Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain
Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain
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DOI：
10.1111/j.1471-4159.2006.03968.x
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发表时间：
2006-09-01
影响因子：
4.7
通讯作者：
Lucas, Jose J.
中科院分区：
文献类型：
--
作者：
Diaz-Hernandez, Miguel;Valera, Adriana G.;Lucas, Jose J.
在亨廷顿氏病（HD）中，如同在其余CAG三重重复病症中一样，扩增的含多聚谷氨酰胺（polyQ）的蛋白质形成聚集成包涵体（IB）的神经元内纤维状聚集体。由于IB含有泛素和蛋白酶体亚基，因此提出蛋白酶体活性的抑制可能是polyQ疾病的发病机制的基础。最近的体外酶促研究揭示了真核蛋白酶体不能消化扩增的polyQ，从而表明polyQ偶尔未能退出蛋白酶体可能会干扰其蛋白水解功能。然而，最近还发现由合成polyQ制成的体外组装聚集体不能抑制蛋白酶体活性。由于合成的polyQ聚集体缺乏在受影响的神经元内发现的翻译后修饰，如聚泛素化，我们决定研究从泰特/HD 94小鼠模型和人HD脑组织中分离的突变亨廷顿蛋白（htt）聚集体的作用。在这里，我们表明，分离的泛素化丝状htt聚集体，提取从IBs由以前报道的方法，选择性地抑制在体外的26 S，但不是20 S蛋白酶体的肽酶活性在一个非竞争性的方式。一致的是，免疫电子显微镜显示htt细丝与26 S蛋白酶体的19 S遍在蛋白相互作用调节帽直接相互作用。在这里，我们还报告了一种新的方法分离的IBs的基础上磁分选。有趣的是，分离的IBs没有改变蛋白酶体活性。因此，我们的研究结果表明，突变体htt丝状聚集体可以抑制蛋白酶体活性，但只有当不招募到IB，从而加强了IB形成是通过中和分散的丝状htt聚集体的毒性保护的概念。
In Huntington's disease (HD), as in the rest of CAG triplet-repeat disorders, the expanded polyglutamine (polyQ)-containing proteins form intraneuronal fibrillar aggregates that are gathered into inclusion bodies (IBs). Since IBs contain ubiquitin and proteasome subunits, it was proposed that inhibition of proteasome activity might underlie pathogenesis of polyQ disorders. Recent in vitro enzymatic studies revealed the inability of eukaryotic proteasomes to digest expanded polyQ, thus suggesting that occasional failure of polyQ to exit the proteasome may interfere with its proteolytic function. However, it has also recently been found that in vitro assembled aggregates made of synthetic polyQ fail to inhibit proteasome activity. Because synthetic polyQ aggregates lack the post-translational modifications found inside affected neurons, such as poly ubiquitylation, we decided to study the effect of mutant huntingtin (htt) aggregates isolated from the Tet/HD94 mouse model and from human HD brain tissue. Here, we show that isolated ubiquitylated filamentous htt aggregates, extracted from IBs by a previously reported method, selectively inhibited the in vitro peptidase activity of the 26S but not of the 20S proteasome in a non-competitive manner. In good agreement, immuno-electron microscopy revealed a direct interaction of htt filaments with the 19S ubiquitin-interacting regulatory caps of the 26S proteasome. Here, we also report a new method for isolation of IBs based on magnetic sorting. Interestingly, isolated IBs did not modify proteasome activity. Our results therefore show that mutant htt filamentous aggregates can inhibit proteasome activity, but only when not recruited into IBs, thus strengthening the notion that IB formation is protective by neutralizing toxicity of dispersed filamentous htt aggregates.