IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

鉴别

• 批准号: 7722976
• 负责人: BIBHUTI N. SINGH
• 金额: $ 0.58万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722976
• 关键词: Affinity Chromatography; Apoptosis; Biological Assay; Cell Death; Column Chromatography; Complementary DNA; Computer Retrieval of Information on Scientific Projects Database; Coombs' Test; Cysteine Protease; Cysteine Proteinase Inhibitors; Detection; Endopeptidases; Epithelial Cells; Flow Cytometry; Fluorescein; Fluoresceins; Funding; Gel; Grant; Human; Immunity; Infection; Institution; Isothiocyanates; MALDI-TOF Mass Spectrometry; Manuscripts; Mass Spectrum Analysis; Microbiology; Parasites; Pathogenesis; Peptide Hydrolases; 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

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). A related manuscript has recently been published in Infection and Immunity.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 Trichomonad寄生虫阴道阴道引起人类中最常见的非病毒性传播感染之一。寄生虫以及分泌的半胱氨酸蛋白酶（CP）通过亲和力色谱分离，然后是生物凝胶P-60色谱柱色谱法分离出来，可在人类阴道上皮细胞（HVEC）中诱导凋亡，如细胞死亡检测检测ELISA（Plul）ELISA（Plul）Assay v-Fluyanter cyeration cyeration cy cy cy cy cy cy cy cyeration。凋亡的开始与蛋白酶活性相关，因为特定的CP抑制剂E-64抑制了这两种活性。通过SDS-PAGE和质谱法分析了来自阴道的分泌的半胱氨酸蛋白酶（CP）馏分，该阴道被证明在HVEC中诱导了凋亡。 CP馏分的SDS-PAGE分析显示，三重态带约为30 kDa，并且矩阵辅助激光解吸/电离飞行时间MS表示分子质量23.6和23.8 kDa的两个密切相关的峰。蛋白水解消化CP的质谱肽测序导致与先前报道的cDNA克隆，CP2，CP3和CP4（Mallinson，D。J. （www.tigr.org）。最后两个物种是CP部分中最丰富的组成部分。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感染的发病机理，可能对治疗干预具有重要意义。结果最近发表在感染中。免疫。 （72，4151-8，2004）和J. Biol。化学（280，23853-60，2005）。最近在感染和免疫力方面发表了一份相关的手稿。