ROLE OF ERAB/L-3-HYDROXYACYL-COENZYME A DEHYDROGENASE TYPE II ACTIVITY IN ABETA -INDUCED CYTOTOXICITY

Abstract Endoplasmic reticulum-associated amyloid β-peptide (Aβ)-binding protein (ERAB)/l-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer’s disease (AD)-affected brain, binds Aβ, and contributes to Aβ-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a K m of ≈68 μm and aV max of ≈430 μmol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Aβ-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant β-amyloid precursor protein (βAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and βAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2–C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17β-estradiol. Addition of micromolar levels of synthetic Aβ(1–40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (K i ≈ 1.6 μm), as well as oxidation of 17β-estradiol (K i ≈3.2 μm) and (−)-2-octanol (K i ≈ 2.6 μm). Because micromolar levels of Aβ were required to inhibit ERAB/HADH II activity, whereas Aβ binding to ERAB/HADH II occurred at much lower concentrations (K m ≈ 40–70 nm), the latter more closely simulating Aβ levels within cells, Aβ perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and βAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or βAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and βAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Aβ-rich environment.