IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

鉴别

基本信息

批准号：
7955896
负责人：
BIBHUTI N. SINGH
金额：
$ 0.25万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7955896
关键词：
AIDS prevention Affinity Chromatography Apoptosis Biological Assay Biology Cell Death Column Chromatography Complementary DNA Computer Retrieval of Information on Scientific Projects Database Coombs&apos Test Cysteine Protease Cysteine Proteinase Inhibitors Detection Epithelial Cells Flow Cytometry Fluorescein Fluoresceins Funding Gel Glycoconjugates Grant Human Immunity Infection Institution Isothiocyanates Journals MALDI-TOF Mass Spectrometry Manuscripts Mass Spectrum Analysis Medicine Microbiology Parasites Pathogenesis Peptide Hydrolases Publications Publishing Reporting Research Research Personnel Resources Sexually Transmitted Diseases Source Therapeutic Intervention Trichomonas Infections Trichomonas vaginalis Triplet Multiple Birth United States National Institutes of Health Vagina Viral annexin A5 in vivo molecular mass protein aminoacid sequence

项目摘要

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 and prevention of AIDS. The results have been published in Infect. Immun. (72, 4151-8, 2004 ) and J. Biol. Chem. (280, 23853-60, 2005). A related manuscripts was published in Infection and Immunity and another has been submitted for publication in Glycoconjugate Journal.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它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传播感染之一——滴虫病。细胞死亡检测表明，该寄生虫以及通过亲和层析和 Bio-Gel P-60 柱层析分离的分泌型半胱氨酸蛋白酶 (CP) 组分可诱导人阴道上皮细胞 (HVEC) 凋亡ELISA(PLUS) 测定和膜联蛋白 V-异硫氰酸荧光素流式细胞术分析。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。通过 SDS-PAGE 和质谱法分析阴道毛滴虫分泌的半胱氨酸蛋白酶 (CP) 组分，该组分可诱导 HVEC 细胞凋亡。 CP 级分的 SDS-PAGE 分析显示了 30 kDa 左右的三重带，基质辅助激光解吸/电离飞行时间 MS 表明两个密切相关的分子量峰，分别为 23.6 和 23.8 kDa。对蛋白水解消化的 CP 进行质谱肽测序，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. 和 North, M. J. (1994) Microbiology 140, 2725-2735 ），以及另一个与CP4高度同源的序列（www.tigr.org）。最后两个物种是 CP 部分中最丰富的成分。这些结果表明CP诱导的程序性细胞死亡可能参与体内阴道毛滴虫感染的发病机制，可能对艾滋病的治疗干预和预防具有重要意义。研究结果发表在《Infect》杂志上。免疫。 (72, 4151-8, 2004) 和 J. Biol。化学。 (280, 23853-60, 2005)。相关手稿发表在 Infection and Immunity 上，另一篇已提交 Glycoconjugate Journal 上发表。