中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (20): 3773-3778.doi: 10.3969/j.issn.1673-8225.2012.20.037

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

变异亨廷顿蛋白对CBP组蛋白乙酰化酶活性及其蛋白表达的影响**☆

丛树艳，张  威，王  亚，邵  华，冯  娟   

  1. 中国医科大学附属盛京医院神经内科，辽宁省沈阳市  110003
  • 收稿日期:2012-03-21 修回日期:2012-04-12 出版日期:2012-05-13 发布日期:2012-05-13
  • 通讯作者: 丛树艳，博士，副教授，中国医科大学附属盛京医院神经内科，辽宁省沈阳市 110003 congshuyan@hotmail.com
  • 作者简介:丛树艳☆，女，1973年生，辽宁省沈阳市人，汉族，2006年荷兰莱顿大学毕业，博士，副教授，主要从事神经遗传变性疾病分子发病机制的研究。congshuyan@hotmail.com
  • 基金资助:

    国家自然科学基金资助项目(30971017)；辽宁省博士启动基金(20080526)项目。

Effect of mutant huntingtin on the expression levels and histone acetyltransferase activity of CREB-binding protein  

Cong Shu-yan, Zhang Wei, Wang Ya, Shao Hua, Feng Juan   

  1. Department of Neurology, Shengjing Hospital of China Medical University, Shenyang  110003, Liaoning Province, China
  • Received:2012-03-21 Revised:2012-04-12 Online:2012-05-13 Published:2012-05-13
  • Contact: Cong Shu-yan, Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110003, Liaoning Province, China congshuyan@hotmail.com
  • About author:Cong Shu-yan☆, Doctor, Associate professor, Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110003, Liaoning Province, China congshuyan@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30971017*; the Doctoral Start-up Foundation of Liaoning Province, No. 20080526*

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摘要：

背景：目前针对转录因子CBP在亨廷顿舞蹈病发病机制中作用的研究主要集中在包涵体形成对CBP的抑制作用。
目的：在亨廷顿舞蹈病细胞模型中，探讨变异亨廷顿蛋白对CBP组蛋白乙酰基转移酶活性及CBP蛋白表达的影响。
方法：运用可经多西环素诱导表达绿色荧光蛋白标记的HD第一外显子片段含有23个(正常)或74个(变异)多聚谷氨酰胺片段(N-htt-Q23或-Q74)的大鼠肾上腺神经嗜铬细胞瘤PC12细胞模型，用免疫荧光显微镜观察细胞内变异亨廷顿蛋白表达，用免疫沉淀后的内源性CBP进行组蛋白H4乙酰化测定，Western blot检测CBP蛋白水平，通过外源性加入特异性蛋白酶体抑制剂MG-132，观察其对CBP蛋白水平的影响。用Lightcycler PCR检测CBP mRNA转录水平。
结果与结论：多西环素诱导1 d时，N-htt-Q74只在少数细胞中形成包涵体，6 d时大部分细胞含有包涵体(>90%)，而N-htt-Q23始终不形成蛋白包涵体。变异亨廷顿蛋白可抑制CBP组蛋白乙酰基转移酶活性及内源性CBP蛋白水平，但不影响CBP mRNA的转录水平。在表达N-htt-Q74细胞中，MG-132可通过影响蛋白降解部分恢复CBP蛋白水平。结果提示，除了变异亨廷顿蛋白包涵体，可溶性变异亨廷顿蛋白在早期已经开始抑制CBP组蛋白乙酰基转移酶活性和降低CBP蛋白水平，在亨廷顿舞蹈病发病的分子机制中起重要作用。
 

关键词: 亨廷顿舞蹈病, CREB结合蛋白, 蛋白酶体抑制剂, 变异亨廷顿蛋白, 组蛋白乙酰化酶

Abstract:

BACKGROUND: Studies addressing the role of CREB-binding protein (CBP) in the pathogenesis of Huntington's disease are mainly concentrated in the inhibitory effect of aggregate formation on CBP.
OBJECTIVE: To investigate the effect of mutant huntingtin (htt) on the histone acetyltransferase (HAT) activity and expression levels of CBP in the cell model of Huntington’s disease.
METHODS: Neuronal phaeochromocytoma rat PC12 cells, stably inducible for GFP-tagged HD exon 1 with either 23 (normal) or 74 (expanded) glutamines (N-htt-Q23 or -Q74), driven by a doxycycline (dox)-dependent Tet-On promoter were used. N-htt expression was observed via immunofluorescence microscopy. The HAT activities of endogenous CBP were determined via a histone H4 acetylation assay with immunoprecipitated CBP. CBP protein levels were observed via Western blot. The effect of specific proteasome inhibitor on CBP protein level was investigated by adding MG-132. CBP mRNA levels were detected by Lightcycler PCR.
RESULTS AND CONCLUSION: Cells expressing N-htt-Q74 started to form visible aggregates 1 day after dox induction in a small percentage of cells (< 1%), while after 6 days aggregates were present in about 90% of cells. In contrast, N-htt-Q23 distributed evenly throughout the cells and did not form aggregates. Expression of soluble N-htt-Q74 already inhibited the HAT activity of CBP, the inhibitory effect was exacerbated by aggregate formation of N-htt-Q74, while N-htt-Q23 did not affect CBP HAT activity. Expression of soluble N-htt-Q74 already reduced CBP protein levels; the level of CBP was dramatically decreased after 6 days, while N-htt-Q23 did not affect the levels of CBP protein. Neither N-htt-Q74 nor -Q23 affected CBP mRNA levels. The proteasome inhibitor MG-132 partly restored CBP protein levels by affecting protein degradation in the cells expressing N-htt-Q74. The results indicated that decrement of CBP HAT activity and its protein level play important roles in the molecular pathogenesis HD. Aside from aggregated mutant N-htt, soluble mutant N-htt already represses CBP HAT activity and CBP level, providing theoretical evidence for administration of CBP and histone deacetylase inhibitor in the treatment of Huntington's disease in the early stages.

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