The N-myristoylome of Trypanosoma cruzi.
The N-myristoylome of Trypanosoma cruzi.
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DOI：
10.1038/srep31078
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发表时间：
2016-08-05
影响因子：
4.6
通讯作者：
Fairlamb AH
中科院分区：
文献类型：
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作者：
Roberts AJ;Fairlamb AH
蛋白质N-肉豆蔻酰化由N-肉豆蔻酰转移酶（NMT）催化，N-肉豆蔻酰转移酶是克氏锥虫（锥虫病的病原体）中的必需且可药用的靶标。在这里，我们采用全细胞标记叠氮肉豆蔻酸和点击化学，以确定N-肉豆蔻酰化蛋白在不同的生命周期阶段的寄生虫。在分裂形式（昆虫外鞭毛体和哺乳动物无鞭毛体阶段）和非分裂锥鞭毛体阶段之间仅观察到荧光标记的微小差异。在存在和不存在NMT抑制剂DDD 85646的情况下，使用基于蛋白质组学策略的无标记和稳定同位素标记的培养细胞（SILAC）的组合，我们鉴定了56种在三种实验方法中的至少两种中富集的蛋白质。其中，6个可能是假阳性，其余50个在一个或多个T. cruzi基因组其中大多数是功能未知的蛋白质（32），其余的（18）涉及各种关键的细胞和代谢功能，如细胞内转运，细胞信号传导和蛋白质周转。总之，我们已经确定，在T. cruzi接近其他真核生物（0.5-1.7%）。
Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%).