隐球菌漆酶的晶体结构及活性的研究

The basidiomycetous yeast Cryptococcus is an important human pathogen, which has been paid close attention to its pathogenesis, especially the important virulence factor-melanin it produced. Our early clinical and basic research, with recent studies worldwide show that, the pathogenicity of melanin is closely related to certain fungi, including cryptococcus, dark fungi, Aspergillus or Candida albicans. Laccase encoded by LAC1 is the main kinase that catalyze a broad range of substrates to melanin in cryptococcus and other fungi, however, the mechanism of fungi albino mutation caused by the abnormal expression of LAC1 gene is still not clear. Recently, we have demonstrated the importance of melanin induced by phenoloxidase in the resistance to microbial killing mechanisms, found the activation pathway of phenoloxidases and confirmed its structure can affect the activity strongly through the study of insect recombinant PPOs. So we plan to investigate the crystal structures of the laccase from the fungus Cryptococcus neoformans through X-ray crystal diffractometry and photoaffinity labeling technique, get point mutations on important loci, verify vitro expression activity, compare with the other strains of the copper-3 protease, design and choose suitable laccase inhibitors according to the protein structure, then verify the activity and pathogenic changes in vitro and vivo. We will clarify the pathogenic mechanisms of laccase in Cryptococcus and even other fungi which produce melanin, and provide a theoretical basis for searching possible new targets of antifungal agents.

隐球菌是人类重要致病真菌，致病机制备受关注，尤其针对其产黑素这一重要毒力因子。本课题组前期临床及基础研究及国内外研究表明，真菌产黑素的致病性与隐球菌、暗色真菌、曲霉甚至白念珠菌等都密切相关。由LAC1基因编码的漆酶是隐球菌等真菌细胞内酶促产生黑素的主要催化酶类，然而LAC1基因表达异常导致真菌白化突变的机制始终未有明确的阐释。近期我们合作研究小组通过对模式昆虫的研究，佐证了和真菌漆酶很相近的酚氧化酶诱导的产黑素在抵抗微生物杀伤机制中的重要性，证实其结构可强烈影响其活性大小。因此，我们拟经X-射线晶体衍射学和光亲和标记技术等，研究解析漆酶的晶体结构，对重要的位点进行点突变，体外表达活性验证，与其他已知菌种漆酶结构及copper-3蛋白酶比较，据蛋白结构筛选漆酶的抑制剂，再验证体内外活性及致病性变化。以期阐明漆酶在隐球菌等产黑素真菌致病中的作用机制，并为寻找可能的抗真菌药物新靶点提供理论依据。

隐球菌是人类重要致病真菌，致病机制备受关注，尤其针对其产黑素这一重要毒力因子。本课题组前期临床及基础研究及国内外研究表明，真菌产黑素的致病性与隐球菌、暗色真菌、曲霉甚至白念珠菌等都密切相关。由LAC1基因编码的漆酶是隐球菌等真菌细胞内酶促产生黑素的主要催化酶类，然而LAC1基因表达异常导致真菌白化突变的机制始终未有明确的阐释。近期我们研究小组成员通过对模式昆虫的研究，佐证了酚氧化酶诱导的产黑素在抵抗微生物杀伤机制中的重要性，发现了其活化途径，证实其结构可强烈影响其活性大小。因此，我们拟经X-射线晶体衍射学和光亲和标记技术等，研究解析漆酶的晶体结构，对重要的位点进行点突变，体外表达活性验证，与其他已知菌种漆酶结构的copper-3蛋白酶比较，据蛋白结构筛选漆酶的抑制剂，再验证体内外活性及致病性变化。我们发现新生隐球菌漆酶是一含铜的催化酶，含624个氨基酸，14个内含子。主要由Lac1和Lac2编码，黑素主要由Lac1催化产生，同源蛋白主要在铜结合域具有同源性。我们用大肠杆菌成功重组表达了漆酶蛋白，发现其不可溶，通过变复性进行纯化得到纯度较高的蛋白，并进行结晶及结构解析，工作尚在进行中。以期阐明漆酶在隐球菌等产黑素真菌致病中的作用机制，并为寻找可能的抗真菌药物新靶点提供理论依据。