Interaction with Sug1 enables Ipat ubiquitination leading to caspase 8 activation and cell death
Interaction with Sug1 enables Ipat ubiquitination leading to caspase 8 activation and cell death
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DOI：
10.1042/bj20091349
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发表时间：
2010-04-01
影响因子：
4.1
通讯作者：
Swarup, Ghanshyam
中科院分区：
文献类型：
--
作者：
Kumar, Yatender;Radha, Vegesna;Swarup, Ghanshyam
起始半胱天冬酶的激活依赖于相互作用的蛋白质，Ipaf [ICE（白介素-1 β-转化酶）蛋白酶激活因子] {NLRC 4 [NLR（Nod-like receptor）family CARD（半胱天冬酶激活和募集结构域）containing 4]}是一种炎性体组分，参与半胱天冬酶1的激活和凋亡。研究Ipaf的激活机制，我们发现Ipaf的C-末端LRR（leucine-rich repeat）结构域，通过分子内相互作用，负性调节其凋亡诱导功能。在A549肺癌细胞中，Ac-Ipaf（LRR结构域缺失的Ipaf）的表达诱导依赖于caspase 8的细胞死亡，但不依赖于caspase I。使用Ac-Ipaf作为诱饵的酵母双杂交筛选鉴定了人Sug 1（gal 1的抑制子），其为26 S蛋白酶体的组分，作为相互作用蛋白。在哺乳动物细胞中，Sug 1与Ipg相互作用并共定位。Sug 1与Ipaf的氨基酸91-253结合，这也是LRR结构域结合的区域。它增强Ipaf和Ac-Ipaf诱导的细胞死亡，Sug 1和Ipaf的共表达诱导caspase-8依赖性细胞死亡。由Ipaf和Sug 1形成的细胞复合物含有半胱天冬酶8。Ac-Ipaf的表达或Sug 1与Ipaf的共表达导致细胞质聚集体的形成和半胱天冬酶8的活化。Sug 1共表达使Ipaf能够通过泛素化修饰。将泛素分子标记至Ipaf导致聚集体形成，增强半胱天冬酶8的相互作用和活化，导致诱导细胞死亡。使用RNAi（RNA干扰）和显性负性方法，我们已经表明，由Ac-Ipaf表达或用TNF-α（肿瘤坏死因子α）或阿霉素治疗诱导的细胞死亡依赖于Sug 1。我们的研究结果表明，Ipaf的泛素化的作用，使其与Sug 1的相互作用，导致caspase 8激活和细胞死亡。
Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1 beta-converting enzyme)protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase I. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipg. Sug1 binds to amino acids 91-253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-alpha (tumour necrosis factor alpha) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.