Chimeric approach for narrowing a membrane-inserting region within human perforin

Amy E. Neely, Kimberly A Mandigo, R. Robinson, T. Ness, M. H. Weiland
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Abstract

Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved &bgr;-sheet flanked by two clusters of &agr;-helices. Within the CDCs, these helices refold into two transmembrane &bgr;-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.
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缩小人穿孔膜插入区域的嵌合方法
穿孔素是一种形成孔的免疫蛋白,其功能是将凋亡蛋白的混合物传递到目标病原体中。近年来对细菌胆固醇依赖性细胞溶素(CDCs)的研究为穿孔素的成孔机制提供了模型。perforin和CDC家族成员共享一个保守的&bgr;-薄片,两侧是两个&agr;-螺旋簇。在cdc中,这些螺旋重新折叠成两个跨膜发夹,TMH1和TMH2。基于结构守恒和电子显微镜成像,预测穿孔内的类似螺旋也为膜插入;然而,这些区域的长度大约是疾控中心tmh的两倍。为了测试其中一个区域的膜插入潜力,嵌合体是用一种表征良好的CDC,即perfringolysin-O (PFO)作为这些结构的主干来创建的。将PFO的TMH2区替换为穿孔素相应的螺旋区。虽然观察到溶血活性,但嵌合体难溶。第二个嵌合体包含相同的区域,截断以匹配PFO TMH2区域的长度。截断的嵌合体表现出更好的溶解度,显著的溶血活性和形成PFO所产生的孔的能力。这些结果提供了穿孔素螺旋作为TMHs功能的第一个证据,更重要的是缩小了负责膜插入的残基。
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