ISOLATION OF mRNA 5'-CAPPING ENZYME FROM THE PATHOGENIC FUNGUS CANDIDA ALBICANS AND SCREENING FOR SPECIFIC INHIBITORS OF THE ENZYME

从致病真菌白色念珠菌中分离 mRNA 5-加帽酶并筛选该酶的特异性抑制剂

• 批准号: 07670408
• 负责人: DOI Rikuo
• 金额: $ 1.47万
• 依托单位: YOKOHAMA CITY UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670408/
• 关键词: Candida albicans; mRNA 5' capping enzyme; mRNA 5' guanylyltransferase; mRNA 5' triphosphatase; beta-glucan; histidine kinase; 5'-トリフォスファターゼ

We isolated mRNA 5' capping enzyme gene from the pathogenic fungus Candida albicans. The enzyme which was expressed in E.coli and purified has an activity for mRNA 5' guanylyltransferase and was used for screening of the specific inhibitors. 2 candidates inhibitors were selected. We also isolated a human cDNA for mRNA 5' capping enzyme from HeLa cDNA library. This cDNA encoded both mRNA 5' triphosphatase at 5' half and mRNA 5' guanylyltransferase at 3' half unlike C.albicans mRNA 5' capping enzyme encodes only mRNA 5' guanylyltransferase. From these results, it was suggested that mRNA 5' capping enzyme of C.albicans has a different conformation from that of human despite the enzymes of C.albicans and human have the same activity, and mRNA 5' capping enzyme is a feasible target for drug discovery. X-ray crystallography is underway using purified enxyme from E.coli containing mRNA 5' guanylyltransferase of Schizosaccharomyces pombe.To search for more insight about C.albicans, we investigated the enzymes for beta-glucan synthesis. beta-glucan is a component of cell wall in yeast. We demonstrated that CaKRE6 gene encode beta-1,6-glucan (one of beta-glucan) synthesis, is essential for growth, and CaGSC1, CaGSL1, CaGSL2 gene products are needed for beta-1,3-glucan synthesis. Furthermore, CaSLN1 and CaNIK1 encode histidine kinase which was not found in higher eukaryotes and the both gene products are need for pathogenicity in C.albicans.

我们从病原真菌白色念珠菌中分离出 mRNA 5' 加帽酶基因。在大肠杆菌中表达并纯化的酶具有mRNA 5'鸟苷基转移酶活性，可用于筛选特异性抑制剂。选出2个候选抑制剂。我们还从 HeLa cDNA 文库中分离出了 mRNA 5' 加帽酶的人类 cDNA。该 cDNA 在 5' 半部编码 mRNA 5' 三磷酸酶，在 3' 半部编码 mRNA 5' 鸟苷基转移酶，与白色念珠菌 mRNA 5' 加帽酶仅编码 mRNA 5' 鸟苷基转移酶不同。这些结果表明，尽管白色念珠菌和人类的酶具有相同的活性，但白色念珠菌的mRNA 5'加帽酶具有与人类不同的构象，并且mRNA 5'加帽酶是药物发现的可行靶标。正在使用来自大肠杆菌的纯化酶进行 X 射线晶体学分析，该酶含有粟酒裂殖酵母 mRNA 5' 鸟苷基转移酶。为了寻找有关白色念珠菌的更多信息，我们研究了用于 β-葡聚糖合成的酶。 β-葡聚糖是酵母细胞壁的组成部分。我们证明CaKRE6基因编码β-1,6-葡聚糖（β-葡聚糖之一）的合成，对于生长至关重要，而CaGSC1、CaGSL1、CaGSL2基因产物是β-1,3-葡聚糖合成所必需的。此外，CaSLN1和CaNIK1编码在高等真核生物中未发现的组氨酸激酶，并且这两种基因产物是白色念珠菌致病性所必需的。

项目成果

Nagahashi et al: "Isolation of a functional homelog of Saccharomyces cerevisiae SLN1 from the pathogenic fungus candida albicans" Microbiology. 144. 425-432 (1998)

Nagahashi 等人：“从致病真菌白色念珠菌中分离酿酒酵母 SLN1 的功能同源物”微生物学。

Shigehisa Nagahashi, et.al: "Isolation of CaSLNI and CaNIKI,the genes for osmosensing histidine kinase homologues from the pathogenic fungus Candida albicans" Microbiology. 144. 425-432 (1998)

Shigehisa Nagahashi 等人：“CaSLNI 和 CaNIKI 的分离，这是来自致病真菌白色念珠菌的渗透传感组氨酸激酶同源物的基因”微生物学。

Tomio Yabe: "HKR1 encodes a cell surface protein that regulates both cell wall B-glucan synthesis and budding pattern in the yeast Saccharomyces cerevsue" Journal of Bacteridogy. 178. 477-483 (1996)

Tomio Yabe：“HKR1 编码一种细胞表面蛋白，可调节酿酒酵母的细胞壁 B-葡聚糖合成和出芽模式”《细菌学杂志》。

Yabe et al: "HKR1 encodes a cell surface protein that regulates both cell wall β-glucan synthesis and buciding pattern in yeast saccharomyces cerevisiae" Journal of Bacteriology. 178. 477-483 (1996)

Yabe 等人：“HKR1 编码一种细胞表面蛋白，可调节酿酒酵母细胞壁 β-葡聚糖的合成和发酵模式”《细菌学杂志》178. 477-483 (1996)。

MIO et al: "Cloning of CaGSC1, the Candida albicans homologue of FKS1, and its involvement in beta-1,3-glucan synthesis" Journal of Bacteriology. 179. 4096-4105 (1997)

MIO 等人：“CaGSC1（FKS1 的白色念珠菌同源物）的克隆及其参与 β-1,3-葡聚糖合成”《细菌学杂志》。