CHARACTERIZATION OF TRYPANOSOMA BRUCEI GP63 PROTEIN

布氏锥虫 GP63 蛋白的表征

• 批准号: 6446633
• 负责人: Douglas J. LaCount
• 金额: $ 2.26万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6446633
• 关键词: SDS polyacrylamide gel electrophoresis; Trypanosoma brucei rhodesiense; affinity chromatography; complement pathway; confocal scanning microscopy; cytolysis; enzyme activity; epitope mapping; fluorescence microscopy; fungal proteins; gene complementation; gene expression; genetic regulation; host organism interaction; immunoprecipitation; intracellular parasitism; laboratory mouse; laboratory rabbit; metalloendopeptidases; molecular cloning; northern blottings; protein structure function; western blottings

Trypanosoma brucei, the causative agent of African trypanosomiasis, multiplies extracellularly in the bloodstream of its mammalian hosts but is resistant to complement mediated lysis (CML). The objective of this proposed research is to determine the role of T. brucei GP63 (Tb-GP63) genes in resistance to CML. Tb-GP63 is a homolog of leishmania GP63 (L- GP63), a cell surface zinc metalloprotease that is critical for the resistance of leishmania to CML. Two groups of Tb-GP63 genes have been identified A and B. which are approximately 40 % identical and 60 % similar to L- GP63 and to each other. All disulfide bonded cysteines in L-GP63 and nine prolines are conserved in Tb-GP63-A and -B, suggesting that the Tb-GP63 proteins will be structurally similar to L-GP63. To study the role of Tb- GP63 in the resistance of T. brucei to CML Tb-GP63 will expressed in a baculovirus expression system and purified antibodies will be generated against Th-GP63 and cell lines that over-express or that lack Tb-GP63 will be created. With these reagents. the following questions will be addressed: (i) is Tb-Gp63-A is a functional cell surface protease? (ii) is Tb-GP63-B also a functional GP63 homolog? (iii) does Tb-GP63 increase the resistance cells to CML? (iv) is Tb-GP63 is required for growth of T. brucei in mice? and (v) do the Tb-GP63 gene 3' UTRs regulate stage specific expression of Tb-GP63? These studies may identify a new target for anti-trypanosome drugs and will provide insight into the molecular basis for the success of T. brucei as a parasite.

非洲锥虫病的致病药物锥虫锥虫锥虫在其哺乳动物宿主的血液中细胞外繁殖，但对补体介导的裂解（CML）具有抗药性。这项拟议的研究的目的是确定Brucei GP63（TB-GP63）基因在对CML抗性中的作用。 TB-GP63是Leishmania GP63（L-GP63）的同源物，这是一种细胞表面金属蛋白酶，对利什曼尼亚对CML的抗性至关重要。已经鉴定出两组TB-GP63基因A和B。它们相同40％，60％与L-GP63相似。 L-gp63中的所有二硫键键合结合半胱氨酸均在TB-GP63-A和-B中保守，这表明TB-GP63蛋白在结构上与L-GP63的结构相似。为了研究TB-GP63在杆状病毒表达系统中表达的T. Brucei对CML TB-GP63的耐药性，将产生针对TH-GP63的纯化抗体，并且将产生过表达或缺乏TB-GP63的细胞系。使用这些试剂。将解决以下问题：（i）TB-GP63-A是否是功能性细胞表面蛋白酶？ （ii）TB-GP63-B是否也是功能性GP63同源物？ （iii）TB-GP63是否会增加电阻细胞对CML？ （iv）小鼠中T. brucei生长是否需要TB-GP63？ （v）TB-GP63基因3'UTR是否调节了TB-GP63的阶段特异性表达？这些研究可能会确定抗肌体药物的新靶标，并将为布鲁氏菌作为寄生虫成功的分子基础提供洞察力。

项目成果

RNA interference in African trypanosomes.

• DOI: 10.1078/1434-4610-00047
• 发表时间: 2001-07
• 期刊: Protist
• 影响因子: 2.5
• 作者: D. LaCount;J. Donelson