Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors
Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors
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DOI：
10.1128/jvi.00231-08
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发表时间：
2008-08-01
影响因子：
5.4
通讯作者：
Friesen, Paul D.
中科院分区：
文献类型：
--
作者：
Guy, Michael P.;Friesen, Paul D.
杆状病毒蛋白P49和P35是多种生物中有效的细胞凋亡抑制因子。虽然相关，但P49和P35在感染允许昆虫细胞的过程中分别抑制了启动子和效应子半胱氨酸酶。这种新的caspase特异性的分子基础尚不清楚。为了推进选择性抑制细胞死亡半胱氨酸酶的策略，我们研究了这些杆状病毒底物抑制剂之间的生化差异。我们在这里报告P49和P35使用相似的化学计量抑制机制，需要caspase裂解其反应位点环(RSL)和保守的N-末端半胱氨酸的化学贡献来稳定所产生的抑制复合体。我们的数据表明，P49作为一个同源二聚体同时与两个caspase结合。相比之下，P35是一种单体、单价的抑制剂。P49和P35的RSL半胱氨酸天冬氨酸酶识别序列也不同。我们通过监测病毒诱导的对寄主昆虫夜蛾Caspase的主要效应因子SF-caspase-1的蛋白降解过程，测试了P-4-P-1识别基序对caspase特异性的作用。当P49‘S TVTD识别基序被P35’S DQMD基序取代时，P49对裂解和激活前SF-Caspase-1的启动子caspase的抑制作用减弱，而是与活性的SF-caspase-1形成稳定的抑制复合体。相反，当P35‘S的DQMD基序被TVTD取代时，P35的效应器caspase特异性没有改变。我们的结论是，TVTD识别基序是P49抑制启动子caspase所必需的，但不是充分的。我们的发现表明，P-4-P-1识别位点在P49和P35的caspase特异性中起着关键作用，并表明其他决定因素参与了靶标选择。
Baculovirus proteins P49 and P35 are potent suppressors of apoptosis in diverse organisms. Although related, P49 and P35 inhibit initiator and effector caspases, respectively, during infection of permissive insect cells. The molecular basis of this novel caspase specificity is unknown. To advance strategies for selective inhibition of the cell death caspases, we investigated biochemical differences between these baculovirus substrate inhibitors. We report here that P49 and P35 use similar mechanisms for stoichiometric inhibition that require caspase cleavage of their reactive site loops (RSL) and chemical contributions of a conserved N-terminal cysteine to stabilize the resulting inhibitory complex. Our data indicated that P49 functions as a homodimer that simultaneously binds two caspases. In contrast, P35 is a monomeric, monovalent inhibitor. P49 and P35 also differ in their RSL caspase recognition sequences. We tested the role of the P-4-P-1 recognition motif for caspase specificity by monitoring virus-induced proteolytic processing of Sf-caspase-1, the principal effector caspase of the host insect Spodoptera frugiperda. When P49's TVTD recognition motif was replaced with P35's DQMD motif, P49 was impaired for inhibition of the initiator caspase that cleaves and activates pro-Sf-caspase-1 and instead formed a stable inhibitory complex with active Sf-caspase-1. In contrast, the effector caspase specificity of P35 was unaltered when P35's DQMD motif was replaced with TVTD. We concluded that the TVTD recognition motif is required but not sufficient for initiator caspase inhibition by P49. Our findings demonstrate a critical role for the P-4-P-1 recognition site in caspase specificity by P49 and P35 and indicate that additional determinants are involved in target selection.