The unfoldase ClpC1 ofMycobacterium tuberculosisregulates the expression of a distinct subset of proteins having intrinsically disordered termini
The unfoldase ClpC1 ofMycobacterium tuberculosisregulates the expression of a distinct subset of proteins having intrinsically disordered termini
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DOI：
10.1074/jbc.ra120.013456
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发表时间：
2020-07-10
影响因子：
4.8
通讯作者：
Agarwal, Nisheeth
中科院分区：
文献类型：
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作者：
Lunge, Ajitesh;Gupta, Radhika;Agarwal, Nisheeth
人类致病分支杆菌结核分枝杆菌(Mtb)含有一个协调良好的酪蛋白水解酶(CLP)蛋白分解机制，由两个寡聚体片段组成，一个由ClpP1和ClpP2亚基组成的桶状异十四聚体蛋白水解酶核心，以及由ClpX或ClpC1组成的六聚体环状ATP依赖展开酶。ClpP1P2蛋白酶亚单位在Mtb中的作用已得到很好的证实，但相关的解折叠酶，如ClpC1的潜在作用仍然难以捉摸。利用CRISPR干扰介导的基因沉默方法，我们在这里证明了clpC1对于结核分枝杆菌的胞外生长和在巨噬细胞中的生存是不可或缺的。对缺失clpC1和clpP2的Mtb细胞进行的基于相对和绝对定量的等压标记的定量蛋白质组学实验结果表明，ClpC1P1P2复合体在Mtb中关键地维持着各种生长必需蛋白质的动态平衡，其中一些蛋白质的末端含有内在的无序区域。我们发现CLP机制调节剂量敏感的蛋白质，例如以十二聚体构象存在的小热休克蛋白Hsp20。此外，我们观察到Hsp20在WT Mtb中低表达，并且在缺乏clpC1或clpP2的情况下其表达被显著诱导。值得注意的是，尽管hsp20转录本被显著诱导，但在clpc1或clpP2基因敲除菌株中检测到高水平的Hsp20蛋白，而在亲本细菌中检测不到。综上所述，Hsp20等寡聚蛋白的细胞水平是通过ClpC1对它们的识别、分解和降解在翻译后保持的，这需要其蛋白质底物上的无序末端。
The human pathogenMycobacterium tuberculosis(Mtb) harbors a well-orchestrated Clp (caseinolytic protease) proteolytic machinery consisting of two oligomeric segments, a barrel-shaped heterotetradecameric protease core comprising the ClpP1 and ClpP2 subunits, and hexameric ring-like ATP-dependent unfoldases composed of ClpX or ClpC1. The roles of the ClpP1P2 protease subunits are well-established in Mtb, but the potential roles of the associated unfoldases, such as ClpC1, remain elusive. Using a CRISPR interference?mediated gene silencing approach, here we demonstrate thatclpC1is indispensable for the extracellular growth of Mtb and for its survival in macrophages. The results from isobaric tags for relative and absolute quantitation?based quantitative proteomic experiments withclpC1- andclpP2-depleted Mtb cells suggested that the ClpC1P1P2 complex critically maintains the homeostasis of various growth-essential proteins in Mtb, several of which contain intrinsically disordered regions at their termini. We show that the Clp machinery regulates dosage-sensitive proteins such as the small heat shock protein Hsp20, which exists in a dodecameric conformation. Further, we observed that Hsp20 is poorly expressed in WT Mtb and that its expression is greatly induced upon depletion ofclpC1orclpP2. Remarkably, high Hsp20 protein levels were detected in theclpC1(?)orclpP2(?)knockdown strains but not in the parental bacteria, despite significant induction ofhsp20transcripts. In summary, the cellular levels of oligomeric proteins such as Hsp20 are maintained post-translationally through their recognition, disassembly, and degradation by ClpC1, which requires disordered ends in its protein substrates.