The Plasmodium 2TM and PHIST protein families

Plasmodium 2TM 和 PHIST 蛋白家族

• 批准号: 7895033
• 负责人: THOMAS J TEMPLETON
• 金额: $ 41.35万
• 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 2011-07-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895033
• 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; Face; Family; Furosemide; Gene Expression; Gene Family; Gene Targeting; Generations; Genes; Genomics; Human; Immune; Immune Sera; Immune response; Infection; Injection of therapeutic agent; Integral Membrane Protein; Ion Channel; Malaria; Mediating; Membrane; Methodology; Methods; Modeling; Modification; Molecular; Muscle Rigidity; 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; extracellular; knock-down; knockout gene; member; paralogous gene; patch clamp; pressure; protein expression; protein function; protein protein interaction; protein transport; public health relevance; 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拥有广泛的蛋白质，根据这些蛋白质中是否存在信号肽和随后的 Pexel/ HT 红细胞运输基序。该目录包括本提案主题的两大蛋白质家族：预测的可能在红细胞膜内发挥作用的 2 种跨膜 (2TM) 蛋白质，包括 RIFIN、STEVOR 和 Pfmc-2TM 家族，以及可能在红细胞膜内发挥作用的 PHIST 结构域家族在红细胞胞浆内发挥作用。人类疟疾寄生虫、间日疟原虫和啮齿动物疟疾寄生虫拥有大家族的蛋白质，这些蛋白质在拓扑上与 2TM 蛋白质相似，并且还含有 Pexel/HT 基序，表明 2TM 蛋白质的功能在疟原虫中广泛保守。我们使用恶性疟原虫分离株 NF54 的第一代和第二代克隆系证明，stevor 和 Pfmc-2TM 家族的表达存在克隆变异并经历转换。此外，STEVOR 和 Pfmc-2TM 家族在 2TM 结构域之间具有一个环，该环在旁系同源物之间和跨分离边界均高度可变，表明它在红细胞表面暴露于宿主免疫压力。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考虑它们可能编码受感染红细胞的新通透性途径 (NPP)。我们在这里建议使用基因操纵的寄生虫系来测试这一假设，这些寄生虫系要么显性表达表位标记的2TM蛋白版本，要么通过特定转录因子的征募而在2TM蛋白表达中被整体敲低。这些寄生虫系将用于表型测定，例如山梨醇裂解和全细胞膜片钳。我们还提供了证据，表明通过转录因子征兵进行基因敲低的方法可用于解决恶性疟原虫中其他亚端粒基因家族的功能，从而可能有助于我们了解感染的寄生虫编码的修饰红细胞。我们对选定的 PHIST 结构域蛋白的基因表达和细胞定位进行了初步研究，并建议通过靶向基因破坏以及确定红细胞细胞质内预测的蛋白质-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，这些功能压力驱动恶性疟原虫中 PHIST 结构域家族扩增至超过 6 个成员。公共卫生相关性：疟疾仍然是世界上约 40% 人口的公共卫生问题，特别是在耐药性不断增加和有效疫苗持续缺乏的情况下。该提案重点关注两个扩增的亚端粒基因家族，第一个编码统一具有信号肽、Pexel/HT 红细胞运输基序的 2TM 蛋白，以及表明完整膜拓扑结构的 2 个跨膜 (2TM) 区域。第二个基因家族编码 PHIST 结构域蛋白，其中包括 60 多个成员，预计与红细胞细胞质一起发挥作用。 2TM 蛋白在所有疟原虫物种中都是保守的，不是作为直系同源家族，而是作为拓扑保守的完整膜蛋白。因此，预计它们的功能（可能在红细胞表面）在整个疟原虫分支中都是保守的。在本提案中，我们认为 2TM 基因家族的显着扩增、它们在旁系同源物之间和跨分离边界的多样性、红细胞膜的定位以及与离子通道亚基的拓扑相似性，结合起来使它们成为构成受感染红细胞中的新渗透途径。我们描述了一系列研究来检验这一假设，包括敲低基因家族表达的新方法。将研究选定的 PHIST 结构域蛋白的表达、细胞定位和靶向基因破坏后的表型，以了解它们在红细胞细胞质中的功能。