Peptide ligands for pro-survival protein Bfl-1 from computationally guided library screening.
Peptide ligands for pro-survival protein Bfl-1 from computationally guided library screening.
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DOI：
10.1021/cb300679a
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发表时间：
2013-04-19
影响因子：
4
通讯作者：
Keating, Amy E.
中科院分区：
文献类型：
--
作者：
Dutta, Sanjib;Chen, T. Scott;Keating, Amy E.
Bcl-2蛋白家族的促存活成员通过结合促凋亡蛋白中的短螺旋BH 3基序来抑制细胞死亡。哺乳动物促存活蛋白Bcl-xL、Bcl-2、Bcl-w、Mcl-1和Bfl-1以不同的亲和力和特异性与天然BH 3基序、工程化肽和小分子结合。生物物理学研究已经确定了这些蛋白质的相互作用模式，特别是研究最多的家族成员Bcl-xL和Mcl-1。Bfl-1是一种促生存蛋白，参与预防白血病、淋巴瘤和黑色素瘤的细胞凋亡。虽然Bfl-1是一个有前途的治疗靶点，但对其结合偏好知之甚少。我们通过筛选肽文库探索了Bfl-1与BH 3样肽的结合，所述肽文库被设计为对高度相关序列多样性进行采样。使用酵母表面展示进行筛选，产生了几种新型高亲和力Bfl-1结合剂，并通过深度测序鉴定了数千种推定的结合剂。进一步筛选特异性导致鉴定出与Bfl-1结合的肽，其Kd < 1 nM，并且与其他促存活Bcl-2家族成员相比，与Bfl-1的解离非常慢。该序列中的点突变产生对Bfl-1具有约50 nM亲和力的肽，其在平衡结合测定中对Bfl-1具有选择性。对工程化Bfl-1结合剂的分析加深了我们对促生存蛋白的结合特征如何不同的理解，并可能指导靶向Bfl-1抑制剂的开发。
Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-xL, Bcl-2, Bcl-w, Mcl-1 and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-xL and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with Kd < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ, and may guide the development of targeted Bfl-1 inhibitors.
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