Regulation of latent stage differentiation through central carbon metabolism and ubiquitination

通过中心碳代谢和泛素化调节潜伏期分化

• 批准号: 10563243
• 负责人: Chris Tonkin
• 金额: $ 52.2万
• 依托单位: WALTER AND ELIZA HALL INST MEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-05 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563243
• 关键词: Acute; Amino Acids; Biochemical; Biology; Blindness; Brain; CRISPR screen; Carbon; Central Nervous System; Clinical; Complex; Country; Cues; Development; Disease; Drug resistance; Environment; Enzymes; Exposure to; Genes; Genetic; Genetic Screening; Genetic Transcription; Glutamine; Human; Immunocompromised Host; In Vitro; Individual; Life; Link; Metabolic; Metabolic Pathway; Metabolism; Methodology; Muscle; Nutritional; Parasites; Pathogenesis; Patients; Pharmaceutical Preparations; Play; Population; Prevalence; Proteomics; Publishing; Refractory; Regulation; Role; Signal Transduction; Source; Structure; Techniques; Tissues; Toxoplasma; Translations; Ubiquitin; Ubiquitination; Work; brain tissue; genome-wide; in vivo; insight; latent infection; metabolomics; model organism; programs; therapy resistant; ubiquitin-protein ligase; whole genome

Project summary Toxoplasma latently infects approximately 30% of the human population and is refractory to acute stage therapies. Latent Toxoplasma can act as a reservoir for acute stage reactivation in immunocompromised individuals and can cause progressive blindness in otherwise healthy individuals. Thus, latent stages are clinically important, but currently untreatable. Drug resistance is likely due to changes in the metabolism of latent stages, but what these are remain a complete mystery. Furthermore, latent Toxoplasma is exposed to different nutritional environments when infecting its target tissue types – muscle and the central nervous system (CNS) – and the impact this has on Toxoplasma latency is not understood. We have found that changes in carbon sources, that mimic muscle and CNS trigger Toxoplasma differentiation into latent forms. We then performed a whole-genome CRISPR screen and identified select Toxoplasma carbon catabolic enzymes that are required for differentiation and furthermore, identified a role for ubiquitination in metabolic adaptation and differentiation into latent forms. These background studies form the basis for developing a comprehensive understanding of Toxoplasma metabolism during latency and understanding how metabolic rewiring is linked to differentiation through regulation by ubiquitination. The specific aims of this program are to: Aim 1: Define the metabolic pathways required for differentiation and persistence of latent Toxoplasma. We will determine the role of select enzymes (identified in CRISPR screen) required for utilization of carbon sources that mimic brain and muscle and their impact on differentiation in vitro and in vivo. This will determine how metabolism of latent and acute stages differs and how this is linked to environmental sensing and differentiation. Aim 2: Determine the role of ubiquitination in the regulation of differentiation into latent forms. Define the composition, biochemical activity and structure of an E3 ubiquitin ligase complex that is important for differentiation into latent forms, thus determining how ubiquitination regulates differentiation. Aim 3: Define how ubiquitination regulates metabolism and differentiation into latent forms. We have identified likely substrates of the E3 ubiquitination machinery and will define their function in regulation of both metabolism and differentiation.

项目摘要 弓形虫潜伏性感染约30%的人口，并且急性期难治 治疗潜伏性弓形虫可作为免疫功能低下患者急性期再激活的储存库 个体，并可能导致其他健康个体的渐进性失明。因此，潜伏期是 临床上很重要但目前无法治愈 耐药性可能是由于潜伏期代谢的变化，但这些变化仍然是一个完整的 谜此外，潜伏的弓形虫在感染其宿主时暴露于不同的营养环境中。 靶组织类型-肌肉和中枢神经系统（CNS）-及其对弓形虫的影响 延迟是不被理解的。我们发现碳源的变化，模拟肌肉和中枢神经系统触发 弓形虫分化为潜伏型。然后，我们进行了全基因组CRISPR筛选， 选择分化所需的弓形虫碳分解代谢酶，并进一步鉴定了 泛素化在代谢适应和分化成潜在形式中的作用。 这些背景研究构成了全面了解弓形虫的基础 代谢在潜伏期和理解如何代谢重新布线与分化， 通过泛素化调节。该方案的具体目标是： 目的1：明确潜伏期弓形虫分化和持续存在所需的代谢途径。 我们将确定利用碳所需的选择酶（在CRISPR筛选中鉴定）的作用， 模拟脑和肌肉的来源及其对体外和体内分化的影响。这将决定 潜伏期和急性期的代谢如何不同，以及这与环境感知的联系， 分化 目的2：确定泛素化在分化成潜在形式的调节中的作用。定义 E3泛素连接酶复合物的组成、生物化学活性和结构， 分化为潜在形式，从而决定了泛素化如何调节分化。 目的3：定义泛素化如何调节代谢和分化成潜在形式。我们有 确定了E3泛素化机制的可能底物，并将确定它们在调控E3泛素化机制和E3泛素化机制中的功能。 代谢和分化。