The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

疟原虫表面阴离子通道与疟原虫营养获取

基本信息

批准号：
8946347
负责人：
SANJAY A DESAI
金额：
$ 104.52万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8946347
关键词：
Accounting Affect Animal Model Anions Antimalarials Basic Science Binding Biological Assay Biology Calcium Cations Cell physiology Cell surface Cells Cellular biology Characteristics Chemicals Cleaved cell DNA Dehydration Divalent Cations Dyes Enzymes Erythrocyte Membrane Erythrocytes Exclusion Extracellular Domain Future Gene Family Gene Silencing Genes Genetic Genetic Crosses Genotype Goals Growth Human Infection Informatics International Ion Channel Ion Transport Ions Kinetics Lipid Biochemistry Malaria Mediating Membrane Modeling Molecular Molecular Genetics Molecular and Cellular Biology Multigene Family Nutrient Organism Parasites Pathway interactions Peptide Hydrolases Permeability Pharmacology Physiological Physiology Plasma Plasmodium falciparum Protein Biochemistry Proteins Proteolysis Reporting Research Role Site-Directed Mutagenesis Specificity Structure Structure-Activity Relationship Surface Transfection Transmembrane Transport Variant Work base biophysical properties calcium indicator chymotrypsin drug development drug discovery extracellular gene cloning guanidinium high throughput screening inhibitor/antagonist insight novel pathogen protein function research study small molecule libraries sodium ion solute uptake

项目摘要

In 2014, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis and physiological role of increased erythrocyte permeability after infection with malaria parasites. In one study, we examined how an unusual parasite multigene family contributes to formation of nutrient channels at the host erythrocyte membrane. Previous studies implicated the plasmodial surface anion channel (PSAC) and the clag multigene family in the increased uptake of nutrients and monovalent ions at the erythrocyte surface. An important question in the field at present is how CLAG proteins contribute to channel activity. In one model, these proteins function as enzymes that activate quiescent channels already present on the host cell surface; in another, the CLAG proteins contribute directly to formation of the channel, either in isolation or through interactions with unrelated proteins. This uncertainly is especially important because the clag genes do not have detectable homology to known ion channels in other organisms. In this reporting year, we used proteases to examine the channel's composition. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3xDd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings suggest that surface-exposed CLAG3 contributes directly to channel function; they also provide early structural insights into the PSAC pore. PLoS ONE 9: e93759 (2014). In a second study, we examined the unusual ability of PSAC to identify and distinguish solutes for uptake. This question is important because there are many nutrients and antimalarial drugs that enter infected erythrocytes primarily via PSAC. Despite the broad range of permeant solutes, the channel stringently excludes sodium ions; this exclusion is essential for survival of the intracellular parasite in host plasma. Here, we explored mechanisms for this remarkable solute selectivity and identified guanidinium as an organic cation with high permeability into erythrocytes infected with malaria parasites, but negligible uptake by uninfected cells. Transport characteristics and pharmacology indicated that this uptake is specifically mediated by PSAC. We also examined organic and inorganic cation permeabilities and proposed that cation dehydration is the rate-limiting step in transport through the channel. The high guanidinium permeability of infected cells also allows rapid and stringent synchronization of parasite cultures, as required for molecular and cellular studies of this pathogen. This study provides a framework for nutrient and ion permeation through PSAC. Understanding the structural and molecular basis of permeation is critical to knowing the channel's role in host-parasite interactions and to developing inhibitors that may be future antimalarial drugs. BioMed Research International, in press (2014). In a third study, we examined the increased permeability of infected cells to calcium, an essential divalent cation. We used nondestructive loading of a fluorescent calcium indicator dye (Fluo-8) into human erythrocytes to quantify Ca++ uptake kinetics. Our studies revealed that infection with malaria parasites produces marked increases in erythrocyte Ca++ permeability. Pharmacological studies revealed that this uptake is not mediated by PSAC or by typical mammalian Ca++ channels. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++. These findings suggest a novel pathway for Ca++ uptake after infection. Inhibitors of this pathway may be excellent starting points for antimalarial drug development. Malaria J. 13:184 (2014).

2014年，Apicomplexan分子生理部门继续研究分子基础和生理作用，在感染疟原虫寄生虫后增长的红细胞渗透性的生理作用。在一项研究中，我们研究了一个不寻常的寄生虫多基因家族如何在宿主红细胞膜上形成营养通道。先前的研究暗示了质质表面阴离子通道（PSAC）和扎拉克多基因家族在养分增加和红细胞表面的单价离子的摄取增加。目前，该领域的一个重要问题是粘液蛋白如何促进通道活性。在一个模型中，这些蛋白质是激活宿主细胞表面上已经存在的静态通道的酶。在另一种中，粘液蛋白直接在隔离或与无关蛋白的相互作用的情况下直接促进了通道的形成。这是不确定的尤其重要的，因为锁骨基因与其他生物体中已知的离子通道没有可检测的同源性。在这个报告年度，我们使用蛋白酶来检查频道的组成。尽管具有不同特异性的蛋白酶都在clag3的细胞外域内裂解，但它们产生了不同程度的转运抑制。胰凝乳蛋白酶诱导的抑制取决于寄生虫基因型，其通道受HB3寄生虫诱导的抑制作用，比DD2克隆的降低程度更大。 HB3XD2遗传交叉，DNA转染和基因沉默实验中功能性蛋白水解的遗传均指向CLAG3基因，为这些基因的作用提供了独立的证据。具有DD2特异性抑制剂和定点诱变的蛋白酶保护测定法表明，clag3细胞外环上的变体L1115F残基有助于抑制剂的结合，并说明功能性蛋白水解的差异。这些发现表明，表面暴露的锁骨3直接有助于通道功能。它们还为PSAC孔提供了早期的结构见解。 PLOS ONE 9：E93759（2014）。在第二项研究中，我们研究了PSAC识别和区分吸收溶质的异常能力。这个问题很重要，因为有许多营养和抗疟药主要通过PSAC进入感染的红细胞。尽管有广泛的渗透溶质，但该通道还是严格排除了钠离子。该排除对于宿主血浆中细胞内寄生虫的存活至关重要。在这里，我们探索了这种非凡的溶质选择性的机制，并确定鸟嘌呤是一种有机阳离子，具有很高的渗透性对感染疟原虫寄生虫感染的红细胞，但未感染的细胞可以忽略不足。运输特征和药理学表明，这种摄取是由PSAC专门介导的。我们还检查了有机和无机阳离子的渗透率，并提出阳离子脱水是通过通道运输的速率限制步骤。受感染细胞的高鸟巢渗透性还允许寄生虫培养物的快速和严格同步，这是该病原体的分子和细胞研究所必需的。这项研究为通过PSAC提供了营养和离子渗透的框架。了解渗透的结构和分子基础对于了解该通道在宿主 - 寄生虫相互作用中的作用以及发展可能是未来抗疟药的抑制剂至关重要。 Biomed Research International，出版社（2014年）。在第三项研究中，我们检查了感染细胞对钙的渗透性增加，这是一种必不可少的二价阳离子。我们将荧光钙指示剂染料（Fluo-8）的非破坏性负载用于人红细胞来量化Ca ++摄取动力学。我们的研究表明，疟疾寄生虫的感染产生的红细胞Ca ++渗透性显着增加。药理学研究表明，这种摄取不是由PSAC或典型的哺乳动物CA ++通道介导的。寄生虫生长抑制研究表明，对细胞外Ca ++的保守要求。这些发现表明感染后CA ++摄取的新途径。该途径的抑制剂可能是抗疟药开发的绝佳起点。 Malaria J. 13：184（2014）。