Primaquine-binding proteins identified in Pneumocystis

在肺孢子虫中鉴定出伯氨喹结合蛋白

• 批准号: 6947493
• 负责人: Sherry F Queener
• 金额: $ 22.73万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6947493
• 关键词: Escherichia coli; Pneumocystis; RNA; affinity chromatography; binding proteins; bioinformatics; fungal genetics; fungal proteins; intermediate filaments; laboratory rat; mass spectrometry; molecular biology information system; molecular cloning; pharmacokinetics; primaquine; protein purification; protein sequence; recombinant proteins

DESCRIPTION (provided by applicant): Primaquine, an 8-aminoquinoline, with clindamycin is the most effective clinical alternative to standard therapy for patients with pneumonia caused by Pneumocystis, but the mechanism of action of 8-aminoquinolines against the organism and against others such as Plasmodium remains unknown. Therefore, using primaquine as the immobilized ligand we have used affinity chromatography to isolate proteins from Pneumocystis with high affinity for primaquine. Preliminary identification of one of these proteins suggests it is an intermediate filament protein, which may relate to the profound cytoplasmic disruption produced in Pneumocystis by 8-aminoquinolines. To establish the role of primaquine binding proteins in the mechanism of action of primaquine against Pneumocystis, we propose the following specific aims. Specific Aim 1: Isolate and identify additional primaquine binding proteins from Pneumocystis. 1a. Purify proteins from Pneumocystis using primaquine affinity chromatography. 1b. Sequence peptide fragments of the proteins using mass spectrometry. 1c. Use bioinformatics and mass spectrometry data to search for homologs. 1d. Clone: full-length gene product of putative primaquine-binding proteins. Specific Aim 2: Use existing protein sequence data to identify the coding sequences for Pneumocystis intermediate filament protein(s). 2a. Clone by degenerate PCR/RACE based on alignments, codon bias in the organism, and available Pneumocystis genome database information. 2b. Identify the full length transcript from Pneumocystis RNA. 2c. Express and purify full length recombinant proteins from Escherichia coli. Achieving a clear understanding of the mechanism of action of 8-aminoquinolines toward Pneumocystis will inform similar studies in the future with Plasmodium. Thus, this project could have significant impact upon therapy of both an opportunistic infection affecting thousands of patients and a world-wide disease that affects millions of persons each year.

说明（由申请人提供）：伯氨喹（一种 8-氨基喹啉）与克林霉素联合使用，是肺孢子虫引起的肺炎患者标准治疗的最有效的临床替代方案，但 8-氨基喹啉针对该生物体以及其他生物体（如疟原虫）的作用机制仍不清楚。因此，使用伯氨喹作为固定配体，我们使用亲和层析从肺孢子虫中分离出对伯氨喹具有高亲和力的蛋白质。对这些蛋白质之一的初步鉴定表明它是一种中间丝蛋白，这可能与 8-氨基喹啉在肺孢子虫中产生的深度细胞质破坏有关。为了确定伯氨喹结合蛋白在伯氨喹抗肺孢子虫作用机制中的作用，我们提出以下具体目标。具体目标 1：从肺孢子虫中分离并鉴定其他伯氨喹结合蛋白。 1a.使用伯氨喹亲和层析纯化肺孢子虫中的蛋白质。 1b.使用质谱法对蛋白质的肽片段进行测序。 1c.使用生物信息学和质谱数据来搜索同系物。 1d。克隆：假定的伯氨喹结合蛋白的全长基因产物。具体目标 2：使用现有的蛋白质序列数据来鉴定肺孢子虫中间丝蛋白的编码序列。 2a.根据生物体中的比对、密码子偏差和可用的肺孢子虫基因组数据库信息，通过简并 PCR/RACE 进行克隆。 2b.鉴定肺孢子虫 RNA 的全长转录本。 2c.从大肠杆菌中表达并纯化全长重组蛋白。清楚地了解 8-氨基喹啉对肺孢子虫的作用机制将为今后针对疟原虫的类似研究提供信息。因此，该项目可能对影响数千名患者的机会性感染和每年影响数百万人的世界范围疾病的治疗产生重大影响。