CBAP蛋白於T細胞發育之功能研究

Abstract

T細胞受器所傳遞之訊息對胸腺細胞於CD4與CD8分子雙陽性之發育時期是非常關鍵的，但是參與其中的分子尚未完全被鑑定與分析。於先前的研究中，本實驗室已發現GM-CSF/IL-3/IL-5 receptor common β-chain associated protein (CBAP)會調控由受體缺乏所引起的細胞凋亡與由ZAP70所傳達的T細胞移動，而本研究旨在進一步探討CBAP於T細胞譜系中的功能。基於CBAP在胸腺細胞中的高表現量，本研究使用CBAP基因剃除小鼠探討該基因於胸腺發育時的功能。CBAP基因剃除小鼠的早期胸腺細胞發育與正向篩選能力無明顯缺陷；然而，在數種檢測負向篩選能力的動物模式中(包含利用T細胞受器轉基因鼠、施打抗CD3抗體與施打超抗原staphylococcal enterotoxin B等)，T細胞受器所誘導之胸腺細胞死亡則顯著地下降。這與在CBAP基因剃除之胸腺細胞中，T細胞受器所誘導之BIM蛋白累積量較少有高度的關聯。進一步的研究發現在缺少CBAP時，T細胞受器近端如ZAP70、LAT與PLCγ1與遠端如JNK之訊息傳遞皆變弱，而且LAT訊息傳遞複合體之形成也較不完全。這些證據顯示CBAP是一個參與在T細胞受器訊息傳遞中的新穎分子，並在胸腺細胞的負向篩選中調控其死亡。此外，藉由酵母菌雙雜合系統找尋與CBAP有交互作的蛋白顯示，CBAP有可能參與許多類型的生物進程，這項結果也呼應了本研究與本實驗室先前的研究：CBAP在數種不同的生理反應中均扮演調控的角色。

T cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. Our laboratory previously demonstrated that GM-CSF/IL-3/IL-5 receptor common β-chain associated protein (CBAP) modulates cytokine withdrawal-induced apoptosis in vitro and ZAP70-mediated T cell migration/adhesion in vivo. In this study, the function of CBAP in T cell lineage was further investigated. Based on the high expression of CBAP during thymocyte development, a CBAP knockout mouse was utilized to investigate the function of CBAP in thymocyte development. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Furthur investigation on TCR proximal signaling revealed that TCR-induced association of LAT signalosome components is reduced in CBAP-deficient thymocytes. These data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection. Moreover, CBAP-interacting proteins identified by a yeast two-hybrid system implied that CBAP is involved in divergent biological processes, echoing multiple functions of CBAP characterized in the previous and present studies.