熱休克蛋白27與周邊神經退化性疾病

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Title:	熱休克蛋白27與周邊神經退化性疾病
Other Titles:	Hsp27 In Peripheral Neuron Degenerative Diseases
Authors:	溫珮吟 Wen, Pei-Yin
Contributors:	謝明麗 Hsieh, Ming-Li 東海大學生命科學系
Keywords:	熱休克蛋白27;周邊神經退化性疾病;細胞質堆積物;點突變 Hsp27;Peripheral Neuron Degenerative Diseases;aggregation;mutation;CMT2;HMN
Date:	2007
Issue Date:	2011-03-24T05:34:58Z (UTC)
Abstract:	Charcot-Marie-Tooth (CMT) 是一種顯性遺傳的運動及感覺神經退化的疾病。有研究指出，神經細胞軸突不正常的第二型CMT疾病，是由於熱休克蛋白27一段高度保留的α- crystallin 作用區上突變所引起。熱休克蛋白27是小分子熱休克蛋白的一員，具有伴護蛋白 (chaperon) 功能。不但可以保護處在壓力下的細胞、降低細胞凋亡，還會與細胞骨骼間交互作用，以維持細胞特性。根據先前研究，我們假設：熱休克蛋白27中α- crystallin作用區具有重要的蛋白功能，位於其中的高度保留胺基酸若發生點突變，會影響小分子熱休克蛋白27正常功能，在細胞質形成聚合物，並使神經間質絲混亂，最後導致神經退化性疾病。首先我們檢查周邊神經遺傳退化性病人熱休克蛋白27基因，利用聚合?連鎖反應，增幅放大熱休克蛋白27 α- crystallin作用區，接著用核?酸定序來確定是否帶有點突變。目前為止，我們篩選了十四個病人，但在熱休克蛋白27 α- crystallin作用區基因上都是正常。另外我們成功的利用定點突變方法，在α- crystallin作用區上構築六個帶有不同點突變的熱休克蛋白27質體: K112W、 E119L、K123W、 R127W、S135F、R140E，以及在α- crystallin作用區外的P182L。前三者將帶有電荷的胺基酸轉變為不帶電，後四者則根據患有疾病病人所帶有的致病點突變。我們利用短暫轉染，將正常或突變熱休克蛋白27載體送入老鼠神經腫瘤細胞株 (N2a)，用西方墨點法確定蛋白可完整表達並且確認表達量相似後，做一些分析來研究對細胞的影響。然而從細胞免疫染色的結果，我們觀察到表達P182L突變蛋白細胞株內，產生細胞質堆積物比例明顯高於表達正常蛋白的細胞。另外在DAPI染色結果顯示，表達R140E突變蛋白細胞的細胞凋亡情況，會比表達正常蛋白細胞更嚴重，但是同樣可以致病的R127W細胞凋亡情況卻沒有顯著增加。另一方面，表達E119L和P182L突變蛋白的細胞，其細胞凋亡情況則比表達LacZ蛋白對照組來得低。然而表達其他突變熱休克蛋白27細胞，相較於表達正常蛋白的細胞則沒有顯著差異。根據以上兩個分析的結果可以知道，並不是所有位於熱休克蛋白27上α- crystallin 作用區的帶電胺基酸都是同樣重要的。然而我們也不能排除這些胺基酸的改變，會影響熱休克蛋白27其他未知功能的可能性。最近我們正在構築穩定表達正常或致病突變 (R127W、S135F、R140E及P182L) 蛋白的細胞株，希望在未來對於神經退化性疾病的致病成因可以做進一步研究。 Charcot-Marie-Tooth (CMT) is a dominantly hereditary motor and sensory neurodegenerative disease. Previous studies showed that mutations of highly conserved α- crystallin domain in heat shock protein 27 (Hsp27) are responsible for axonal CMT type 2F. Hsp27, a member of small Hsps, has chaperone activity and anti-apoptotic function. Additionally, Hsp27 maintains the cell property by interacting with cytoskeleton. Therefore, we propose that because α-crystallin domain of Hsp27 is an important functional domain, missense mutation on the conserved residues of this region may disrupt normal functions of Hsp27, resulting in cytoplasmic aggregation and abnormal neuronal intermediate filament network . To look for novel mutation, we sequenced the α-crystallin domain of Hsp27 gene from CMT patients in Taiwan. Up to date, 14 patients with CMT disease have been examined but none of them contain mutations in α-crystallin domain of Hsp27. On the other hand, we successfully made Hsp27 constructs carrying various mutations on α- crystallin domain or other region by site-directed mutagenesis, including K112W, E119L, K123W, R127W, S135F, R140E, and P182L. The last four mutants were made according to pathogenic mutations and the others were changed from charged amino acids to non-charged ones. After transfecting different constructs into a mouse neuroblastoma cell line (N2a), similar expression levels of normal and mutant Hsp27 were confirmed by Western blot analysis. In addition, results from immunocytochemical staining demonstrated that only P182L led to severe cytoplasmic aggregates in N2a cells. Furthermore, DAPI staining was used to examine cell viability. Our results showed that pathogenic mutant R140E, but not R127W, significantly reduced cell viability, as compared with wild type Hsp27. However, other mutants do not have significant impacts on cell viability in our assay conditions. We conclude that different specific amino acid may be required for different functions of Hsp27. According to our results, we suggest that these charged residues on α-crystallin domain are not equally important for the functions we examined in the current study. However, we can not exclude the possibility that some amino acids may be involved in unidentified cellular functions of Hsp27. Currently, stable cell lines expressing normal or pathogenic mutant Hsp27, including R127W, S135F, R140E and P182L, are under construction. These cell models will be used to study the pathogenesis of CMT in the future.
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