Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, USA.
Biochim Biophys Acta Biomembr. 2019 Apr 1;1861(4):713-728. doi: 10.1016/j.bbamem.2018.12.020. Epub 2019 Jan 10.
Psd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCer) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp quantum yield of Psd2 in the presence of GlcCer and Erg, individually or in simultaneously enriched membranes. The partition coefficient (K) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.
Psd2 是一种豌豆防御素，由 47 个氨基酸残基组成，通过一种未被描述的机制抑制真菌物种的生长。在这项工作中，评估了 Psd2 与模拟不同生物体脂质组成的模型膜的相互作用。蛋白脂质覆盖分析表明，Psd2 除了识别磷脂酰胆碱 (POPC) 和一些磷脂酰肌醇种类（如 PtdIns(3)P、(5)P 和 (3,5)P）外，还识别镰刀菌属葡萄糖神经酰胺 (GlcCer) 和麦角固醇 (Erg)，表明这些脂质可能作为 Psd2 的靶标发挥重要作用。还使用脂质囊泡进行了测定，以研究肽-膜相互作用后发生的行为和动力学。表面等离子体共振分析表明，Psd2 对纯 POPC 和含有 70:30 比例的 GlcCer 和 Erg 的基于 POPC 的囊泡具有更高的亲和力，而对含有胆固醇 (Chol) 的囊泡的亲和力较低。荧光光谱的分区实验表明，在 GlcCer 和 Erg 单独或同时存在于富含膜中时，Trp 量子产率的 Psd2 降低。通过使用丙烯酰胺和 5/16-二氧代-硬脂酸进行猝灭实验获得的分配系数 (K) 表明 Psd2 对这些囊泡的分配偏好。此外，我们表明 GlcCer 神经酰胺骨架的 C8C9 双键和 C9 位的甲基的存在与 Psd2 对构巢曲霉的活性相关。这些结果与 Psd2 对真菌的选择性及其在人红细胞中无毒性一致。Psd2 代表一种有前途的天然化合物，可用于治疗真菌感染。
