The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease
The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease
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DOI：
10.1128/jb.00174-20
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发表时间：
2020-08-01
影响因子：
3.2
通讯作者：
Darwin, Andrew J.
中科院分区：
文献类型：
--
作者：
Chung, Sammi;Darwin, Andrew J.
细菌羧基末端加工酶(CTPs)广泛存在，并与信号转导、细胞壁代谢和毒力等重要过程有关。然而，CTP依赖的切割靶蛋白的功能尚不清楚。对大肠杆菌CTP PRC的研究表明，它能与非极性和/或结构不受限制的C ter-mini切割蛋白质，但尚不清楚这是否适用于广泛应用。铜绿假单胞菌有一个潜伏的CTP，CTPA是毒力所必需的。CTPA与外膜脂蛋白LbcA形成复合体，降解细胞壁水解酶。在这项研究中，我们研究了两种不同的CTPA底物的C末端是否对它们的降解起重要作用。我们确定这些底物与它们最接近的大肠杆菌同系物相比延长了C末端。从这些延伸物中去除7个氨基酸足以减少CTPA在体内和体外的降解。一个截短的底物的降解通过添加来自另一个的C终端而不是通过添加不相关的序列来恢复。然而，通过添加底物的C-末端氨基酸对非底物的C末端进行修饰，不会引起CTPA对它们的降解。因此，CTPA底物的C末端是其有效降解所必需的，但不是充分的。虽然C末端截断的底物不被降解，但它们在体内仍与LbcA-CTPA复合体结合。因此，CTPA对蛋白质的降解需要与LbcA-CTPA复合体进行不依赖于C末端的相互作用，然后是依赖于C末端的降解，这可能是因为CTPA通常在C末端启动切割。重要的羧基末端处理蛋白(CTPs)存在于生命的所有三个领域，但人们对它们的确切工作方式知之甚少，包括它们如何识别底物。细菌CTP与毒力有关，包括铜绿假单胞菌的CTPA，它与外膜脂蛋白LbcA形成复合体，降解潜在危险的肽聚糖水解酶。我们报告了一个重要的进展，揭示了CTPA的有效降解需要至少两个可分离的现象，其中一个依赖于底物C末端编码的信息。与LbcA-CTPA复合体的C末端不依赖的结合之后是CTPA依赖于C末端的切割。由于CTPs与毒力、肽聚糖重塑和其他重要过程有关，因此增加了对CTPs如何靶向蛋白质的重要理解。
Bacterial carboxyl-terminal processing proteases (CTPs) are widely con-served and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc sug-gested that it cleaves proteins with nonpolar and/or structurally unconstrained C ter-mini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a diver-gent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C termi-nus from the other but not by adding an unrelated sequence. However, modifica-tion of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associ-ated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally ini-tiates cleavage at a C-terminal site. p IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We re-port an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. In-creased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.