New enzymatic pathway(s) in 4-hydroxynonenal metabolism

The main effect of lipid peroxidation, which often occurs in response to oxidative stress, is the production of different toxic aldehydes. In particular, over the years, the lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE) has received much attention for its dual role in the pathogenesis of several diseases and as signaling molecule. HNE metabolism is reported to mainly occur through its conjugation with glutathione (GSH) and the subsequent formation of 3-glutathionyl-4-hydroxynonanal (GSHNE) [1, 2]. This molecule is susceptible to both oxidative and reductive transformations, which occur through the action of either the NADPH-dependent activity of aldose reductase (AKR1B1) [1] or through the NAD(P)+ -dependent activity of aldehyde dehydrogenase, respectively [3, 4]. Recently, we have demonstrated the implication of a new NADP+-dependent enzymatic activity able to oxidize GSHNE to its corresponding acid 3-glutathionyl-nonanoic-γ-lactone (GSHNA-γ-lactone) [5]. The enzyme was purified from a human astrocytoma cells line (ADF) to electrophoretic homogeneity as protein doublet in SDS-PAGE, with an apparent molecular weight of 31-32 kDa. Proteomic analysis identified both proteins as human CBR1, also known as NADP+ 15-hydroxyprostaglandine dehydrogenase with 74% of homology and proved their migration differences due to the occurrence of a carboxyethyl moiety at Lys239 [5]. This modification has been already described for the human enzyme and has been demonstrated to have no effect on the protein activity and specificity [6, 7]. The enzyme efficiently catalyzes the oxidation of GSHNE, while it is practically inactive towards 4-hydroxy trans-2-nonenal and other HNE-S-thiolated adducts containing an incomplete glutathionyl moiety [5]. Nucleotide sequence analysis of hCBR1 cDNA from ADF cells completely matched with the human wild type counterpart [5], excluding any gain-of-function mutations in the cDNA-derived protein sequence of hCBR1 [8, 9]. Highly purified human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1), which preserves its ability to oxidize specifically GSHNE, is also shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal [10]. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates [10]. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding 3-glutathionyl-1,4-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-γ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor [10]. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.