Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2
Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2
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DOI：
10.1186/1471-2091-12-61
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发表时间：
2011-12-01
期刊：
影响因子：
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通讯作者：
Ortiz-Lombardia, Miguel
中科院分区：
文献类型：
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作者：
Benaroudj, Nadia;Raynal, Bertrand;Ortiz-Lombardia, Miguel
背景：酪蛋白水解酶(CLPP)是一种桶形的自我分隔的多肽酶，参与清除受损或短暂的调节蛋白。结核分枝杆菌(MTB)基因组包含两个编码ClpPs的基因，分别为ClpP1和ClpP2，这两个基因可能在结核杆菌的毒力中发挥作用。结果：我们首次报道了结核分枝杆菌ClpP1和ClpP2多肽酶的生化特性。这两种蛋白都是在大肠杆菌中生产和纯化的。使用不同特性的荧光模型肽不能显示重组分枝杆菌ClpP1或ClpP2的肽酶活性。然而，我们发现ClpP1在Arg8残基之后具有自身的裂解活性，而ClpP2在Ala12残基之后具有裂解活性。此外，我们还表明，对模型多肽没有任何肽酶活性的原因并不是由于蛋白质的N端柔性末端堵塞了进入孔道，也不是因为对ClpX或ClpC ATPase复合体的绝对要求。最后，我们还发现，去除ClpP1和ClpP2的前肽并不会导致模型肽的裂解。我们还发现，重组的ClpP1和ClpP2不是以传统的功能十四聚体的形式组装，而是以从单体到七聚体的低阶齐聚形式组装。ClpP1和ClpP2的同时存在并没有导致十四聚体组装。删除ClpP1和ClpP2(可能的前肽或进入门)的氨基末端促进了高阶低聚物种的组装，这表明分枝杆菌ClpPs灵活的N端参与了七聚体之间相互作用的不稳定。结论：尽管分枝杆菌ClpP1和ClpP2的初级序列中保守了一个丝氨酸蛋白酶催化三联体，但它们对肽模型不具有常规的肽酶活性，并显示了一种不寻常的自组装机制。因此，它们的肽酶和蛋白分解活性的机制可能不同于其他ClpP蛋白降解复合体。
Background: Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium.Results: We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides.We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers.Conclusion: Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.