CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis
CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis
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CGA-N9，一种源自嗜铬粒蛋白 A 的抗菌肽：热带念珠菌的直接细胞渗透和内吞作用
DOI：
10.1042/bcj20180801
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发表时间：
2019-02-14
影响因子：
4.1
通讯作者：
Shi, Jiaofan
中科院分区：
文献类型：
--
作者：
Li, Ruifang;Chen, Chen;Shi, Jiaofan
CGA-N9是衍生自人嗜铬粒蛋白A的N-末端的肽，其包含氨基酸47-55。最小抑菌浓度（MIC）试验表明，CGA-N9具有抗微生物活性，对热带念珠菌表现出时间依赖性抑制活性，在人红细胞（HRBC）和小鼠脑微血管内皮细胞中具有高安全性（bEnd. 3）。根据透射电子显微镜（TEM）、流式细胞术和共聚焦显微镜的结果，CGA-N9在细胞中积累而不破坏细胞膜的完整性;肽最初定位于细胞膜，随后内化到胞质溶胶中。对细胞内化机制的研究表明，即使在4摄氏度和叠氮化钠（NaN 3）存在的情况下，大多数CGA-N9分子也会进入酵母细胞，这两种情况都会阻断所有能量依赖性转运机制。此外，肽内化受到内吞抑制剂5-（N-乙基-N-异丙基）-阿米洛利（EIPA）、细胞松弛素D（CyD）和肝素的影响;氯丙嗪（CPZ）也对CGA-N9内化有一定影响。在MIC测定中获得了类似的结果，其中在EIPA、CyD、肝素或CPZ的存在下，CGA-N9的抗念珠菌活性被不同程度地阻断。因此，大多数CGA-N9通过C. Tropicalis细胞膜通过直接细胞渗透，而其余部分通过巨胞饮和硫酸蛋白聚糖介导的内吞作用进入，网格蛋白介导的内吞作用有轻微的贡献。
CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47-55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4 degrees C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.