Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
FASEB J. 2019 May;33(5):6470-6482. doi: 10.1096/fj.201802540R. Epub 2019 Feb 22.
Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, defensin 173 (NoD173), from NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αβ motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth .-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of defensin NoD173 and protein engineering to enhance oncolysis.
防御素是一类广泛存在于动植物物种中的宿主防御肽家族。除了抵御病原微生物感染外，一些防御素还对肿瘤细胞具有选择性细胞毒性。因此，防御素作为潜在的抗菌和抗癌治疗药物引起了人们的兴趣。防御素作用于微生物和肿瘤细胞的机制似乎是保守的，涉及靶向和破坏细胞膜。这在植物防御素中得到了最好的定义，植物防御素与特定的磷脂，如磷脂酰肌醇 4,5-二磷酸（PIP2）和磷脂酸结合后，形成防御素-脂质寡聚体复合物，使膜不稳定，导致细胞裂解。在这项研究中，为了进一步确定植物防御素的抗癌和治疗特性，我们对一种新型植物防御素 NoD173 进行了表征，发现低微摩尔浓度的 NoD173 选择性杀伤一系列肿瘤细胞系，而对正常原代细胞没有影响。为了提高 NoD173 的抗癌活性，我们通过突变探索了增加正电荷的方法，用赖氨酸取代 NoD173 的 Q22 得到 NoD173(Q22K)，使抗肿瘤细胞活性提高了 2 倍。NoD173 和 NoD173(Q22K)突变体仅表现出低水平的溶血活性，并且在血清中都保持对肿瘤细胞的活性。在 B16-F1 小鼠模型中测试了 NoD173 抑制实体肿瘤生长的能力，结果表明，将 NoD173 注射到已建立的皮下肿瘤中，可显著抑制肿瘤生长。最后，我们表明 NoD173 特异性靶向 PIP2，并通过 X 射线晶体学确定了 NoD173 的高分辨率结构，该结构形成了一个保守的家族定义的半胱氨酸稳定的αβ基序，具有二聚体脂质结合构象，配置成 4 个二聚体的拱形寡聚体。这些数据提供了关于防御素如何靶向膜杀死肿瘤细胞的机制的见解，并提供了防御素能够抑制肿瘤生长的概念验证。
