鳥糞嘌呤核苷酸交換因子 BIG1 和 BIG2 (Brefeldin A-Inhibited Guanine Nucleotide-Exchange Protein 1 and 2, BIG1 and BIG2)藉由Sec7 domain 使腺嘌呤核苷二磷酸核糖化因子(ADP-ribosylation factors, Arfs)與GTP結合, 來活化腺嘌呤核苷二磷酸核糖化因子,而參與細胞膜與高基氏體(Golgi)間的囊泡運輸。在我們實驗室先前的研究使用酵母菌雙雜交系統，發現TNLG4A可能會與BIG1有交互作用。TNLG4A 身為腫瘤壞死因子(TNF, tumor necrosis factor)的一員，會藉由和細胞表層的接受蛋白結合, 活化眾多生物反應，例如組織再生、創傷修復、刺激細胞生長與血管新生，並有文獻報導過TNLG4A和它的受體(receptor)結合後會影響神經膠質母細胞瘤的轉移及存活能力。TNLG4A 會以兩種型態存在細胞：全長的第二型穿膜蛋白以及被蛋白酶furin切割後釋放到外界的的水溶性型態。對於內源性的TNLG4A如何從細胞內運送至細胞外去刺激下游反應目前尚不清楚。在本篇論文中，我們利用酵母菌雙雜交系統等實驗方法，確認了BIG1 和TNLG4A彼此直接交互作用的區域。此外，為了進一步了解他們形成複合體的功能，我們藉由RNA干擾降解BIG1/2表現以及過度表現TNLG4A來觀察細胞的反應。綜合實驗結果，這些新發現給予BIG1/2是否藉由參與TNLG4A的運輸來調控細胞生理反應有初步的了解。

Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein BIG1 and its paralog BIG2 are well known to activate small GTPase Arf1 (ADP-ribosylation factor 1), the crucial regulator of vesicular trafficking between Golgi and plasma membrane. BIG1 and BIG2 would replace Arf1-GDP to Arf1-GTP by their Sec7 domain. TNLG4A, a member of TNF cytokine superfamily, was identified as a BIG1-interacting protein by using yeast two-hybrid library screening. TNLG4A that acts via binding to a cell surface receptor has multiple biological activities that implicated in tissue regeneration, wound repair, stimulation of cell growth, angiogenesis, and invasion, migration and survival of giloblastoma. TNLG4A-producing cells can co-express two forms, as a cell surface-associated type II transmembrane protein and a soluble form that released from the membrane-anchored TNLG4A by furin protease into the extracellular milieu. Little is known about the membrane transport pathway of TNLG4A from intracellular to plasma membrane and the mechanisms of how TNLG4A is released to the extracellular milieu. We showed here that BIG1 via its Sec7 domain interacted directly with TNLG4A and their interaction were confirmed by GST pull down assay and immunoprecipitation. To characterize the function of the interaction, we observed the subcellular location and expressions of TNLG4A in giloblastoma U251 cells that depletion of BIG proteins transiently or stably. We also investigated the downstream respond of giloma in cytokine secretion and migration ability. Our data revealed that BIG1 and BIG2 participate in transport of TNLG4A to the cell surface and secretion to extracellular medium that may involve in regulation of cell physiological responds, such as cell migration.

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