於HEK293細胞中破壞β-tubulin/CCT-β蛋白質複合體引發細胞凋亡的分子機制

Abstract

先前我們發現一個藉iodoacetyl基團和具碳端保護的色氨酸的氨端結合而成的iodoacetamide可造成HEK293細胞停至sub-G0 期及造成HEK293細胞凋亡 (Lin et al., 2009)。由於 Cys354位於β-tubulin和chaperonin containing TCP1 subunit β (CCT-β) 此兩蛋白的蛋白質複合體之結合部位，此化合物可藉其攜帶碘的碳原子和β-tubulin的Cys354的硫原子作共價性結合以破壞此複合體。另一方面，HEK293用此化合物處理也造成除了caspase-1之外所有caspases的活化。在本論文中，我首先藉對細胞處理I-Trp的濃度越高及時間越久，cytochrome c由粒線體釋放到細胞質的量也越多，這個發現同時也顯示了倚賴粒線體一系列反應的內源性細胞凋亡訊息傳遞之參與。另外，由於CCT-β在I-Trp所引發地細胞凋亡當中的重要性已被提出 (Lin et al., 2009)，若將CCT-β進行knockdown可望降低细胞凋亡的程度。因此在由shRNA轉染的HEK293細胞中測得cytochrome c釋放到細胞質中的量的確減少，符合預期中β-tubulin / CCT-β 複合體對細胞生存的重要性。 有趣的是，藉由使用IETD-CHO去抑制caspase-8的活性，發現cytochrome c 釋放到細胞質的程度也趨緩；由於caspase-8與caspase-10都已被證明會在外源性細胞凋亡中活化 (Juo et al., 1998; Wang et al., 2001)，而在某些情況下內源性和外源性細胞凋亡途徑會發生crosstalk的現象(Gewies, 2003)，因此這個發現除了顯示β-tubulin / CCT-β 複合體被破壞將導致外源性細胞凋亡途徑的活化之外，也證明了在此情況下內源性與外源性的細胞凋亡訊息傳遞路徑之間彼此會有crosstalk的現象。 再者，當細胞受到I-Trp刺激引發細胞凋亡之際，藉由阻止內質網內鈣離子的釋放，發現到caspase-4及caspase-5的活性也會隨之明顯減低。由於鈣離子的釋放，以及caspase-4的活化，都和內質網中壓力累積所引起的細胞凋亡有所關聯 (Hitomi et al., 2004)，這個結果代表內質網也在此β-tubulin/CCT-β複合體毀壞所引發的細胞凋亡機制中扮演了重要的調節角色。 接著，為了初步篩選和細胞凋亡的路徑相關的結合夥伴，使用重組的CCT-β蛋白來捕捉和CCT-β有結合的蛋白質，結果發現了Hsp90及VCP這兩種已知對調控細胞生存及死亡占有一席之地的蛋白質，並做進一步探討。 總括而論，本篇研究嘗試去釐清β-tubulin/CCT-β複合體毀壞所引發的細胞凋亡反應中的詳細分子機制，發現三種細胞凋亡途徑彼此之間的交互作用，嘗試去找出β-tubulin/CCT-β複合體下游其他重要的因子，並且為一個可針對tubulin-binding agent有抗性的，或者是CCT-β會過度表現的癌症種類有所療效的標靶治療作機制上的探討。

Previously, we identified an iodoacetamide with iodoacetyl group attached to the N-terminal of C-terminal protected Trp residue to cause migration of HEK293 cells into sub-G0 phase and cell apoptosis (Lin et al., Cancer Res. 2009). This compound can form a covalent bond through its iodo-bearing carbon with the thiol atom of Cys354 of β-tubulin to disrupt the complex of β-tubulin and chaperonin containing TCP1 subunit β (CCT-β) since Cys354 is located at the interface of the two interacting proteins. Treatment of HEK293 cell with the compound triggered apoptosis by activation of all caspases except caspase-1. In this thesis, I first identified the release of cytochrome c from mitochondria to cytosol after the cells were treated with I-Trp, and the release level showed the time and dosage dependence, which indicated the involvement of mitochondria-dependent intrinsic apoptotic signaling. Since the importance of β-tubulin/CCT-β complex to cell survival has been described (Lin et al., 2009), knockdown of CCT-β may reduce the subsequent apoptotic reaction. Transient knockdown by shRNA had been performed in order to confirm this hypothesis, and the cytochrome c releasing was thus decreased to the expectation. Interestingly, the leaking of cytochrome c into cytosol had also been prevented by inhibiting caspase-8 activity using IETD-CHO, a caspase-8 inhibitor. This proved the existence of extrinsic apoptotic signaling, as well as the crosstalk between intrinsic and extrinsic apoptotic pathway in this case, since caspase-8 and caspase-10 have been mentioned to be activated in extrinsic apoptotic pathway (Juo et al., 1998; Wang et al., 2001) and the crosstalk between intrinsic and extrinsic apoptotic pathway may happen under some kinds of circumstances (Gewies, 2003). Moreover, the rise of caspase-4 and caspase-5 activities due to I-Trp treatment could be reversed by blocking the Ca2+ release from intracellular reservoir. Due to the fact that Ca2+ release is an upstream event of ER-stress induced apoptosis, and caspase-4 has been described as ER stress-specific caspases (Hitomi et al., 2004), the data implicated ER played as a mediator among the reaction launched by CCT-β and β-tubulin complex destruction, and the crosstalk included the ER-stress induced apoptosis as well here. Then His-tagged CCT-β was used to pull down and identify several possible binding partners of CCT-β. This lead to the discover of Hsp90 and VCP as candidate interaction proteins of CCT-β, which both of the two proteins have been noticed of their mediating role in cell survival and cell death. Overall, these studies elucidated the possible mechanisms underlying the destruction of β-tubulin/CCT-β complex, found out the crosstalk between the several apoptotic pathway in this case, discovered the essential components in the downstream of β-tubulin/CCT-β complex and to suggest chemotherapeutic target for treating the clinical tubulin-binding agent-resistant or CCT-β–overexpressing tumors.