Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.
Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.
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DOI：
10.1016/j.brainres.2014.08.018
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发表时间：
2014-10-02
期刊：
影响因子：
2.9
通讯作者：
Wang, Michael M.
中科院分区：
文献类型：
--
作者：
Zhang, Xiaojie;Lee, Soo Jung;Young, Kelly Z.;Josephson, David A.;Geschwind, Michael D.;Wang, Michael M.
伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病CADASIL是由100多种NOTCH 3突变引起的。几乎所有编码的突变蛋白质都含有奇数个半胱氨酸。因此，NOTCH 3的结构变化可能是CADASIL的主要分子异常。因此，我们寻找CADASIL组织中NOTCH 3蛋白结构改变的证据。在兔中产生了针对两个非重叠N-末端NOTCH 3序列的四种抗体。这些试剂用于免疫组织化学实验，以检测死后CADASIL脑（n=8）、对照脑和过表达NOTCH 3的细胞中的表位。为了确定NOTCH 3表位的生物化学性质，我们使用这些抗体来探测用酸、尿素、胍、离子去污剂、丙烯酰胺以及基于巯基和磷的还原剂处理的纯NOTCH 3-Fc融合蛋白。在8个CADASIL脑样本中，所有抗体均对动脉进行了强烈染色。最显著的染色是在变性的软脑膜动脉和穿膜血管中。对照组大脑的正常血管没有反应。抗体不与过表达NOTCH 3的培养细胞或纯化的NOTCH 3-Fc蛋白反应。此外，用酸、离液变性剂、烷化剂和去污剂处理纯蛋白未能暴露N-末端NOTCH 3表位。然而，抗体识别用三种不同还原剂（DTT、β-巯基乙醇和TCEP）处理的纯化的N 0 TCH 3-Fc蛋白中的新的N-末端表位。我们得出结论，CADASIL动脉具有潜在的N-末端NOTCH 3表位，这表明体内NOTCH 3结构改变的第一个证据。
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue. Four antibodies were raised in rabbits against two non-overlapping N-terminal NOTCH3 sequences. These reagents were used in immunohistochemical experiments to detect epitopes in post-mortem CADASIL brains (n=8), control brains, and cells overexpressing NOTCH3. To determine the biochemical nature of NOTCH3 epitopes, we used these antibodies to probe pure NOTCH3-Fc fusion proteins treated with acid, urea, guanidinium, ionic detergents, acrylamide, and thiol- and phosphorus-based reductants. All antibodies avidly stained arteries in 8 of 8 CADASIL brain samples. The most prominent staining was in degenerating media of leptomeningeal arteries and sclerotic penetrating vessels. Normal appearing vessels from control brains were not reactive. Antibodies did not react with cultured cells overexpressing NOTCH3 or with purified NOTCH3-Fc protein. Furthermore, treatment of pure protein with acid, chaotropic denaturants, alkylators, and detergents failed to unmask N-terminal NOTCH3 epitopes. Antibodies, however, recognized novel N-terminal epitopes in purified NOTCH3-Fc protein treated with three different reductants (DTT, beta-mercaptoethanol, and TCEP). We conclude that CADASIL arteries feature latent N-terminal NOTCH3 epitopes, suggesting the first evidence in vivo of NOTCH3 structural alterations.
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影响因子：
4.8
作者：
Meng, He;Zhang, Xiaojie;Wang, Michael M.
通讯作者：
Wang, Michael M.
影响因子：
168.9
作者：
Joutel, A;Vahedi, K;TournierLasserve, E
通讯作者：
TournierLasserve, E
影响因子：
3.7
作者：
Meng H;Zhang X;Yu G;Lee SJ;Chen YE;Prudovsky I;Wang MM
通讯作者：
Wang MM
影响因子：
8.3
作者：
Dong H;Ding H;Young K;Blaivas M;Christensen PJ;Wang MM
通讯作者：
Wang MM
影响因子：
15.9
作者：
Joutel, A;Andreux, F;Tournier-Lasserve, E
通讯作者：
Tournier-Lasserve, E