Marcum Jennifer L, Mathenia Jeremy K, Chan Roy, Guttmann Rodney P
Department of Physiology, University of Kentucky, Lexington, KY, USA.
Biochem Biophys Res Commun. 2005 Aug 26;334(2):342-8. doi: 10.1016/j.bbrc.2005.06.089.
The hippocampus of Alzheimer's disease brain has been shown to be highly oxidized compared to age-matched controls. One of the most sensitive targets of oxidation is anionic sulfur which can be found within the active site of members of the cysteine-protease family. Thus, while members of the cysteine-protease family such as the calpains and caspases have been found to be in an active conformation in vulnerable brain regions in AD it is possible that their proteolytic activity is hampered due to the robust oxidative stress found at these locations. To address this issue, the amount of caseinolytic activity present in the hippocampus from post-mortem brain samples of AD and age-matched controls was determined. No difference in caseinolytic activity in the absence of exogenous reducing agent was observed between AD and control. However, after addition of the thiol-specific reducing agent, dithiothreitol (DTT), the amount of caseinolytic activity was significantly increased in AD compared to the DTT-mediated increase in control. This suggests that the cysteine proteases are more oxidized in AD brain and that latent proteolytic activity in AD brain can be released by antioxidants. Further testing revealed that the calcium-dependent caseinolytic activity was significantly lower in AD brain compared to controls. This is despite the fact that the major calcium-dependent thiol protease, calpain, is threefold more activated in AD brain based on autolytic activation measured by Western blotting. This calcium-dependent protease difference between AD and control brains was negated by addition of DTT. These data suggest that cysteine protease activity in AD brain is inactivated by oxidants, which is evident by the ability of thiol-specific reducing agents such as DTT to rescue and recover activity.
与年龄匹配的对照组相比，阿尔茨海默病患者大脑的海马体显示出高度氧化。氧化作用最敏感的靶点之一是阴离子硫，它存在于半胱氨酸蛋白酶家族成员的活性位点内。因此，虽然已发现半胱氨酸蛋白酶家族成员，如钙蛋白酶和半胱天冬酶，在阿尔茨海默病患者大脑的易损区域处于活性构象，但由于这些部位存在强烈的氧化应激，它们的蛋白水解活性可能受到阻碍。为了解决这个问题，研究测定了阿尔茨海默病患者和年龄匹配的对照组死后脑样本海马体中酪蛋白水解活性的量。在没有外源还原剂的情况下，未观察到阿尔茨海默病患者和对照组之间酪蛋白水解活性的差异。然而，添加硫醇特异性还原剂二硫苏糖醇（DTT）后，与DTT介导的对照组增加相比，阿尔茨海默病患者的酪蛋白水解活性显著增加。这表明半胱氨酸蛋白酶在阿尔茨海默病患者大脑中被氧化程度更高，并且抗氧化剂可以释放阿尔茨海默病患者大脑中的潜在蛋白水解活性。进一步测试表明，与对照组相比，阿尔茨海默病患者大脑中钙依赖性酪蛋白水解活性显著降低。尽管通过蛋白质印迹法测量的自溶激活显示，主要的钙依赖性硫醇蛋白酶钙蛋白酶在阿尔茨海默病患者大脑中的激活程度是对照组的三倍。添加DTT消除了阿尔茨海默病患者和对照组大脑之间这种钙依赖性蛋白酶的差异。这些数据表明，阿尔茨海默病患者大脑中的半胱氨酸蛋白酶活性被氧化剂灭活，硫醇特异性还原剂如DTT能够挽救和恢复活性，这一点很明显。
