The Plasmodium 2TM and PHIST protein families

Plasmodium 2TM 和 PHIST 蛋白家族

• 批准号: 8257765
• 负责人: THOMAS J TEMPLETON
• 金额: $ 41.83万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-18 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257765
• 关键词: Address; Adhesions; Affinity; Antibodies; Antigenic Diversity; Architecture; Benzoates; Biological Assay; Cataloging; Catalogs; Cells; Chloride Channels; Cleaved cell; Coupled; Cystic Fibrosis Transmembrane Conductance Regulator; Cytolysis; Cytoplasm; Destinations; Drug resistance; Epitopes; Erythrocyte Membrane; Erythrocytes; Exhibits; Family; Furosemide; Gene Expression; Gene Family; Gene Targeting; Generations; Genes; Genomics; Health; Human; Immune; Immune Sera; Immune response; Infection; Injection of therapeutic agent; Integral Membrane Protein; Ion Channel; Malaria; Mediating; Membrane; Methodology; Methods; Modeling; Modification; Molecular; Orthologous Gene; Parasites; Pathway interactions; Peptide Signal Sequences; Permeability; Phenotype; Plasmodium; Plasmodium falciparum; Population; Predisposition; Protein Family; Proteins; Public Health; Regulation; Rodent; Shapes; Sorbitol; Structural Protein; Structure; Surface; Switch Genes; System; Tertiary Protein Structure; Testing; Time; Titrations; Transcription Coactivator; Transmembrane Domain; Vaccines; Variant; Xenopus oocyte; Yeasts; base; blasticidin S; extracellular; knock-down; knockout gene; member; paralogous gene; patch clamp; pressure; protein expression; protein function; protein protein interaction; protein transport; response; solute; trafficking; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a signal peptide followed by a Pexel/HT erythrocyte trafficking motif. This catalog includes two large families of proteins that are the subject of this proposal: the predicted 2 transmembrane (2TM) proteins that likely function within the erythrocyte membrane, including the RIFIN, STEVOR and Pfmc-2TM families, and the PHIST domain family which likely function within the erythrocyte cytoplasm. The human malaria parasite, P. vivax, and the rodent malaria parasites possess large families of proteins which are topologically similar to the 2TM proteins and also harbor Pexel/HT motifs, suggesting that the function of the 2TM proteins is widely conserved in Plasmodium. We have shown using primary and second generation clonal lines of the P. falciparum isolate, NF54 that expression of stevor and Pfmc-2TM families is clonally variant and undergoes switching. Moreover, the STEVOR and Pfmc-2TM families possess a loop between the 2TM domains that is hypervariable both between paralogs and across isolate boundaries, suggesting that it is exposed to host immune pressure at the erythrocyte surface. Thus host immune pressure has likely driven the amplification and antigenic diversification of the 2TM protein families, and a mechanism of gene expression switching insures that immune responses do not eliminate parasite infections. The topological and sequence similarity of the 2TM proteins to subunits of ion channels prompted us to consider that they might encode the new permeability pathways (NPPs) of infected erythrocytes. We propose here to test this hypothesis using genetically manipulated parasite lines that either dominantly express epitope-tagged versions of 2TM proteins or are globally knocked down in 2TM protein expression via conscription of specific transcription factors. These parasite lines will be used phenotypic assays, such as sorbitol lysis and whole-cell patch clamp. We also present evidence that the methodology of gene knockdown via transcription factor conscription can be used to address the function of other sub-telomeric gene families in P. falciparum, and thereby are likely to contribute to our understanding of parasite-encoded modifications of the infected erythrocyte. We present preliminary studies on the gene expression and cellular localization of select PHIST domain proteins and propose to pursue their function via targeted gene disruption as well as determination of predicted protein-protein interactions within the erythrocyte cytoplasm. In this manner we hope to generate hypotheses on the identity of the functional pressures which have driven the amplification of the PHIST domain family to over 6 members in P. falciparum. PUBLIC HEALTH RELEVANCE: Malaria remains a public health concern to approximately 40% of the world's population, particularly in the face of increasing drug resistance and the continued absence of an effective vaccine. This proposal focuses on two amplified sub-telomeric gene families, the first encoding the 2TM proteins which uniformly possess signal peptides, Pexel/HT erythrocyte trafficking motifs, and 2 transmembrane (2TM) regions that indicate an integral membrane topology. The second gene family encodes the PHIST domain proteins which include over 60 members and are predicted to function with the erythrocyte cytoplasm. The 2TM proteins are conserved in all species of Plasmodium, not as orthologous families but rather as topologically conserved integral membrane proteins. Thus their function, likely at the erythrocyte surface, is predicted to all be conserved across the Plasmodium clade. In this proposal we argue that the profound amplification of the 2TM gene families, their diversity between paralogs and across isolate boundaries, localization to the erythrocyte membrane, and topological similarity to sub-units of ion channels, combine to make them premier candidates for comprising the new permeability pathways in infected erythrocytes. We describe a spectrum of studies in which to test this hypothesis, including new methods to knockdown expression of gene families. The expression, cellular localization, and phenotype following targeted gene disruption will be studied for select PHIST domain proteins toward an understanding of their function within the erythrocyte cytoplasm.

