PARASITE CYSTEINE PROTEASES AND HOST CELL APOPTOSIS

寄生虫半胱氨酸蛋白酶和宿主细胞凋亡

• 批准号: 6599464
• 负责人: BIBHUTI N. SINGH
• 金额: $ 38万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599464
• 关键词: SDS polyacrylamide gel electrophoresis; Trichomonas vaginalis; antibody; apoptosis; chromatography; communicable disease transmission; complementary DNA; cysteine endopeptidases; cytotoxicity; flow cytometry; host organism interaction; molecular cloning; parasite infection mechanism; polymerase chain reaction; recombinant proteins; sexually transmitted diseases; tissue /cell culture; virus infection mechanism; western blottings

DESCRIPTION (provided by applicant): Trichomonas vaginalis, a parasite, causes one of the most common non-viral sexually transmitted disease in humans, trichomoniasis. Trichomoniasis is a serious disease that causes vaginitis in women and urethritis in men. It has been linked to infertility, preterm delivery, low birth weight infants and cervical cancer. T. vaginalis may be emerging as one of the most important cofactors in amplifying HIV transmission, especially in African-American communities of the United States. The long-term goal of this proposal is to elucidate the mechanisms of T. vaginalis pathobiology at the molecular and cellular levels. We have developed an in vitro culture system of human vaginal epithelial cells (HVECs) to study parasite-host cell interaction. T. vaginalis infection of host cells in vitro induces cell destruction. The soluble fraction (SF) elaborated (secreted/released) by the parasites induces apoptosis in the HVECs in a species-specific manner. The partially purified active agent(s) from T. vaginalis-SF contains a cysteine protease(s) that appears to be responsible for inducing apoptosis in HVECs. The specific aims are: 1) to purify and characterize the protease(s) that induce apoptosis; including, cloning, sequencing, expression of recombinant protein and express mutants of the active molecule(s); 2) study the physiology/cell biology of the parasite molecules with respect to biosynthesis, secretion, and activation of the apoptotic protease(s) by T. vaginalis; and 3) identify the initial events leading to HVEC apoptosis triggered by the parasite molecule (s), including cell surface death receptors. The active toxic agents will be purified by a series of chromatographic procedures. Each step will be monitored using both cytotoxicity and protease assays. The purified material will be subjected to proteolytic digestion and isolated peptides will be sequenced. Sequence information will be used to clone the cDNA. The cDNA will then be used to produce recombinant protein, which will be tested in functional assays to demonstrate that the cloned protein induces HVEC apoptosis. Antibodies (Abs) will be generated against active protease(s) to study the optimal release of cytotoxic agent under a variety of conditions and to show if it is synthesized in a soluble form or associated with membranes. Antagonist Abs against death receptors will be utilized with the purified agent to elucidate which of the receptors is involved in triggering apoptotic pathways. Activation of caspases-8, -9, and -3 will be monitored by Western blot/flow cytometry analyses using specific Abs and quantified using specific peptide substrates. Knowledge of the molecular understanding of host cell pathology induced by parasite will reveal new strategies/effective treatment for trichomoniasis.

描述（由申请人提供）：阴道毛滴虫是一种寄生虫，会引起人类最常见的非病毒性传播疾病之一——滴虫病。滴虫病是一种严重的疾病，会导致女性阴道炎和男性尿道炎。它与不孕症、早产、低出生体重儿和宫颈癌有关。阴道毛滴虫可能正在成为扩大艾滋病毒传播的最重要的辅助因素之一，尤其是在美国的非裔美国人社区。该提案的长期目标是在分子和细胞水平上阐明阴道毛滴虫的病理生物学机制。我们开发了人阴道上皮细胞（HVEC）的体外培养系统来研究寄生虫与宿主细胞的相互作用。阴道毛滴虫在体外感染宿主细胞会诱导细胞破坏。寄生虫精心制作（分泌/释放）的可溶性部分（SF）以物种特异性方式诱导 HVEC 细胞凋亡。来自阴道毛滴虫-SF的部分纯化的活性剂含有半胱氨酸蛋白酶，其似乎负责诱导HVEC细胞凋亡。具体目标是：1）纯化和表征诱导细胞凋亡的蛋白酶；包括重组蛋白的克隆、测序、表达以及活性分子的表达突变体； 2) 研究寄生虫分子的生理学/细胞生物学，涉及阴道毛滴虫凋亡蛋白酶的生物合成、分泌和激活； 3) 确定由寄生虫分子（包括细胞表面死亡受体）触发的导致 HVEC 凋亡的初始事件。活性有毒物质将通过一系列色谱程序进行纯化。将使用细胞毒性和蛋白酶测定来监测每个步骤。纯化的材料将进行蛋白水解消化，并对分离的肽进行测序。序列信息将用于克隆 cDNA。然后，该 cDNA 将用于生产重组蛋白，并在功能测定中对其进行测试，以证明克隆的蛋白可诱导 HVEC 凋亡。将产生针对活性蛋白酶的抗体 (Ab)，以研究细胞毒性剂在各种条件下的最佳释放，并显示其是否以可溶形式合成或与膜结合。针对死亡受体的拮抗剂抗体将与纯化的试剂一起使用，以阐明哪些受体参与触发细胞凋亡途径。 caspase-8、-9 和 -3 的激活将通过使用特定 Ab 的蛋白质印迹/流式细胞术分析进行监测，并使用特定的肽底物进行定量。对寄生虫引起的宿主细胞病理学的分子理解将揭示滴虫病的新策略/有效治疗。