Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei, 10617, Taiwan, ROC.
Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei, 10617, Taiwan, ROC.
Microbiol Res. 2019 Mar;220:21-31. doi: 10.1016/j.micres.2018.12.003. Epub 2018 Dec 17.
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ for degradation. However, it is not clear how the natural substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ proteases can degrade SulA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139∼149 aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142 and 147 residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.
在大肠杆菌中，ClpYQ（HslUV）是一种由两个组件组成的 ATP 依赖性蛋白酶，其中 ClpQ 是肽酶亚基，ClpY 是 ATP 酶和展开酶。ClpY 的功能是识别蛋白质底物，并使未折叠的多肽变性和移位到 ClpQ 的蛋白水解位点进行降解。然而，目前尚不清楚天然底物如何被 ClpYQ 蛋白酶识别，以及底物如何被 ClpY 选择、展开并移位到 ClpQ 六聚体的内部位点。Lon 和 ClpYQ 蛋白酶都可以在细菌细胞中降解细胞分裂抑制剂 SulA。在这项研究中，我们首先使用酵母双杂交和体内降解分析，证明了 SulA 的 C 端内部疏水区（139∼149 aa）对于与 ClpYQ 的结合和降解是必需的。在各种革兰氏阴性菌中，我们鉴定了 SulA 中 142 和 147 残基之间的保守区域 GFIMRP。通过使用 MBP-SulA(F143Y)（苯丙氨酸被酪氨酸取代）作为底物，我们的结果表明，SulA 的这个保守残基对于 ClpYQ 的识别和降解是必需的。支持这些数据，MBP-SulA(F143Y)、MBP-SulA(F143N)（苯丙氨酸被天冬酰胺取代）在体内与 ClpYQ 蛋白酶的半衰期更长。相比之下，MBP-SulA(F143D)和 MBP-SulA(F143S)的半衰期都更短。因此，在大肠杆菌 ClpYQ 蛋白酶复合物中，ClpY 识别 SulA 的 C 端区域，而 SulA 的 F143 对于 ClpYQ 蛋白酶的识别和降解起着重要作用。
