Ribosome Heterogeneity in Cryptococcus neoformans

新型隐球菌的核糖体异质性

• 批准号: 10687190
• 负责人: John C Panepinto
• 金额: $ 39.15万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687190
• 关键词: 5' Untranslated Regions; Affect; Antifungal Agents; Antifungal Therapy; Basidiomycota; Binding; Biology; Catalytic Domain; Cellular Stress; Cessation of life; Complex; Cryptococcus; Cryptococcus neoformans; Data; Defect; Development; Drug Targeting; Eukaryota; Exhibits; Fungal Drug Resistance; Future; Goals; Growth; Heterogeneity; Homologous Gene; Human; Hypoxia; Immune system; Immunologic Deficiency Syndromes; Impairment; In Vitro; Intervention; Investigation; Lung; Maps; Messenger RNA; Molecular Chaperones; Mycoses; Names; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Peptide Initiation Factors; Persons; Pharmacologic Substance; Phenotype; Phosphorylation; Process; Prokaryotic Cells; Protein Biosynthesis; Protein Isoforms; Proteins; Proteomics; RNA; RNA Recognition Motif; RNA-Binding Proteins; RNA-Protein Interaction; Regulon; Resistance; Ribosomes; Role; Sedimentation process; Signal Pathway; Specificity; Stress; Structure; Techniques; Temperature; Testing; Therapeutic; Translating; Translation Initiation; Translational Regulation; Translations; Transplantation; Virulence; Work; antimicrobial; antimicrobial drug; biological adaptation to stress; comorbidity; experimental study; fungus; human pathogen; knock-down; mRNA Decay; mRNA capping; mortality; mutant; novel; pathogen; pathogenic fungus; recruit; scaffold; stress tolerance; translation factor; translatome

Invasive fungal infections are a major cause of co-morbidity and mortality in patients living with immunodeficiency and transplantation. A limited antifungal arsenal and emergence of antifungal drug resistance have made the need for novel antifungal therapies paramount. Cellular protein synthesis carried out by the ribosome is a known and well-vetted target for pharmaceutical intervention in prokaryotes and eukaryotes, but has not been exploited in antifungal development because of the presumed inability to target the core catalytic function of the ribosome with specificity in fungi. Using the human pathogen Cryptococcus neoformans, we have leveraged two approaches to identify factors that associate with the fungal ribosome under stress conditions encountered in the host. Using a homology based approach, we have identified Cryptococcus-specific homologues of the conserved eukaryotic mRNA cap-binding complex (eIF4F) that we have names alternative eIF4G (AFG1) and alternative eIF4E (AFE1). Deletion of AFG1 results impairs hypoxia adaptation, which is a major pathogenesis-associated pathway in the major human fungal pathogens. Further work on the function of Afg1 and Afe1 as components of a novel eIF4F complex are outlined in Aim 1, and the identification of the mRNA targets they bind is outlined in Aim 3. Our second approach developed a novel proteomic pipeline that we’ve termed RiboPROT to identify the factors associating with translating ribosomes during adaptation to temperature stress and oxidative stress. Aim 2 of this proposal will investigate the role of these proteins in stress-responsive translation and pathogenesis. We have prioritized hits from our initial RiboPROT experiments to investigate further, and propose to identify additional factors under additional conditions relevant to cryptococcal pathogenesis and biology. For those with RNA-binding domains, cognate mRNA targets will be identified in Aim 3, identifying stress responsive translational regulons. At the conclusion of these studies, we will have a comprehensive set of ribosome-associating factors to investigate in future work as antifungal targets. These unique stress-specific and fungi-specific ribosome-associating factors may provide a novel pathway to targeting the fungal ribosome with specificity.

侵袭性真菌感染是免疫缺陷和移植患者合并症和死亡的主要原因。有限的抗真菌库和抗真菌耐药性的出现使得对新型抗真菌疗法的需求变得至关重要。由核糖体进行的细胞蛋白质合成是原核生物和真核生物药物干预的已知且经过充分审查的目标，但尚未在抗真菌开发中得到利用，因为推测无法在真菌中特异性靶向核糖体的核心催化功能。使用人类病原体新型隐球菌，我们利用两种方法来识别在宿主遇到的应激条件下与真菌核糖体相关的因素。使用基于同源性的方法，我们鉴定了保守真核 mRNA 帽结合复合物 (eIF4F) 的隐球菌特异性同源物，我们将其命名为替代 eIF4G (AFG1) 和替代 eIF4E (AFE1)。 AFG1 结果的缺失会损害缺氧适应，这是主要人类真菌病原体的主要发病机制相关途径。目标 1 概述了对 Afg1 和 Afe1 作为新型 eIF4F 复合物的组成部分的功能的进一步研究，目标 3 概述了它们结合的 mRNA 靶点的鉴定。我们的第二种方法开发了一种新型蛋白质组管道，我们将其称为 RiboPROT，以识别在适应温度应激和氧化应激过程中与核糖体翻译相关的因素。该提案的目标 2 将研究这些蛋白质在应激反应翻译和发病机制中的作用。我们优先考虑了最初 RiboPROT 实验中的命中结果以进行进一步研究，并建议在与隐球菌发病机制和生物学相关的其他条件下确定其他因素。对于那些具有 RNA 结合域的蛋白，将在目标 3 中鉴定同源 mRNA 靶标，从而鉴定应激反应性翻译调节子。在这些研究结束时，我们将拥有一套全面的核糖体相关因子，可以在未来的工作中作为抗真菌靶点进行研究。这些独特的应激特异性和真菌特异性核糖体相关因子可能提供一种特异性靶向真菌核糖体的新途径。