Ribosome Heterogeneity in Cryptococcus neoformans

新型隐球菌的核糖体异质性

• 批准号: 10494146
• 负责人: John C Panepinto
• 金额: $ 38.44万
• 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/10494146
• 关键词: 5' Untranslated Regions; Affect; Antifungal Agents; Antifungal Therapy; 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; 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; Regulation; Regulon; Resistance; Ribosomes; Role; Sedimentation process; Signal Pathway; Specificity; Stress; Structure; Techniques; Temperature; Testing; Therapeutic; Translating; Translation Initiation; Translations; Transplantation; Virulence; Work; antimicrobial; antimicrobial drug; base; 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共发病和死亡的主要原因。有限的抗真菌武器库和抗真菌耐药性的出现使得对新型抗真菌疗法的需求至关重要。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和经过充分审查的靶标，但尚未在抗真菌开发中利用，因为假定无法特异性靶向真菌中核糖体的核心催化功能。利用人类病原体新型隐球菌，我们利用两种方法来确定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，我们鉴定了保守的真核mRNA帽结合复合体（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的缺失损害了缺氧适应，这是主要人类真菌病原体的主要发病相关途径。进一步研究Afg1和Afe1作为新型eIF4F复合物的组成部分的功能的工作在Aim 1中概述，它们结合的mRNA靶标的鉴定在Aim 3中概述。我们的第二种方法开发了一种新的蛋白质组学管道，我们将其命名为RiboPROT，以确定在适应温度应激和氧化应激过程中与翻译核糖体相关的因素。本研究的目的2将探讨这些蛋白在应激反应翻译和发病机制中的作用。我们已经优先考虑了我们最初的RiboPROT实验，以进一步研究，并提出在其他条件下确定与隐球菌发病机制和生物学相关的其他因素。对于那些具有rna结合结构域的，同源mRNA靶标将在Aim 3中识别，识别应激反应的翻译调控。在这些研究的结论中，我们将有一套全面的核糖体相关因子来研究未来的抗真菌靶点。这些独特的胁迫特异性和真菌特异性核糖体相关因子可能为特异性靶向真菌核糖体提供了新的途径。