Mitsui Kenichi, Nakayama Hiroshi, Akagi Takumi, Nekooki Munenori, Ohtawa Kenji, Takio Koji, Hashikawa Tsutomu, Nukina Nobuyuki
Laboratories for CAG Repeat Diseases, The Institute of Physical and Chemical Research (RIKEN) Brain Science Institute, Wako-shi, Saitama 351-0198, Japan.
J Neurosci. 2002 Nov 1;22(21):9267-77. doi: 10.1523/JNEUROSCI.22-21-09267.2002.
Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.
通过使用荧光激活细胞分选仪，从蜕皮激素诱导型突变神经2A细胞系（HD150Q - 28）中纯化出含有异常长聚谷氨酰胺序列（150个谷氨酰胺残基重复）的绿色荧光蛋白（GFP）融合截短型N端亨廷顿蛋白聚集体。为了分析与聚集体相互作用的蛋白质，我们将纯化的聚集体进行SDS - PAGE；凝胶中的显著蛋白条带用嗜麦芽窄食单胞菌赖氨酰内肽酶消化，随后进行高效液相色谱 - 质谱（MS）分析。串联质谱分析的结果表明，除了泛素和广泛报道的伴侣蛋白，如热休克同源蛋白70（HSC70）、人DNA J - 1（HDJ - 1）和HDJ - 2外，翻译延伸因子 - 1α（EF - 1α）和热休克蛋白84（HSP84）也被识别为与聚集体相互作用的蛋白质。通过几种免疫化学方法进一步证实了这些蛋白质被隔离到聚集体中。我们证实，除了其他已知蛋白质外，EF - 1α和HSP84也与细胞内聚集体共定位。对HD150Q - 28细胞中EF - 1α和HSP84的瞬时表达分析表明，这两种蛋白质都提高了细胞活力。此外，两种转染细胞中的聚集体形成率均降低。我们的研究表明，EF - 1α和HSP84都参与了聚谷氨酰胺疾病的神经退行性过程。
