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.

摘要 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100万新的利什曼病病例。最严重的利什曼病形式(例如黑热病或 内脏利什曼病)如果不治疗是致命的。几乎没有可用的治疗方法，而且有显著的副作用和寄生虫 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疾病 开发新的治疗方法是高度优先考虑的疾病。大多数基因在体内的表达 利什马尼亚是在翻译层面上受到监管的。在哺乳动物细胞中，翻译启动是很好的特征。 这一过程中的关键步骤包括由帽结合蛋白eif4e识别m7gtp部分。 位于信使RNA(MRNA)的5‘端。EIF4E，通过与其他翻译启动的互动 因子，最终协调小核糖体亚基的招募。 到目前为止，已鉴定出六种利什曼原虫mRNA帽结合蛋白亚型(LIF4E-1至-6)。 利什曼原虫的mrna帽结构是独一无二的，它由真核m7gtp帽和4个 高甲基化的核苷酸(“CAP-4”)。保守的39个微小外显子拼接前导RNA(SL RNA) 核苷酸也通过反式剪接添加到每个转录本中。我们之前确定了X射线晶体 LIF4E-1的结构，LIF4E-1是在利什曼原虫无鞭毛体(人感染性)中表达的唯一帽结合亚型 阶段)，与抑制其帽子结合活性的相互作用伙伴(L4E-IP1)结合。LIF4E-1也是 最近发现与利什曼原虫启动因子3(LIF3)的“a”亚基直接相互作用，LIF3是一个大的(~800 kDa) 结合小核糖体亚基的多蛋白复合体。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IF3亚基，并且LIF4E-1/LIF3a分子的细节 相互作用是未知的。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一个合格的预起始复合体(PIC)。这个 LIF4E-1/LIF3a的相互作用将绕过对eIF4G样支架蛋白的需要。在这项提案中，我们将 追求两个特定的目标：1)确定LIF3a与帽结合蛋白相互作用的分子基础 LIF4E-1.2)确定与LIF3a或LIF3a结合的LIF4E-1/CAP-4 SL RNA的高分辨率冷冻EM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- 利什曼原虫中的蛋白质相互作用，而这些信息反过来可以指导特定的 针对这些寄生虫的翻译起始抑制物。