The P-loop region of Schlafen 3 acts within the cytosol to induce differentiation of human Caco-2 intestinal epithelial cells.
The P-loop region of Schlafen 3 acts within the cytosol to induce differentiation of human Caco-2 intestinal epithelial cells.
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DOI：
10.1016/j.bbamcr.2014.09.017
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发表时间：
2014-12
期刊：
影响因子：
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通讯作者：
Basson MD
中科院分区：
文献类型：
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作者：
Chaturvedi L;Sun K;Walsh MF;Kuhn LA;Basson MD
Schlafen 3 （Slfn3）介导啮齿动物肠细胞在体内和体外的分化，是肠道功能所必需的。目前对Schlafen蛋白的结构-功能关系知之甚少。为了确定促进分化的Slfn3结构域，我们研究了在转染全长大鼠Slfn3 DNA或截断构建体的Slfn3缺失的人cco - 2bbe细胞中绒毛蛋白和蔗糖酶异芽糖酶（SI）启动子的活性。共聚焦显微镜和免疫印迹显示，Slfn3主要是细胞质。野生型Slfn3增加了绒毛蛋白启动子活性，并通过添加核排斥序列进一步增强了绒毛蛋白启动子活性，这表明Slfn3不通过核直接作用影响转录。因此，我们试图剖析Slfn3刺激启动子活性的区域。由于对Slfn3 n端区域的检查显示序列类似于氨基肽酶（App）和与NTPases相似的发散p环，我们最初将Slfn3分为包含App和p环区域的n端结构域和c端区域。只有n端结构刺激启动子活性。进一步的截断表明，App和较小的p环结构都增强了启动子活性，类似于n端序列。n端区域的点突变（R128L，改变了App结构域的一个关键活性位点残基，L212D，在Schlafens中保守，但在p环蛋白中可变）不影响活性。这些结果表明，Slfn3在细胞质中触发二级信号级联，引发分化标记的表达，并将活性区域缩小到与P-loop NTPases同源的Slfn3序列的第三个区域，这是了解其作用机制的第一步。
Schlafen 3 (Slfn3) mediates rodent enterocyte differentiation in vitro and in vivo, required for intestinal function. Little is known about Schlafen protein structure-function relationships. To define the Slfn3 domain that promotes differentiation, we studied villin and sucrase isomaltase (SI) promoter activity in Slfn3-null human Caco-2BBE cells transfected with full-length rat Slfn3 DNA or truncated constructs. Confocal microscopy and Western blots showed that Slfn3 is predominantly cytosolic. Villin promoter activity, increased by wild type Slfn3, was further enhanced by adding a nuclear exclusion sequence, suggesting that Slfn3 does not affect transcription by direct nuclear action. We therefore sought to dissect the region in Slfn3 stimulating promoter activity. Since examination of the Slfn3 N-terminal region revealed sequences similar to both an aminopeptidase (App) and a divergent P-loop resembling those in NTPases, we initially divided Slfn3 into an N-terminal domain containing the App and P-loop regions, and a C-terminal region. Only the N-terminal construct stimulated promoter activity. Further truncation indicated that both the App and the smaller P-loop constructs enhanced promoter activity similarly to the N-terminal sequence. Point mutations within the N-terminal region (R128L, altering a critical active site residue in the App domain, and L212D, conserved in Schlafens but variable in P-loop proteins) did not affect activity. These results show that Slfn3 acts in the cytosol to trigger a secondary signal cascade that elicits differentiation marker expression and narrow the active domain to the third of the Slfn3 sequence homologous to P-loop NTPases, a first step in understanding its mechanism of action.