Cleavage-dependent activation of a chlamydia-secreted protease
Cleavage-dependent activation of a chlamydia-secreted protease
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DOI：
10.1111/j.1365-2958.2004.04072.x
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发表时间：
2004-06-01
影响因子：
3.6
通讯作者：
Zhong, GM
中科院分区：
文献类型：
--
作者：
Dong, F;Pirbhai, M;Zhong, GM
衣原体分泌的蛋白质命名为CPAF（衣原体蛋白酶体样活性因子）先前显示降解宿主转录因子（如RFX5）所需的主要组织相容性（MHC）基因激活。尽管CPAF由衣原体基因组中的单个开放阅读框（ORF）编码，但两个命名为CPAFn和CPAFc的片段是纯化的主要产物。本研究旨在测试CPAF裂解成CPAFn和CPAFc是否是CPAF蛋白水解活性所需的生理过程。脉冲追踪实验表明，CPAF最初在衣原体感染的细胞中合成为70 kDa的全长蛋白，并迅速裂解成CPAFn和c片段。通过转基因在哺乳动物细胞中表达的全长CPAF保持未切割，没有蛋白水解活性，而在大肠杆菌细胞中表达的CPAF被加工，并具有RFX5降解活性。即使在E. coli不能降解RFX5。更重要的是，当人工诱导突变体CPAF进行切割时，RFX5降解活性部分恢复。这些观察结果一起证明了CPAF的切割对于CPAF活性是必要的和足够的。
A chlamydia-secreted protein designated CPAF (chlamydial proteasome-like activity factor) was shown previously to degrade host transcriptional factors (e.g. RFX5) required for major histocompatibility (MHC) gene activation. Although CPAF is encoded by a single open reading frame (ORF) in the chlamydial genome, two fragments designated CPAFn and CPAFc were the main products purified. The current study was designed to test whether cleavage of CPAF into CPAFn and CPAFc is a physiological process required for CPAF proteolytic activity. Pulse-chase experiments revealed that CPAF was initially synthesized in chlamydia-infected cells as a 70 kDa full-length protein and rapidly cleaved into CPAFn and c fragments. Full-length CPAF expressed via a transgene in mammalian cells remained uncleaved and had no proteolytic activity, whereas CPAF expressed in Escherichia coli cells was processed and possessed RFX5 degradation activity. CPAF mutants deficient in processing even when expressed by E. coli failed to degrade RFX5. More importantly, the RFX5 degradation activity was partially restored when the mutant CPAF was artificially induced to undergo cleavage. These observations together have demonstrated that cleavage of CPAF is both necessary and sufficient for CPAF activity.