-) mice resulted from increased MDA- and 4-HNE- protein adduct levels and decreased proteasome activity (Lassen et al., 2007). ALDH3A1 could arrest or retard corneal opacification by detoxifying MDA and 4-HNE, recycling glutathione, inhibiting the aggregation of oxidized and/or damaged proteins through a chaperone holdase activity or even by directly absorbing UV radiation. Experimental in vitro studies on the effect of UVB on recombinant human ALDH3A1, initially by Manzer et al. (2003) and later by Estey et al. (2010) revealed that UVB exposure causes structural transitions of ALDH3A1 through both covalent and non-covalent post-translational modifications that result in the formation of inactive, non-native, soluble ALDH3A1 L-693,403 maleate with no evident precipitation (Estey et al., 2010; Manzer et al., 2003). The UV-absorption properties of ALDH3A1 could contribute to preventing the development of corneal precipitates through a mechanism of “suicide response” (Estey et al., 2010). In addition, this stress-dependent oligomerization and structural transition of ALDH3A1 is characteristic for proteins with holdase chaperone activity, like α-crystallin (Raman and Rao, 1997). Along these lines, Estey et al. (2007) demonstrated that ALDH3A1 partially unfolds and consequently loses its native tertiary structure also under thermal stress conditions. However, in this study ALDH3A1 failed to prevent the thermal aggregation of lactate dehydrogenase in vitro and managed to prevent the UV-induced inactivation of G6PD only at relatively high concentrations (Estey et al., 2007). In contrast, our experiments showed that both the MBP and 6xHis fusion ALDH3A1 recombinant proteins displayed significant efficiency in maintaining the enzymatic activity of SmaI after incubation at 37°C for 90min. Furthermore, the 6xHis-tagged ALDH3A1 was able to prevent the heat-induced aggregation and inactivation of CS. The discrepancies observed between the two studies could be attributed to the utilization of different target proteins, the usage of different relative concentrations of ALDH3A1 or even the diverse experimental conditions used. According to our results, 6xHis-tagged ALDH3A1 showed to have enhanced chaperone-like activity in comparison to the MBP-tagged ALDH3A1. In addition, human 6xHis-tagged ALDH3A1 was also shown previously to exhibit higher specific enzymatic activity when compared to MBP-tagged ALDH3A1, most likely due to the large size of the MBP tag and its conformational impact on the proper folding of ALDH3A1 (Voulgaridou et al., 2013). However, the possibility that the enzymatic activity of ALDH3A1 contributes to the chaperone-like function of the protein has proved to be invalid as indicated by our experiments with the catalytically mutant form of 6xHis-tagged ALDH3A1. Dot heat shock assays revealed that expression of both wild-type and mutant 6xHis-tagged ALDH3A1 results in higher colony formation efficiencies in BL21(DE3) E. coli cells following incubation for 1h at 65°C. The same observations were confirmed with thermotolerance assays, where expression of both wildtype and mutant human 6xHis-tagged ALDH3A1 was associated with increased tolerance to thermal stress.