Transmembrane pore formation by the carboxyl terminus of Bax protein
Transmembrane pore formation by the carboxyl terminus of Bax protein
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DOI：
10.1016/j.bbamem.2012.08.006
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发表时间：
2013-02-01
影响因子：
3.4
通讯作者：
Tatulian, Suren A.
中科院分区：
文献类型：
--
作者：
Garg, Pranav;Nemec, Kathleen N.;Tatulian, Suren A.
Bax 是一种胞质蛋白，通过与线粒体外膜结合来响应各种细胞凋亡信号，导致膜透化、细胞色素 c 释放和 caspase 介导的细胞死亡。目前讨论的膜穿孔机制包括 Bax 与其他促凋亡蛋白（如 Bak）形成异源寡聚复合物，或 Bax 的多个疏水螺旋的膜插入，或在线粒体膜插入蛋白的物理帮助下形成脂质孔。我们和其他研究小组提供了令人信服的证据，表明 Bax 的 C 端“螺旋 9”介导膜结合和孔形成，但 Bax C 端的孔形成能力的机制仍不清楚。在这里，我们表明，对应于 Box C 末端的 20 个氨基酸肽 (VTIFVAGVLTASLTIWKKMG) 和两个赖氨酸被谷氨酸或亮氨酸取代的两个突变体在两性离子和阴离子磷脂膜中具有有效的膜孔形成活性。通过分析钙黄绿素从脂质囊泡中释放的动力学，可以确定孔形成的速率常数、膜内肽的亲和力、跨膜孔的寡聚状态以及赖氨酸残基的重要性。这些数据提供了对 Bax 衍生肽形成膜孔的分子细节的深入了解，并为基于肽的细胞毒剂的设计提供了新的机会。 (C) 2012 Elsevier B.V. 保留所有权利。
Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal "helix 9" of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Box C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents. (C) 2012 Elsevier B.V. All rights reserved.