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: 10373100
• 负责人: Melissa Leger-Abraham
• 金额: $ 25.4万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373100
• 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; drug development; experimental study; inducible gene expression; inhibitor; medical schools; neglected tropical diseases; protein protein interaction; rational design; 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各地并引起 每年大约有 100 万新利什曼病病例。最严重的利什曼病（例如黑热病或黑热病） 内脏利什曼病）如果不治疗的话是致命的。可用的治疗方法很少，且副作用和寄生虫显着 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病 开发新疗法是当务之急的疾病。大部分基因表达于 利什曼原虫在翻译层面受到监管。在哺乳动物细胞中，翻译起始已得到充分表征。 此过程中的一个关键步骤包括 eIF4E（一种帽子结合蛋白）对 m7GTP 部分的识别 位于信使 RNA (mRNA) 的 5' 端。 eIF4E，通过与其他翻译起始的相互作用 因素，最终协调小核糖体亚基的招募。 迄今为止，已鉴定出六种利什曼原虫 mRNA 帽结合蛋白亚型（LIF4E-1 至 -6）。 利什曼原虫 mRNA 帽结构是独特的，由真核 m7GTP 帽和后面的四个组成 高度甲基化的核苷酸（“cap-4”）。保守的迷你外显子剪接前导 RNA (SL RNA) 为 39 核苷酸也通过转拼添加到每个转录物中。我们之前确定了X射线晶体 LIF4E-1 的结构，是在利什曼原虫无鞭毛体（人类感染性 阶段），与抑制其帽结合活性（L4E-IP1）的相互作用伙伴结合。 LIF4E-1 也被 最近显示与利什曼原虫起始因子 3 (LIF3) 的亚基“a”直接相互作用，LIF3 是一个大的 (~ 800 kDa) 结合小核糖体亚基的多蛋白复合物。 IF4E 帽结合蛋白之间的相互作用 并且在其他系统中从未观察到过 IF3 亚基，并且 LIF4E-1/LIF3a 分子的详细信息 相互作用未知。我们假设 LIF4E-1/cap-4 SL RNA/LIF3a 相互作用影响 LIF3 的组织以及它如何在无鞭毛体中组装有效的预起始复合物 (PIC)。这 LIF4E-1/LIF3a 相互作用将绕过对 eIF4G 样支架蛋白的需要。在本提案中，我们将 追求两个具体目标：1) 定义 LIF3a 与帽结合蛋白相互作用的分子基础 LIF4E-1。 2) 确定与 LIF3a 或 LIF4E-1/cap-4 SL RNA 结合的高分辨率冷冻电镜结构 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，而这些信息反过来可以指导特定的发展 针对这些寄生虫的翻译起始抑制剂。