Yan S D, Shi Y, Zhu A, Fu J, Zhu H, Zhu Y, Gibson L, Stern E, Collison K, Al-Mohanna F, Ogawa S, Roher A, Clarke S G, Stern D M
Departments of Pathology, Physiology and Surgery, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
J Biol Chem. 1999 Jan 22;274(4):2145-56. doi: 10.1074/jbc.274.4.2145.
Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-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 Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-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 beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(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 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta 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 betaAPP(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 betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.
内质网相关淀粉样β肽（Aβ）结合蛋白（ERAB）/II型L-3-羟基酰基辅酶A脱氢酶（HADH II）在阿尔茨海默病（AD）受累脑内高水平表达，可结合Aβ，并导致Aβ诱导的细胞毒性。纯化的重组ERAB/HADH II催化S-乙酰乙酰辅酶A的NADH依赖性还原反应，Km约为68微摩尔，Vmax约为430微摩尔/分钟/毫克。通过催化结构域（Y168G/K172G）的定点诱变研究了ERAB/HADH II酶活性对Aβ介导的细胞功能障碍的作用。虽然共转染以过表达野生型ERAB/HADH II和变异β淀粉样前体蛋白（βAPP（V717G））的COS细胞出现DNA片段化，但共转染Y168G/K172G突变的ERAB和βAPP（V717G）则无此效应。因此，我们询问该酶是否可能识别其醛产物具有细胞毒性的醇类底物；ERAB/HADH II在NAD+存在下催化多种简单醇类（C2-C10）氧化为各自的醛类，并催化17β-雌二醇的NAD依赖性氧化。向纯化的ERAB/HADH II中添加微摩尔水平的合成Aβ（1-40），可同时抑制S-乙酰乙酰辅酶A的还原反应（Ki约为1.6微摩尔）、17β-雌二醇的氧化反应（Ki约为3.2微摩尔）和（-）-2-辛醇的氧化反应（Ki约为2.6微摩尔）。由于需要微摩尔水平的Aβ才能抑制ERAB/HADH II活性，而Aβ与ERAB/HADH II的结合发生在低得多的浓度下（Km约为40-70纳摩尔），后者更接近细胞内Aβ的水平，因此Aβ对ERAB/HADH II的干扰可能是由酶活性直接调节以外的机制引起的。共转染以过表达ERAB/HADH II和βAPP（V717G）的细胞产生丙二醛-蛋白和4-羟基壬烯醛-蛋白表位，仅在单独过表达ERAB/HADH II或βAPP（V717G）的细胞中可检测到最低水平。在共转染以过表达Y168G/K172G突变的ERAB和βAPP（V717G）的细胞中未观察到此类有毒醛类的产生。我们得出结论，ERAB/HADH II的广义醇脱氢酶活性是在富含Aβ的环境中观察到的细胞毒性的核心。
