Role of Red Cell Membrane in Malaria Parasite Release

恶性疟原虫是人类最严重的疟疾病原体。在人类红细胞(RBC)内48小时的生命周期中，寄生虫复制成具有感染性的裂殖子，然后裂殖子必须离开宿主细胞入侵新的红细胞。越来越多的证据表明，蛋白酶参与了宿主细胞的破裂和寄生虫的释放，尽管这一过程背后的分子机制在很大程度上仍不清楚。最近，我们发现了一种新的恶性疟原虫半胱氨酸水解酶，命名为FulciPain-2(FP-2)，它能裂解宿主红细胞膜上的骨架蛋白、4.1蛋白、内收蛋白和去甲素，在寄生虫发育的后期具有最高的蛋白分解活性。根据我们的发现，我们认为FP-2介导了对红细胞膜稳定性至关重要的红细胞膜骨架蛋白的裂解，从而调节体内寄生虫的释放。这项建议的目的是确定FP-2在体内的功能作用。具体地说，将解决以下问题：(1)FP-2介导的红细胞Anyrin、Adducin和Dematin的切割位点是什么？我们最近确定了蛋白质4.1被FP-2切割的位置，并在体外分离出了一个能阻断FP-2所有已知功能的抑制肽(PI)。类似的策略将被用来确定锚蛋白、内收蛋白和降钙素的切割位点的准确序列。此外，我们还将使用来自PI的一系列肽来确定FP-2的特异性，其中关键残基根据其他木瓜酶家族酶的底物特异性而变化。将选择有效的多肽(S)来确定其对恶性疟原虫红细胞内生长和最终释放的影响。(2)寄生虫来源的胞浆恶性疼痛-2能否在体内与红细胞膜骨架结合？这将通过对寄生虫感染的红细胞和亚细胞部分中FP-2的免疫检测，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)恶性疼痛-2在恶性疟原虫红细胞内生命周期中的作用是什么？为了直接证实FP-2在体内的功能，并确定FP-2本身是否足以释放寄生虫，将利用基因打靶技术破坏FP-2基因，建立一个模型。总之，对FP-2的拟议研究可能开始揭示宿主红细胞释放疟疾寄生虫的分子机制。

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.