Molecular mechanism underlying β-tubulin/CCT-β complex destruction-induced cell apoptosis in HEK293 cells
Date Issued
2010
Date
2010
Author(s)
Lee, Ya-Fei
Abstract
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.
Subjects
CCT-β
β-tubulin
apoptosis
caspase
ERAD
SDGs
Type
thesis
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