Primaquine-binding proteins identified in Pneumocystis

描述（由申请人提供）：伯氨喹是一种8-氨基喹啉，与克林霉素联合使用是肺囊虫引起的肺炎患者最有效的临床替代标准治疗方案，但8-氨基喹啉对机体和其他如疟原虫的作用机制尚不清楚。因此，我们以伯氨喹为固定配体，采用亲和层析法分离了对伯氨喹具有高亲和性的肺孢子虫蛋白。其中一种蛋白的初步鉴定表明，它是一种中间丝蛋白，可能与8-氨基喹啉在肺囊虫中产生的深刻的细胞质破坏有关。为了确定伯氨喹结合蛋白在伯氨喹抗肺囊虫作用机制中的作用，我们提出以下具体目标。特异性目标1：从肺囊虫中分离并鉴定额外的伯氨喹结合蛋白。用伯氨喹亲和层析纯化肺囊虫蛋白。1 b。用质谱法对蛋白质的肽片段进行测序。1 c。使用生物信息学和质谱数据搜索同源物。1 d。克隆：假定的伯氨喹结合蛋白的全长基因产物。具体目标2：利用现有的蛋白质序列数据鉴定肺囊虫中间丝蛋白的编码序列。2 a。通过退化PCR/RACE克隆基于比对，密码子偏倚的生物体，和可用的肺囊虫基因组数据库信息。2 b。鉴定肺囊虫RNA的全长转录本。2 c。表达和纯化大肠杆菌全长重组蛋白。清楚了解8-氨基喹啉类药物对肺囊虫的作用机制将为今后对疟原虫的类似研究提供信息。因此，该项目可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性疾病的治疗产生重大影响。

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.