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，与克林霉素联用是治疗由肺孢子虫引起的肺炎患者的最有效的临床替代疗法，但8-氨基喹啉对微生物和其他如疟原虫的作用机制尚不清楚。因此，我们以伯喹为固定化配体，利用亲和层析法从肺孢子虫中分离出与伯喹具有高亲和力的蛋白质。其中一个蛋白的初步鉴定表明它是一种中间丝蛋白，这可能与8-氨基喹啉类化合物对肺孢子虫细胞质的深刻破坏有关。为了确定伯喹结合蛋白在伯喹抗肺孢子虫作用机制中的作用，我们提出了以下具体目标。具体目标1：从肺孢子虫中分离和鉴定更多的伯喹结合蛋白。1A.伯喹亲和层析纯化肺孢子虫蛋白。1B.利用质谱仪对蛋白质的多肽片段进行测序。1C。使用生物信息学和质谱学数据搜索同源物。1D。克隆：推测为伯喹结合蛋白的全长基因产物。具体目的2：利用已有的蛋白质序列数据确定肺孢子虫中间丝蛋白(S)的编码序列。2A。根据序列比对、生物体内的密码子偏向和可用的肺孢子虫基因组数据库信息，通过简并的PCR/RACE进行克隆。2B。鉴定肺孢子虫RNA的全长转录本。2C。在大肠杆菌中表达和纯化全长重组蛋白。清楚了解8-氨基喹啉类药物对肺孢子虫的作用机制将有助于今后对疟原虫的类似研究。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全球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.