Product of Side-chain Cleavage of Cholesterol, Isocaproaldehyde, Is an Endogenous Specific Substrate of Mouse Vas Deferens Protein, an Aldose Reductase-like Protein in Adrenocortical Cells

Abstract

Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD 50 . In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

Rat Aldose Reductase-Like Protein (AKR1B14) Efficiently Reduces the Lipid Peroxidation Product 4-Oxo-2-nonenal

“…As AKR1B7 shows dual coenzyme specificity for NADH and NADPH, 10,12) the rat enzyme exhibited NADH-linked activity. However, the K m and k cat /K m values for NADH determined in the presence of 0.8 mM 4-nitrobenzaldehyde were 220 mM and 0.25 min…”

“…The two mouse ARLPs reduce aldehydes, but are clearly distinct from mouse AR in their poor efficiency in reducing glucose. 12,13) AKR1B7 and AKR1B8 differ in their substrate specificity and inhibitor sensitivity. Compared to AKR1B7, 10,12) AKR1B8 exhibits high catalytic efficiency for cytotoxic 4-hydroxynonenal (HNE) derived from lipid peroxidation, 13) but has poor reactivity towards isocaproaldehyde resulting from the side chain cleavage of cholesterol during steroidogenesis.…”

“…12,13) AKR1B7 and AKR1B8 differ in their substrate specificity and inhibitor sensitivity. Compared to AKR1B7, 10,12) AKR1B8 exhibits high catalytic efficiency for cytotoxic 4-hydroxynonenal (HNE) derived from lipid peroxidation, 13) but has poor reactivity towards isocaproaldehyde resulting from the side chain cleavage of cholesterol during steroidogenesis. 10) AKR1B7 is insensitive to AR inhibitors (ARIs) 10) and exhibits prostaglandin (PG) F 2a synthase activity, which is not detected in the ARI-sensitive AKR1B8.…”

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…Like other oxidoreductases, the AKRs enzymes seem to be able to differentiate between oxidized and reduced cofactor and bind reduced coenzyme with higher affinity. Some AKRs, however, can use NADH as coenzyme and even exhibit higher k cat with NADH than NADPH (e.g., AKR1C12, MVDP) (Lefrancois-Martinez et al, 1999;Ikeda et al, 1999). Weaker cofactor binding, however, results in higher k cat , but is inevitably accompanied by higher K m for carbonyl substrate and a decrease in the specificity of reduction versus oxidation.…”

“…Neither 1B7 nor 1B8 is a homolog of the human 1B10 according to their tissue distribution and catalytic properties (Cao et al, 1998). 1B7 has very low enzymatic activity and limited tissue distribution (it is abundant in the adrenal gland) (Lau et al, 1995;Lefrancois-Martinez et al, 1999). The FR-1 protein (1B8) has very similar catalytic properties to AR, but it has a higher K m for DLglyceraldehyde and lacks activity with glucose (Srivastava et al, 1998b).…”