中間絲巢蛋白之柱狀結構域片斷缺失異構物 Nes-SΔ107-254 之鑑定

Abstract

巢蛋白（nestin）為一種第六類中間絲蛋白，主要表現在神經與肌肉組織之幹細胞和前驅細胞中。本實驗室先前曾於大鼠背根神經元中鑑定出巢蛋白的第一個蛋白異構物，Nes-S，其具有抗細胞自裁和促細胞增生之功能。在此篇研究中，吾人鑑定出巢蛋白的第二個蛋白異構物，Nes-SΔ107-254。該異構物之分子量為 29 kD ，其蛋白結構於柱狀結構域含有大片斷的缺失，其餘部份皆與 Nes-S 相同。中間絲蛋白之柱狀結構域對其纖維組裝極為重要。透過本實驗室先前製備之兔多株抗體 anti-AY14 操作西方墨點實驗，吾人得知 Nes-SΔ107-254 於大鼠背根神經元中低量表現，且不與神經元表現之其他中間絲蛋白共同形成纖維。此外，基因轉染實驗結果顯示， Nes-SΔ107-254 不但自己無法形成中間絲纖維，也無法與其他中間絲蛋白共同組成中間絲纖維。於細胞內高量表現時， Nes-SΔ107-254 傾向形成蛋白堆疊體。 MTT細胞存活率分析和細胞增殖率分析顯示， Nes-SΔ107-254 與 Nes-S 不同，並不具有抗細胞自裁和促細胞增生之功能。吾人進一步使用免疫螢光染色法得知，帶有由外源表現 Nes-SΔ107-254蛋白堆疊體之 HEK293T 細胞，其細胞凋亡蛋白 caspase-3 之活化量顯著上升，暗示該蛋白堆疊體具有細胞毒性。總言之， Nes-SΔ107-254 是一個新發現的柱狀結構域片斷缺失之巢蛋白異構物，該蛋白缺乏纖維組裝能力，亦不具有抗細胞自裁和促細胞增生之能力，於高量表現時傾向形成有細胞毒性之蛋白堆疊體。

The type VI intermediate filament (IF) protein nestin is predominantly expressed in neurogenic and myogenic stem cells. We have previously identified the first isoform of nestin, Nes-S, in DRG neurons of adult rats. In this study, we report a previously unidentified nestin isoform,Nes-SΔ107-254, that expressed at lower levels in DRG neurons of adult rats. The 29-kD Nes-SΔ107-254 is identical to Nes-S, except that an additional region was removed in its rod domain, leading to the expression of a rod domain-truncated form of Nes-S. Nes-SΔ107-254 is assembly compromised and can not form filaments with either vimentin in the vimentin-positive SW13 cells and HEK293T cells, or with peripherin proteins in the N2a neuroblastoma cells. In addition, Nes-SΔ107-254 was also unable to form filaments by itself when expressed in the vimentin-negative SW13 cells. While the expression of nestin and Nes-S exerts cytoprotective function through the Thr316 residue, our MTT results revealed that Nes-SΔ107-254 did not exert the cytoprotective effect. The statistical analysis demonstrated that Nes-SΔ107-254 was more inclined to form aggregates as compared with Nes-S. Furthermore, elevated caspase-3 activation was observed in HEK293T cells expressing EGFP-Nes-SΔ107-254 protein. Taken together, Nes-SΔ107-254 is a rod domain-truncated isoform of nestin that is susceptible to form aggregates when highly expressed.