Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
FASEB J. 2010 Jul;24(7):2201-10. doi: 10.1096/fj.09-143008. Epub 2010 Feb 24.
Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC(50)=0.9 microM) than Bak BH3 (IC(50)=11.8 microM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins.
Bcl-X(L) 是 Bcl-2 家族的一种抗凋亡成员，是一种位于线粒体的蛋白，能够抑制 Bax 和 Bak 的激活，而 Bax 和 Bak 的激活会导致细胞凋亡，因此它是药物发现的潜在靶点。肽类具有成为治疗性分子的潜力，因为它们可以被设计成与目标蛋白的更大部分结合，具有更高的特异性。在本研究中，我们使用 mRNA 显示技术从随机和简并肽文库中选择与 Bcl-X(L) 相互作用的 16 肽。所选肽与 Bcl-2 家族 BH3 结构域具有序列相似性，其中一种肽与 Bak BH3（IC50=11.8 microM）相比，对 Bcl-X(L) 的体外亲和力更高（IC50=0.9 microM）。我们还发现，所选肽的 GFP 融合物特异性地与 Bcl-X(L) 相互作用，定位于线粒体，并诱导细胞死亡。此外，Bak 蛋白的 BH3 结构域被替换为所选肽的嵌合分子保留了与 Bcl-X(L) 特异性结合的能力。这些结果表明，该所选肽特异性拮抗 Bcl-X(L) 的功能，并克服了完整细胞中 Bcl-X(L) 的作用。我们建议 mRNA 显示是一种强大的技术，可以识别与疾病相关蛋白具有高亲和力和特异性的肽抑制剂。
