Alcalde Juan, González-Muñoz María, Villalobo Antonio
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Madrid, Spain.
Department of Cancer Biology, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.
Heliyon. 2020 May 7;6(5):e03922. doi: 10.1016/j.heliyon.2020.e03922. eCollection 2020 May.
The growth factor receptor bound protein 7 (Grb7) is a Ca-dependent calmodulin (CaM)-binding adaptor protein implicated, among other functions, in cell proliferation, migration and tumor-associated angiogenesis. The goal of this study was to determine whether a peptide based on the CaM binding site of Grb7 disrupts cellular processes, relevant for the malignancy of tumor cells, in which this adaptor protein is implicated. We designed synthetic myristoylated and non-myristoylated peptides corresponding to the CaM-binding domain of human Grb7 with the sequence RKLWKRFFCFLRRS and a variant peptide with the mutated sequence RKLERFFCFLRRE (W246E-ΔK247-S256E). The two non-myristoylated peptides bind dansyl-CaM with higher efficiency in the presence than in the absence of Ca and they enter into the cell, as tested with 5(6)-carboxytetramethylrhodamine (TAMRA)-labeled peptides. The myristoylated and non-myristoylated peptides inhibit the proliferation, migration and invasiveness of A431 tumor cells while they enhance their adhesion to the substrate. The myristoylated peptides have stronger inhibitory effect than the non-myristoylated counterparts, in agreement with their expected higher cell-permeant capacity. The myristoylated and non-myristoylated W246E-ΔK247-S256E mutant peptide has a lesser inhibitory effect on cell proliferation as compared to the wild-type peptide. We also demonstrated that the myristoylated peptides were more efficient than the CaM antagonist -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibiting cell migration and equally efficient inhibiting cell proliferation.
生长因子受体结合蛋白7（Grb7）是一种依赖钙的钙调蛋白（CaM）结合衔接蛋白，除其他功能外，还参与细胞增殖、迁移和肿瘤相关血管生成。本研究的目的是确定基于Grb7的CaM结合位点的肽是否会破坏与肿瘤细胞恶性相关的细胞过程，而该衔接蛋白参与了这些过程。我们设计了与人Grb7的CaM结合域相对应的合成肉豆蔻酰化和非肉豆蔻酰化肽，其序列为RKLWKRFFCFLRRS，以及具有突变序列RKLERFFCFLRRE（W246E-ΔK247-S256E）的变体肽。两种非肉豆蔻酰化肽在有钙存在时比无钙时更有效地结合丹磺酰-CaM，并且它们能进入细胞，这通过用5(6)-羧基四甲基罗丹明（TAMRA）标记的肽进行测试得以证实。肉豆蔻酰化和非肉豆蔻酰化肽抑制A431肿瘤细胞的增殖、迁移和侵袭，同时增强它们与底物的粘附。肉豆蔻酰化肽比非肉豆蔻酰化肽具有更强的抑制作用，这与其预期的更高的细胞穿透能力一致。与野生型肽相比，肉豆蔻酰化和非肉豆蔻酰化的W246E-ΔK247-S256E突变肽对细胞增殖的抑制作用较小。我们还证明，肉豆蔻酰化肽在抑制细胞迁移方面比CaM拮抗剂-(6-氨基己基)-5-氯-1-萘磺酰胺（W-7）更有效，在抑制细胞增殖方面同样有效。
