动物源抗真菌肽CGA-N46降低真菌线粒体膜电位分子机制研究

In the light of the drug resistance of fungi, the investigations of the molecular action mechanism of novel antifungal peptides are very important for the development of the antifungal drugs. CGA-N46, an innate derived peptide from chromogranin A, is an antifungal peptide found by our research group. From the results of the recently accomplished project "The action mechanism of the innate antifungal peptide CGA-N46" supported by the National Natural Science Foundation of China (NNSFC), our research group found that CGA-N46 could reduce the mitochondrial membrane potential of Candida krusei, but had no effect on the permeability of the cell membrane. A conclusion could be made that the antifungal action mechanism of CGA-N46 was specific and novel. Based on the results, the molecular action mechanism of CGA-N46 on the decrease of fungal mitochondrial membrane potential need to be further investigated. In the present project, The anisotropy changes of hematoporphyrin will be researched to reveal the effects of the binding of CGA-N46 with the mitochondrial membrane proteins on the mitochondrial fluidity. TEM will be used to study the osmotic volume changes of mitochondria to clarify the effects of CGA-N46 on the opening of mitochondrial permeability transition pore (mPTP). And fluorescent spectroscopy, atomic force microscope and circular dichroism will be used to research the effect of the structure of CGA-N46 on the permeability of mitochondria. In the end, the molecular action mechanism of CGA-N46 on the decrease of yeast mitochondrial membrane potential will be brought to life from the three aspects listed as above. The research of the project will support theoretical bases for the development of CGA-N46 to be a novel clinical candidate to cure fungal infections.

针对病原真菌耐药性问题，研究新型抗真菌肽作用机制对新药开发具有重要意义。嗜铬粒蛋白A（CGA）N-端衍生肽CGA-N46是申请人发现的具有较高抗真菌活性的动物源抗菌肽。最近结题的国家自然科学基金项目“动物内源性多肽CGA-N46抗真菌作用机制研究”，发现CGA-N46能够降低克柔念珠菌线粒体膜电位，但对细胞膜没有影响，推测其作用位点新颖。抗菌肽降低线粒体膜电位分子机制迄今未见报道。该项目采用血卟啉荧光各向异性研究CGA-N46与线粒体膜蛋白结合对线粒体膜流动性的影响；采用透射电镜研究CGA-N46对线粒体通透性转换孔（mPTP）打开程度的影响；采用荧光光谱法、原子力显微镜和圆二色谱技术研究CGA-N46构型对线粒体膜通透性的影响，从线粒体膜流动性、mPTP打开程度和CGA-N46构型三方面阐明CGA-N46降低真菌线粒体膜电位的分子机制，为将CGA-N46开发成新型抗真菌药物奠定理论基础。

嗜铬粒蛋白A N端衍生肽CGA-N46是项目负责人发现的具有抗真菌活性的动物源抗菌肽。在前期主持完成的国家自然科学基金面上项目“动物内源性多肽CGA-N46抗真菌作用机制研究”中，发现CGA-N46能够降低念珠菌线粒体膜电位，但其分子机制未见报道。在国家自然科学基金资助下，通过筛选获得了生物活性高、安全性好的CGA-N46衍生肽CGA-N12和CGA-N9。本研究采用荧光光谱、原子力显微镜、透射电镜、激光共聚焦显微镜等生物物理学和生物化学的技术与方法，系统研究了抗菌肽CGA-N46及其衍生肽CGA-N12和CGA-N9降低真菌线粒体膜电位分子机制。研究发现：（1）CGA-N46及其衍生肽CGA-N12和CGA-N9是非破膜性抗菌肽。该抗菌肽通过不依赖能量的直接细胞膜渗透和依赖能量的巨胞饮作用、硫酸蛋白聚糖受体介导的内吞作用、网格蛋白介导的内吞作用跨膜，其内化过程不破坏细胞膜完整性；（2）CGA-N46及其衍生肽CGA-N12和CGA-N9，通过提高膜渗透性，降低线粒体膜电位。该抗菌肽能够提高磷脂膜流动性，诱导膜形成非选择性离子通道，提高膜渗透性，造成离子渗漏，线粒体膜电位降低。（3）CGA-N46衍生肽CGA-N12作用于组成线粒体膜通透性转换孔（mPTP）的蛋白质，使mPTP不可逆打开，降低线粒体膜电位。CGA-N12通过影响mPTP蛋白复合物中ANT蛋白m-态的稳定构象、维持mPTP蛋白复合体内膜线粒体膜间隙侧硫位点的二硫键结构、氧化mPTP的游离巯基，使mPTP打开，提高线粒体膜通透性，引起线粒体膜质子梯度消失，线粒体膜电位降低；（4）CGA-N46衍生肽CGA-N12影响ATP合成代谢途径中的酶活性，降低线粒体ATP水平，促进mPTP打开，引起线粒体膜电位降低。CGA-N12提高念珠菌线粒体膜上的ATPase水解活性，降低线粒体脱氢酶活性，使ATP耗竭，进一步促进线粒体通透性转换孔打开，线粒体膜电位降低。（5）CGA-N46衍生肽CGA-N12和CGA-N9降低念珠菌线粒体膜电位是一个多靶点、多方式的作用过程。（6）CGA-N46衍生肽CGA-N12和CGA-N9能够诱导念珠菌细胞凋亡，但不引起正常哺乳动物细胞凋亡。研究成果为将CGA-N46、CGA-N12、CGA-N9开发成新型抗真菌药物奠定理论基础，进一步完善和丰富了抗菌肽作用机制理论体系。