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**☆

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。 

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

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.