关键词： angiogenin apoptosis cytolytic fusion protein molecular dynamics mutagenesis replica exchange ribonuclease inhibitor targeted therapy

Mesh ： Antineoplastic Agents / chemistry Binding Sites Carrier Proteins / chemistry genetics Gene Expression Humans Immunotoxins / chemistry genetics Molecular Dynamics Simulation Mutation Protein Binding Protein Engineering Protein Interaction Domains and Motifs Protein Structure, Secondary Recombinant Fusion Proteins / chemistry genetics Ribonuclease, Pancreatic / chemistry genetics

来  源：   DOI：10.1002/pro.2941   PDF(Sci-hub)   PDF(Pubmed)

Abstract：
Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang\'s affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.
摘要：
靶向的人溶细胞融合蛋白(hCFP)代表了用于特异性靶向和消除恶性细胞群的新一代免疫毒素(IT)。与传统的IT不同，hCFP包含人/人源化靶细胞特异性结合部分(例如，抗体或其片段）融合到人促凋亡蛋白作为细胞毒性结构域（效应子结构域）。因此,hCFP是预期具有低免疫原性的人源化IT。这减少了副作用并允许长期应用。人核糖核酸酶血管生成素（Ang）已被证明是有前途的效应子结构域候选物。然而,基于Ang的hCFP的应用在很大程度上受到细胞内胎盘核糖核酸酶抑制剂（RNH1）的阻碍。它迅速结合并使Ang失活。改变Ang对RNH1亲和力的突变调节基于Ang的hCFP的细胞毒性。在这里，我们进行了总共2.7µs的复制交换分子动力学模拟，以研究其中一些突变-G85R/G86R（GGRRmut），Q117G(QGmut),和G85R/G86R/Q117G（GGRR/QGmut）。GGRRmut大大扰乱了Ang-RNH1的整体相互作用，而QGmut优化它们。将QGmut与GGRRmut相结合可以补偿后者的影响。我们的结果解释了体外发现，虽然基于AngGGRRmut的hCFP显著抵抗RNH1抑制，基于AngWT和AngQGmut的蛋白同样对RNH1抑制敏感，而基于AngGGRR/QGmut的仅有轻微的抗性。这项工作可能有助于设计对RNH1的亲和力降低并改善细胞毒性的新型Ang突变体。
公众号