Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Microb Pathog. 2010 Oct;49(4):164-73. doi: 10.1016/j.micpath.2010.05.008. Epub 2010 May 25.
The Chlamydia-secreted protease/proteasome-like activity factor (CPAF) is synthesized as a proenzyme (proCPAF) and requires processing for proteolytic activity. Recent structural studies have further demonstrated that CPAF is a serine protease that can undergo autoprocessing and self-activation in a concentration-dependent manner in vitro. However, it is not known how CPAF is processed and activated during chlamydial infection. In the current study, we used a mutant CPAF designated as CPAF(E558A) that is deficient in processing by itself as a substrate to search for putative CPAF activation factor(s) in Chlamydia-infected cells. CPAF(E558A) was processed by the lysates made from Chlamydia-infected cells and the processing activity correlated with the presence of endogenous active CPAF in the fractionated lysate samples. CPAF produced in the Chlamydia-infected cells is required for processing the mutant CPAF(E558A) since the processing activity was removed by depletion with anti-CPAF but not control antibodies. Furthermore, a purified and activated wild type CPAF alone was sufficient for processing CPAF(E558A) and no other chlamydial proteases are required. Finally, fusion tag-induced oligomerization can lead to autoprocessing and self-activation of the wild type CPAF in mammalian cells. These observations together have demonstrated that CPAF undergoes autoprocessing and self-activation during chlamydial infection.
沙眼衣原体分泌的蛋白酶/蛋白酶体样活性因子（CPAF）作为一种前酶原（proCPAF）合成，需要进行加工才能具有蛋白水解活性。最近的结构研究进一步表明，CPAF 是一种丝氨酸蛋白酶，可在体外以浓度依赖的方式进行自加工和自我激活。然而，尚不清楚 CPAF 在衣原体感染过程中是如何被加工和激活的。在本研究中，我们使用一种突变的 CPAF（命名为 CPAF[E558A]）作为自身加工缺陷的底物，以寻找衣原体感染细胞中可能的 CPAF 激活因子。CPAF[E558A]可被来自衣原体感染细胞的裂解物加工，并且加工活性与裂解物样品中内源性活性 CPAF 的存在相关。衣原体感染细胞中产生的 CPAF 是加工突变型 CPAF[E558A]所必需的，因为该加工活性可被抗 CPAF 抗体耗尽，但不能被对照抗体耗尽。此外，单独纯化和激活的野生型 CPAF 足以加工 CPAF[E558A]，而不需要其他衣原体蛋白酶。最后，融合标签诱导的寡聚化可导致野生型 CPAF 在哺乳动物细胞中进行自加工和自我激活。这些观察结果共同表明，CPAF 在衣原体感染过程中经历自加工和自我激活。
