Interactions of mouse paneth cell α-defensins and α-defensin precursors with membranes
Interactions of mouse paneth cell α-defensins and α-defensin precursors with membranes
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DOI：
10.1074/jbc.m212115200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Jelinek, R
中科院分区：
文献类型：
--
作者：
Satchell, DP;Sheynis, T;Jelinek, R
小鼠α-防御素（cryptdins）的杀菌活性需要基质金属蛋白酶-7（matrilysin，EC 3.4.24.23，NEAP-7（a））对无活性前体的蛋白水解活化。研究与膜双层的cryptdin-4（Crp 4）肽相互作用的机制，并确定是否NMP-7介导的蛋白水解激活Crp 4的膜破坏活性，协会Crp 4和蜂毒肽与仿生脂质/聚丁二炔染色囊泡的特点。这些肽在它们对囊泡脂质组成的敏感性和它们的双层渗透深度方面不同。与蜂毒肽不同，Crp 4与脂质双层发生强界面结合，破坏双层头基区域，蜂毒肽更深入地插入双层的疏水核心。比色法和色氨酸荧光研究表明，Crp 4插入有利于带负电荷的磷脂和两性离子和大肠杆菌磷脂促进更强的界面结合，蜂毒肽膜相互作用是独立的变量。与Crp 4的膜破坏活性相反，Pro-Crp 4没有扰乱囊泡膜，这与前体缺乏杀菌活性一致，并且在抑制Crp 4杀菌活性的浓度下，Crp 4与前片段反式孵育阻断了Crp 4和G1 W-Crp 4膜相互作用。CD测量结果表明，Crp 4具有预期的β折叠结构，该结构在pro-Crp 4 CD迹线中或当Crp 4与前片段孵育时不明显，表明β折叠信号被前区相互作用减弱或可能被前片段破坏。总的来说，这些结果表明，前片段通过阻断肽介导的靶细胞膜扰动来抑制Crp 4杀菌活性，当NMP-7切割前片段时，这种约束得到缓解。
The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC 3.4.24.23, NEAP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether NMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when NMP-7 cleaves the prosegment.