異生性淋巴瘤激酶相關蛋白KIAA1618的研究

Abstract

KIAA1618/ALO17是在異生性大細胞淋巴瘤 (Anaplastic large cell lymphoma, ALCL) 病例中所發現的功能未知蛋白，由1063個胺基酸組成，在這些ALCL病例中，KIAA1618的N端 (胺基酸序列1-1008) 與異生性淋巴瘤激酶 (Anaplastic lymphoma kinase, ALK) 的C端 (胺基酸序列 1058-1620 ) 形成新的融合蛋白，根據先前的研究，融合蛋白與致癌性有密切的關係。為了研究KIAA1618的功能，將含有HA-tag KIAA1618的質體DNA轉染HEK293T細胞，利用免疫沉澱收取表現蛋白，根據銀離子染色與西方墨點法的結果，KIAA1618表現在接近170 kDa的位置，比預測大小118.43 kDa來的高。分層萃取與免疫螢光染色顯示KIAA1618主要分布於細胞質。利用In vivo ubiquitination assay，顯示在MG132 (proteasome inhibitor) 處理下，KIAA1618具有多泛素化的特性，而利用誘發內質網壓力的藥物也可以發現類似的特性。進一步，凋亡細胞偵測顯示大量表現KIAA1618的HeLa細胞對於Tunicamycin (N-glycosylation inhibitor)與Thapsigargin (Ca2+ pump inhibitor) 處理有較高的耐受性；比較在兩種藥物處理下蛋白質的表現量，結果發現在大量表現KIAA1618的HeLa細胞，Grp78的表現量和PARP-1的斷裂都有顯著降低。總結實驗結果，KIAA1618可能會增加細胞對內質網壓力的耐受性。

KIAA1618/ALO17 is a novel protein discovered from the cases of the anaplastic large cell lymphoma (ALCL). A chimeric protein created due to a translocation of the N-terminal KIAA1618 protein (a.a. 1-1008) fused with the C-terminal truncated anaplastic lymphoma kinase (ALK, a.a. 1058-1620). According to the reported cases of ALCL, the oncogenesis is highly correlated with this chimeric protein. Nonetheless, the physiological function of KIAA1618 is still remained unclear. Using HA-tagged KIAA1618 to transfect HEK293 cells, silver-stain and Western blotting results showed that KIAA1618 migrated at approximate 170 kDa on SDS-PAGE which is much higher than an estimated 118.43 kDa from the predicted 1063 amino acids of KIAA1618 cDNA. Subcellular fraction and immunofluorescence staining revealed a predominant cytosolic distribution of KIAA1618. In vivo ubiquitination assay showed that KIAA1618 was poly-ubiquitinated with the addition of MG132, a proteasome inhibitor. A similar result was found when cells were exposed to some ER stress drugs. Moreover, apoptotic detections showed that KIAA1618 overexpressing HeLa cells were resistant to Tunicamycin and Thapsigargin treatment, an N-glycosylation inhibitor and a Ca2+ pump inhibitor, compared with control cells. Finally, a significant decrease in the expression level of Grp78 and PARP-1 cleavage was found in KIAA1618 overexpressing compared with control cells treated with Tunicamycin and Thapsigargin. Collectively, these results suggested that KIAA1618 may render cells resistance to ER stress.