An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila.
An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila.
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DOI：
10.1091/mbc.e14-03-0833
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发表时间：
2014-08-15
影响因子：
3.3
通讯作者：
Turkewitz AP
中科院分区：
文献类型：
--
作者：
Kumar S;Briguglio JS;Turkewitz AP
在动物细胞中，分泌颗粒中致密核心的组装受前蛋白的蛋白水解加工控制。同样的现象也发生在四膜虫中，但涉及的蛋白酶似乎是高度无关的，这表明类似的调节机制有不同的分子起源。在嗜热四膜虫中，通过致密核心颗粒（称为粘液囊）分泌的肽通过前蛋白加工产生。我们使用表达谱来鉴定候选加工酶，其定位为与粘液囊融合的青色荧光蛋白。值得注意的是，乙酰基组织蛋白酶Cth3p在基于粘液囊的分泌中起关键作用，因为该基因的敲低阻断了整套粘液囊前蛋白的蛋白水解成熟，并显著减少了粘液囊积累。Cth3p的活性被消除的突变的两个预测的活性位点突变，和野生型基因的过表达，但不是催化位点突变体，部分挽救了一个孟德尔突变体缺陷的粘液囊前蛋白加工。我们的研究结果提供了第一个直接的证据前蛋白加工在这个系统中的作用。令人感兴趣的是，定位和CTH3破坏表型都表明该酶提供非粘液囊相关的功能。T.的系统发育分析嗜热菌组织蛋白酶的研究，结合先前关于分拣蛋白受体在粘液囊生物发生中的作用的工作，表明溶酶体酶的再利用是纤毛虫分泌颗粒进化中的重要步骤。
In animal cells, the assembly of dense cores in secretory granules is controlled by proteolytic processing of proproteins. The same phenomenon occurs in the ciliate Tetrahymena thermophila, but the proteases involved appear to be highly unrelated, suggesting that similar regulatory mechanisms have different molecular origins. In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non–mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates.