Characterization of the sequence specificity determinants required for processing and control of sex pheromone by the intramembrane protease Eep and the plasmid-encoded protein PrgY
Characterization of the sequence specificity determinants required for processing and control of sex pheromone by the intramembrane protease Eep and the plasmid-encoded protein PrgY
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DOI：
10.1128/jb.01327-07
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发表时间：
2008-02-01
影响因子：
3.2
通讯作者：
Dunny, Gary M.
中科院分区：
文献类型：
--
作者：
Chandler, Josephine R.;Dunny, Gary M.
当供体检测到产毒的cCF 10时，肽信息素cCF 10诱导粪肠球菌质粒pCF 10的接合转移。cCF 10是通过染色体编码的脂蛋白（CcfA）的信号序列的蛋白水解加工产生的。在供体中，小心控制内源性产生的cCF 10，以防止结合功能的组成型表达，这是一种能量浪费的过程，但体内除外，其中内源性cCF 10诱导结合相关的毒力因子。内源性cCF 10由两种质粒编码的产物控制;膜蛋白PrgY降低供体中的信息素水平，而分泌的抑制剂肽iCF 10抑制逃脱PrgY控制的残留内源性信息素。在这项研究中，我们从遗传学上确定了PrgY、cCF 10和cCF 10前体中的氨基酸特异性决定簇，这些决定簇是cCF 10加工和PrgY介导的控制所必需的。我们发现，PrgY的氨基酸残基125至241是特异性识别cCF 10所必需的，并且PrgY识别七肽cCF 10序列内的决定簇，支持PrgY和成熟cCF 10之间的直接相互作用。此外，我们发现，一个受调节的膜内蛋白水解（RIP）家族信息素受体加工蛋白Eep识别氨基酸N-末端的CcfA的信号序列中的cCF 10。这些结果支持Eep直接靶向RIP的信息素前体和PrgY在加工过程中直接与成熟cCF 10肽相互作用的模型。尽管有证据表明PrgY和Eep都与膜内或膜附近的cCF 10相关，但本文提供的结果表明这两种蛋白质独立发挥功能。
Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.