IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。滴虫寄生虫阴道毛滴虫引起人类最常见的非病毒性性传播感染之一——滴虫病。通过细胞死亡检测ELISA（PLUS）和膜联蛋白v -荧光素异硫氰酸酯流式细胞术分析证实，通过亲和层析和Bio-Gel P-60柱层析分离得到的寄生虫和分泌的半胱氨酸蛋白酶（CP）片段可诱导人阴道上皮细胞（HVEC）凋亡。细胞凋亡的启动与蛋白酶活性相关，因为特异性CP抑制剂E-64抑制这两种活性。采用SDS-PAGE和质谱分析了阴道T.分泌的半胱氨酸蛋白酶（CP）在HVEC中诱导凋亡的作用。CP组分的SDS-PAGE分析显示出30 kDa左右的三重带，基质辅助激光解吸/电离飞行时间MS显示出分子质量23.6和23.8 kDa两个紧密相关的峰。蛋白质水解消化的CPs的质谱肽测序结果与先前报道的cDNA克隆CP2， CP3和CP4相匹配（Mallinson， D. J, Lockwood, b.c, Coombs, g.h, and North, m.j.（1994）微生物学140,2725-2735），以及与CP4高度同源的另一个序列（www.tigr.org）。后两种是CP组分中最丰富的成分。这些结果表明，cp诱导的程序性细胞死亡可能参与了体内阴道生殖道感染的发病机制，可能对治疗干预具有重要意义。研究结果最近发表在《感染》杂志上。Immun。[j] .生物工程学报（自然科学版），2004。化学。（280,23853 - 60,2005）

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The trichomonad parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, were shown to induce apoptosis in human vaginal epithelial cells (HVEC) , as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis was correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. The secreted cysteine protease (CP) fraction from T. vaginalis that was shown to induce apoptosis in HVEC was analyzed by SDS-PAGE and mass spectrometry. SDS-PAGE analysis of the CP fraction revealed triplet bands around 30 kDa, and matrix-assisted laser desorption/ionization time-of-flight MS indicated two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs resulted in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. These results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention. The results were recently published in Infect. Immun. (72, 4151-8, 2004 ) and J. Biol. Chem. (280, 23853-60, 2005)