Structural Basis for Translation Initiation in Leishmania Major

大利什曼原虫翻译起始的结构基础

• 批准号: 10225842
• 负责人: Melissa Leger-Abraham
• 金额: $ 21.13万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-16 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225842
• 关键词: Affinity; Austria; Baculoviruses; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biophysics; Bypass; Cells; Cellular biology; Clinical; Complex; Country; Cryoelectron Microscopy; Crystallization; Cutaneous; Data Collection; Development; Disease; Dorsal; EIF4EL3 gene; Effectiveness; Endemic Diseases; Epitopes; Face; Freezing; Future; Gene Expression; Genes; Goals; Guanosine Triphosphate; Human; Individual; Infection; Interferometry; Leishmania; Leishmania major; Leishmaniasis; Lesion; Life Cycle Stages; Mammalian Cell; Messenger RNA; Methods; Mini-Exons; Molecular; Molecular Conformation; Multiprotein Complexes; NMR Spectroscopy; Nucleic Acids; Nucleotides; Outcome; Parasite resistance; Parasites; Pathway interactions; Peptide Initiation Factors; Pharmaceutical Preparations; Play; Preparation; Process; Prokaryotic Initiation Factor-3; Protein Biosynthesis; Protein Isoforms; Protein Synthesis Inhibitors; Proteins; RNA; RNA Binding; RNA Cap-Binding Proteins; RNA Caps; RNA Recognition Motif; Reagent; Recombinants; Resolution; Ribosomes; Roentgen Rays; Role; Sampling; Scaffolding Protein; Scanning; Severities; Side; Site; Spliced Leader RNA; Spliced Leader Sequences; Structure; Surface; System; Tetracyclines; Therapeutic Intervention; Trans-Splicing; Transcript; Translation Initiation; Translations; Trypanosoma cruzi; Visceral Leishmaniasis; Work; World Health Organization; analog; assay development; biophysical properties; design; drug development; experimental study; inducible gene expression; inhibitor/antagonist; medical schools; neglected tropical diseases; protein protein interaction; reconstitution; recruit; side effect; small molecule; small molecule inhibitor; structural biology

ABSTRACT About 20 species of the protozoan parasite Leishmania are distributed across the globe and cause roughly 1 million new cases of leishmaniasis annually. The most severe form of leishmaniasis (e.g., kala-azar or visceral leishmaniasis) is lethal if untreated. Few therapies are available, and significant side effects and parasite resistance limit their effectiveness. The World Health Organization lists Leishmaniasis as a neglected tropical disease for which the development of new treatments is a high priority. Most of the gene expression in Leishmania is regulated at the level of translation. In mammalian cells, translation initiation is well characterized. A crucial step in this process includes the recognition by eIF4E, a cap-binding protein, of an m7GTP moiety located at the 5’ end of messenger RNAs (mRNAs). eIF4E, through its interaction with other translation initiation factors, ultimately coordinates the recruitment of the small ribosomal subunit. To date, six Leishmania mRNA cap-binding protein isoforms (LIF4E-1 through -6) have been identified. The Leishmania mRNA cap structure is unique and consists of the eukaryotic m7GTP cap followed by four nucleotides that are hypermethylated (“cap-4”). A conserved mini-exon spliced leader RNA (SL RNA) of 39 nucleotides is also added to each transcript through trans-splicing. We previously determined the X-ray crystal structure of LIF4E-1, the only cap-binding isoform that is expressed in Leishmania amastigotes (human infective stage), bound to an interacting partner that represses its cap-binding activity (L4E-IP1). LIF4E-1 was also recently shown to interact directly with the subunit “a” of Leishmania initiation factor 3 (LIF3), a large (~ 800 kDa) multiprotein complex that binds the small ribosomal subunit. An interaction between an IF4E cap-binding protein and an IF3 subunit has never been observed in other systems, and the details of the LIF4E-1/LIF3a molecular interaction are unknown. We hypothesize that a LIF4E-1/cap-4 SL RNA/LIF3a interaction influences the organization of LIF3 and how it assembles a competent pre-initiation complex (PIC) in amastigotes. The LIF4E-1/LIF3a interaction would bypass the need for an eIF4G-like scaffolding protein. In this proposal, we will pursue two specific aims: 1) To define the molecular basis for LIF3a interaction with the cap-binding protein LIF4E-1. 2) To determine a high-resolution cryo-EM structure of LIF4E-1/cap-4 SL RNA bound to LIF3a or assembled with LIF3 on the small ribosomal subunit. Our work will reveal the molecular basis for unique protein- protein interactions in Leishmania parasites, and this information, in turn, could guide the development of specific translation initiation inhibitors against these parasites.

抽象的 大约20种原生动物寄生虫利什曼原虫分布在全球范围内，并导致 每年约有100万例新的利什曼病病例。利什曼病的最严重形式（例如，卡拉 - 阿萨尔或 如果未经治疗，内脏利什曼病）是致命的。很少有疗法可用，副作用和寄生虫很大 阻力限制了它​​们的有效性。世界卫生组织将利什曼病列为被忽视的热带 新疗法的发展是高度重点。大多数基因表达 利什曼尼亚受到翻译水平的监管。在哺乳动物细胞中，翻译起始的特征很好。 此过程中的关键步骤包括M7GTP部分的EIF4E识别（帽结合蛋白）的识别 位于Messenger RNA（mRNA）的5'末端。 EIF4E，通过与其他翻译启动的互动 因素最终协调小核糖体亚基的募集。 迄今为止，已经确定了六个Leishmania mRNA帽结合蛋白同工型（LIF4E-1至-6）。 Leishmania mRNA帽结构是独一无二的，由真核M7GTP盖组成，然后是四个 高甲基化的核苷酸（“ CAP-4”）。 39 还通过移媒体添加了核苷酸。我们以前确定了X射线晶体 LIF4E-1的结构，这是唯一在利什曼尼亚杂志（人类感染）中表达的帽结合同工型 阶段），与反映其帽结合活动的相互作用伙伴约束（L4E-IP1）。 lif4e-1也是 最近显示的是直接与利什曼原虫倡议因子3（lif3）的亚基“ a”相互作用，一个大（〜800 kDa） 结合小核糖体亚基的多蛋白复合物。 IF4E盖结合蛋白之间的相互作用 在其他系统中从未观察到IF3亚基，以及LIF4E-1/LIF3A分子的细节 互动是未知的。我们假设LIF4E-1/CAP-4 SL RNA/LIF3A相互作用会影响 LIF3的组织及其如何组装杂物中的合格的预生效复合物（PIC）。这 LIF4E-1/LIF3A相互作用将绕过对EIF4G样脚手架蛋白的需求。在此提案中，我们将 追求两个特定的目的：1）定义与帽结合蛋白相互作用的分子基础 lif4e-1。 2）确定与LIF3A或LIF3A或 在小核糖体亚基上与LIF3组装。我们的工作将揭示独特蛋白质的分子基础 利什曼原虫寄生虫中的蛋白质相互作用，而这些信息又可以指导特定的发展 对这些寄生虫的翻译起始抑制剂。