描述（由申请人提供）：人类疟原虫恶性疟原虫具有广泛的蛋白质曲目，该蛋白质被提议被贩运到红细胞细胞质或表面，基于信号肽的这些蛋白质，然后是A Pexel/ht erytherthrocytherocyte的蛋白质。该目录包括该提案主题的两个大型蛋白质：预测的2个跨膜（2TM）蛋白可能在红细胞膜中起作用，包括Rifin，Stevor和PFMC-2TM家族，以及可能在红细胞中发挥作用的Phist域。人类疟疾寄生虫，维瓦克斯和啮齿动物疟疾寄生虫具有较大的蛋白质家族，在拓扑上与2TM蛋白质相似，也含有PEXEL/HT序列，这表明2TM蛋白的功能在疟原虫中广泛保存。我们已经显示了恶性疟原虫分离菌的一级和第二代克隆线，NF54表明，Stevor和PFMC-2TM家族的表达是克隆的变体，并且经历了切换。此外，Stevor和PFMC-2TM家族在2TM结构域之间具有循环，在旁系同源体和跨分离株边界之间都是高变量的，这表明它暴露于红细胞表面处的宿主免疫压力。因此，宿主免疫压力可能驱动了2TM蛋白家族的扩增和抗原多样化，并且基因表达转换的机制可确保免疫反应不会消除寄生虫感染。 2TM蛋白与离子通道亚基的拓扑和序列相似性促使我们认为它们可能会编码受感染的红细胞的新渗透率（NPP）。我们在这里提议使用遗传操纵的寄生虫线来检验该假设，该寄生虫线主要表达2TM蛋白的表位标记版本，或者通过特定转录因子的征服在2TM蛋白表达中被全局击倒。这些寄生虫线将用于表型测定，例如山梨糖醇裂解和全细胞斑块夹。我们还提供了证据表明，通过转录因子征集的基因敲低方法可用于解决恶性疟原虫中其他亚tel体基因家族的功能，从而可能有助于我们理解对感染的寄生虫的寄生虫编码的修饰。我们介绍了有关精选PHIST结构域蛋白的基因表达和细胞定位的初步研究，并提议通过靶向基因破坏以及确定红细胞细胞质中预测的蛋白质蛋白质相互作用来追求其功能。通过这种方式，我们希望对功能压力的身份产生假设，这些功能压力将Phist域家族的放大到恶性疟原虫的6多名成员。公共卫生相关性：疟疾仍然是全球大约40％人口的公共卫生问题，尤其是面对耐药性增加和继续缺乏有效疫苗的问题。该提案的重点是两个扩增的亚tel体基因家族，这是第一个编码2TM蛋白，该蛋白均匀地具有信号肽，PEXEL/HT红细胞运输基序和2个跨膜（2TM）区域，该区域表明了整体膜拓扑。第二个基因家族编码PHIST结构域蛋白质，其中包括60多个成员，并被预测可与红细胞细胞胞质发挥作用。 2TM蛋白在所有种类的疟原虫中都是保守的，不是直系同源的家族，而是作为拓扑保守的整体膜蛋白。因此，它们的功能（可能在红细胞表面）被预测在整个质量进化枝上都是保守的。在这一提议中，我们认为2TM基因家族的深刻放大，它们之间的多样性，副产品和跨隔离界的多样性，对促红细胞膜的本地化以及与离子频道的子单位相似的拓扑相似性，结合在一起，使它们成为新的候选者，以使其成为新的许可型eRythected Erythrocythrocythrocythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctythroctytythroctythcyt the。我们描述了测试该假设的一系列研究，包括敲低基因家族表达的新方法。将研究针对靶向基因破坏后的表达，细胞定位和表型，以了解某些PHIST结构域蛋白，以了解其在红细胞细胞质中的功能。