Role of Red Cell Membrane in Malaria Parasite Release

红细胞膜在疟疾寄生虫释放中的作用

• 批准号: 6607699
• 负责人: Manjit Hanspal
• 金额: $ 29.14万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607699
• 关键词: Plasmodium falciparum; SDS polyacrylamide gel electrophoresis; actin binding protein; ankyrins; chemical cleavage; cysteine endopeptidases; cytoskeletal proteins; erythrocyte membrane; gene targeting; laboratory mouse; life cycle; malaria; membrane proteins; molecular cloning; molecular site; parasitism; pathologic process; protease inhibitor; protein 4.1; protein biosynthesis; protein degradation; protein structure function; tissue /cell culture; western blottings

Plasmodium falciparum causes the most severe form of human malaria. During its 48-hour life cycle inside human red blood cells (RBCs) the parasite replicates into infective merozoites, which must then exit the host cell to invade new erythrocytes. Mounting evidence suggests that proteases are involved in host cell rupture and parasite release, although the molecular mechanisms underlying this process remain largely uncharacterized. Recently, we identified a novel P. falciparum cysteine protease named falcipain-2 (FP-2) that cleaves host erythrocyte membrane ankyrin, protein 4.1, adducin, and dematin with maximum proteolytic activity at late stages of parasite development. Based on our findings we propose that FP-2 mediates the cleavage of erythrocyte membrane skeletal proteins that are vital to the stability of red cell membrane, thus modulating the parasite release in vivo. The aim of this proposal is to define the functional role of FP-2 in vivo. Specifically, the following issues will be addressed: (1) What are the FP-2-mediated cleavage sites of erythrocyte ankyrin, adducin, and dematin? We have recently identified the site of protein 4.1 cleavage by FP-2, and have isolated an inhibitory peptide (Pi) that blocks all known functions of FP-2 in vitro. Similar strategy will be used to determine the precise sequence of the cleavage sites of ankyrin, adducin, and dematin. Also, we will determine the specificity of FP-2 by using a series of peptides derived from Pi in which critical residues are varied based on substrate specificities of other papain family enzymes. The potent peptide(s) will be selected to determine it's (their) effect on the intraerythrocytic growth and ultimate release of P. falciparum parasite. (2) Is parasite-derived cytosolic falcipain-2 accessible to the erythrocyte membrane skeleton in vivo? This will be achieved by immunodetection of FP-2 within parasite- infected erythrocytes, and in subcellular fractions, and by studying biosynthesis and maturation of FP-2. Furthermore, we will use a cell-permeable cysteine protease inhibitor in P. falciparum cultures as well as in the mouse in vivo model to elucidate the role of cysteine proteases in parasite release. (3) What is the role of falcipain-2 in th intraerythrocytic life cycle of P. falciparum? To directly confirm the function of FP-2 in vivo and to determine whether FP-2 alone is sufficient for parasite release, a model will be developed by disrupting the FP- 2 gene using gene targeting technology. Together, the proposed studies on FP-2 may begin to reveal molecular mechanisms underlying the release of malaria parasite from the host erythrocyte.

恶性疟原虫是最严重的人类疟疾。 在其48小时的生命周期中，寄生虫在人体红细胞（RBC）内复制成感染性裂殖子，然后必须离开宿主细胞侵入新的红细胞。 越来越多的证据表明，蛋白酶参与宿主细胞破裂和寄生虫释放，虽然这一过程的分子机制仍然在很大程度上没有特征。 最近，我们发现了一种新的恶性疟原虫半胱氨酸蛋白酶，命名为falcipain-2（FP-2），切割宿主红细胞膜锚蛋白，蛋白4.1，内收蛋白和dematin在寄生虫发育的后期具有最大的蛋白水解活性。 基于我们的研究结果，我们提出，FP-2介导的红细胞膜骨架蛋白的切割是至关重要的红细胞膜的稳定性，从而调节寄生虫在体内的释放。 该提案的目的是确定FP-2在体内的功能作用。 具体而言，将解决以下问题：（1）什么是FP-2介导的红细胞锚蛋白，内收蛋白和dematin切割位点？ 我们最近已经确定了FP-2切割蛋白4.1的位点，并分离出了一种抑制肽（Pi），它在体外阻断了FP-2的所有已知功能。 将使用类似的策略来确定锚蛋白、内收蛋白和底蛋白的切割位点的精确序列。 此外，我们将通过使用一系列衍生自Pi的肽来确定FP-2的特异性，其中关键残基基于其他木瓜蛋白酶家族酶的底物特异性而变化。 将选择有效肽以确定其对恶性疟原虫寄生虫的红细胞内生长和最终释放的影响。(2)寄生虫源性胞浆型falcipain-2在体内是否可进入红细胞膜骨架？ 这将通过免疫检测寄生虫感染的红细胞内和亚细胞级分中的FP-2以及通过研究FP-2的生物合成和成熟来实现。 此外，我们将在恶性疟原虫培养物和小鼠体内模型中使用细胞渗透性半胱氨酸蛋白酶抑制剂来阐明半胱氨酸蛋白酶在寄生虫释放中的作用。(3)falcipain-2在恶性疟原虫红细胞内生命周期中的作用是什么？ 为了直接确认FP-2在体内的功能并确定单独的FP-2是否足以释放寄生虫，将通过使用基因靶向技术破坏FP- 2基因来开发模型。 总之，对FP-2的拟议研究可能开始揭示疟原虫从宿主红细胞释放的分子机制。