Hunter Jesse M, Lesort Mathieu, Johnson Gail V W
Department of Cell Biology, and Department of Psychiatry, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
J Neurosci Res. 2007 Jun;85(8):1774-88. doi: 10.1002/jnr.21287.
Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disease caused by an abnormally expanded CAG repeat in the HD gene. Ubiquitylated aggregates containing mutant huntingtin protein in neurons are hallmarks of HD. Misfolded mutant huntingtin monomers, oligomers, or aggregates may be a result of, and cause, ubiquitin- proteasome dysfunction. To investigate the ubiquitin-proteasome system we designed a series of firefly luciferase reporters targeted selectively to different points along this pathway. These reporters were used to monitor ubiquitin-proteasome system function in a striatal cell culture model of HD. Ubiquitylation processes were not reduced in mutant huntingtin cells but recognition or degradation of ubiquitylated substrates was decreased. We also found mutant huntingtin expressing cells had slight but significant decreases in chymotrypsin-like and caspase-like activities, and an unexpected increase in trypsin-like activity of the proteasome core. General proteasome core inhibitors, as well as selective caspase-like activity inhibitors, were less effective in mutant cells. Finally, treatment with 3-nitropropionic acid, a succinate dehydrogenase inhibitor, had opposite effects on the ubiquitin-proteasome system with activation in wild-type and decreased activity in mutant huntingtin expressing cells. The results of these experiments show clearly selective disruption of the ubiquitin-proteasome system in this cell culture model of HD. The high throughput tools that we have designed and optimized will also be useful in identifying compounds that alter ubiquitin-proteasome system function and to investigate other neurodegenerative diseases such Alzheimer's disease and Parkinson's disease.
亨廷顿舞蹈症（HD）是一种进行性常染色体显性神经退行性疾病，由HD基因中异常扩增的CAG重复序列引起。神经元中含有突变亨廷顿蛋白的泛素化聚集体是HD的标志。错误折叠的突变亨廷顿单体、寡聚体或聚集体可能是泛素-蛋白酶体功能障碍的结果，并会导致该功能障碍。为了研究泛素-蛋白酶体系统，我们设计了一系列萤火虫荧光素酶报告基因，它们被选择性地靶向该通路的不同位点。这些报告基因用于监测HD纹状体细胞培养模型中的泛素-蛋白酶体系统功能。在突变亨廷顿蛋白细胞中，泛素化过程并未减少，但泛素化底物的识别或降解减少。我们还发现，表达突变亨廷顿蛋白的细胞中，胰凝乳蛋白酶样和半胱天冬酶样活性略有但显著降低，而蛋白酶体核心的胰蛋白酶样活性意外增加。一般的蛋白酶体核心抑制剂以及选择性半胱天冬酶样活性抑制剂在突变细胞中的效果较差。最后，用琥珀酸脱氢酶抑制剂3-硝基丙酸处理对泛素-蛋白酶体系统有相反的影响，在野生型中激活，而在表达突变亨廷顿蛋白的细胞中活性降低。这些实验结果清楚地表明，在这个HD细胞培养模型中泛素-蛋白酶体系统存在选择性破坏。我们设计和优化的高通量工具也将有助于识别改变泛素-蛋白酶体系统功能的化合物，并用于研究其他神经退行性疾病，如阿尔茨海默病和帕金森病。
