Velier J, Kim M, Schwarz C, Kim T W, Sapp E, Chase K, Aronin N, DiFiglia M
Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, 02114, USA.
Exp Neurol. 1998 Jul;152(1):34-40. doi: 10.1006/exnr.1998.6832.
Huntingtin is a cytoplasmic protein that is found in neurons and somatic cells. In patients with Huntington's disease (HD), the NH2-terminal region of huntingtin has an expanded polyglutamine tract. An abnormal protein interaction by mutant huntingtin has been proposed as a mechanism for HD pathogenesis. Huntingtin associates with vesicle membranes and interacts with proteins involved in vesicle trafficking. It is unclear where along vesicle transport pathways wild-type and mutant huntingtins are found and whether polyglutamine expansion affects this localization. To distinguish wild-type and mutant huntingtin, fibroblasts from normals and HD patients with two mutant alleles (homozygotes) were examined. Immunofluorescence confocal microscopy showed that mutant huntingtin localized with clathrin in membranes of the trans Golgi network and in clathrin-coated and noncoated endosomal vesicles in the cytoplasm and along plasma membranes. Separation of organelles in Nycodenz gradients showed that in normal and HD homozygote patient cells, huntingtin was present in membrane fractions enriched in clathrin. Similar results were obtained in fibroblasts from heterozyote juvenile HD patients who had a highly expanded polyglutamine tract in the HD allele. Western blot analysis of membrane fractions from rat brain showed that wild-type huntingtin was present in fractions that contained purified clathrin-coated membranes or a mixture of clathrin-coated and noncoated membranes. Electron microscopy of huntingtin immunoreactivity in rat brain revealed labeling along dendritic plasma membranes in association with clathrin-coated pits and clusters of noncoated endosomal vesicles 40-60 nm in diameter. These data suggest that wild-type and mutant huntingtin can influence vesicle transport in the secretory and endocytic pathways through associations with clathrin-coated vesicles.
亨廷顿蛋白是一种存在于神经元和体细胞中的细胞质蛋白。在亨廷顿舞蹈症（HD）患者中，亨廷顿蛋白的NH2末端区域有一段扩展的聚谷氨酰胺序列。突变的亨廷顿蛋白异常的蛋白质相互作用被认为是HD发病机制之一。亨廷顿蛋白与囊泡膜相关，并与参与囊泡运输的蛋白质相互作用。目前尚不清楚野生型和突变型亨廷顿蛋白在囊泡运输途径的哪个位置被发现，以及聚谷氨酰胺序列的扩展是否会影响这种定位。为了区分野生型和突变型亨廷顿蛋白，对正常人和携带两个突变等位基因（纯合子）的HD患者的成纤维细胞进行了检测。免疫荧光共聚焦显微镜显示，突变型亨廷顿蛋白与网格蛋白共定位于反式高尔基体网络的膜中、细胞质中以及质膜上的网格蛋白包被和未包被的内体囊泡中。在Nycodenz梯度中分离细胞器表明，在正常和HD纯合子患者细胞中，亨廷顿蛋白存在于富含网格蛋白的膜组分中。在携带高度扩展的HD等位基因聚谷氨酰胺序列的杂合子青少年HD患者的成纤维细胞中也获得了类似结果。对大鼠脑膜组分的蛋白质印迹分析表明，野生型亨廷顿蛋白存在于含有纯化的网格蛋白包被膜或网格蛋白包被和未包被膜混合物的组分中。对大鼠脑中亨廷顿蛋白免疫反应性的电子显微镜观察显示，在树突质膜上有标记，与直径为40 - 60 nm的网格蛋白包被小窝和未包被的内体囊泡簇相关。这些数据表明，野生型和突变型亨廷顿蛋白可通过与网格蛋白包被囊泡的结合影响分泌和内吞途径中的囊泡运输。
