Post-translational modification of GlyGly-Cterm Proteins

GlyGly-Cterm 蛋白的翻译后修饰

• 批准号: 10749396
• 负责人: Cameron Roberts
• 金额: $ 7.43万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-18 至 2026-06-17
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749396
• 关键词: Acinetobacter baumannii; Acute Diarrhea; Autophagocytosis; Autophagosome; Bacteria; Biogenesis; Biological Assay; C-terminal; Cell Fractionation; Cell surface; Cells; Charge; Cholera; Cholera Toxin; Code; Complex; Cytoplasm; Data; Disease; Distal; Elements; Environment; Enzymes; Escherichia coli; Eukaryota; Fluorescence Microscopy; GPI Membrane Anchors; Gene Deletion; Generations; Genes; Glycine; Gram-Negative Bacteria; Impairment; Libraries; Lipoproteins; Maintenance; Membrane; Microbial Biofilms; Modeling; Modification; Molecular; Molecular Probes; N-terminal; Nutrient; Organism; Pathogenesis; Pathway interactions; Peptide Hydrolases; Phosphatidylethanolamine; Phospholipids; Physiological; Play; Post-Translational Protein Processing; Poverty; Process; Prokaryotic Cells; Proteins; Proteomics; Role; Site; Specificity; Stress; Surface; Symptoms; System; Tail; Testing; Toxin; Transmembrane Domain; Type II Secretion System Pathway; Ubiquitin; Vibrio cholerae; Work; adenylate; cell growth; diarrheal disease; environmental change; follow-up; gene product; insight; novel; overexpression; pathogen; pathogenic bacteria; rhomboid; technology development

Abstract Cholera continues to be a global burden manifesting as acute diarrheal disease that impacts impoverished and destabilized regions. Bacteria including Vibrio cholerae utilize the Type II Secretion System (T2SS) for the secretion of a diverse array of effector proteins and toxins to adapt to environmental changes, notably cholera toxin the causative agent of cholera symptoms. While a variety of T2SS substrates including cholera toxin are fully secreted, a subset is retained on the cell surface. A recently identified group of diverse substrates found in some gram-negative bacteria including V. cholerae contains a homologous C-terminal domain, called the GlyGly- Cterm, that targets its passenger proteins to the cell surface. This newly identified domain is processed by rhombosortase, a subfamily of rhomboid protease, before T2SS transport. It remains unclear what sequence of the GlyGly-Cterm is required for surface localization, what, if any, enzymes in addition to rhombosortase are responsible for GlyGly-Cterm protein maturation, and importantly, why V. cholerae have this distinct surface- retention system. Model protein, VesB from V. cholerae, will be used to probe the molecular requirements of the GlyGly-Cterm domain by employing fluorescence microscopy, cell fractionation, and activity assays. Additional genes coding for putative enzymes possibly involved in processing of GlyGly-Cterm proteins have been identified in an ordered V. cholerae transposon library screen. This will be followed up on by generating clean gene deletions and assessing VesB surface localization. Quantitative and spatial proteomics as well as enzymatic assays will be used to characterize the role of these gene products. The importance of the GlyGly-Cterm will be probed by expressing GlyGly-Cterm proteins with and without this extension. Currently characterized cell-surface associated bacterial enzymes are almost exclusively retained by N-terminal lipidation, while the GlyGly-Cterm system represents a novel C-terminal cell-surface anchoring mechanism. The enzymes containing a GlyGly-Cterm are involved in a variety of functions contributing to V. cholerae environmental persistence and pathogenesis including nutrient acquisition, biofilm formation, and potentially maintenance of membrane integrity. The importance of GlyGly-Cterm proteins is demonstrated by disruption of rhombosortase, which results in impaired cell growth, reduced biofilm formation, and sensitivity to membrane stress. Characterization of this system has the potential to explain why some bacteria have evolved this unique cell-surface retention system and may provide insight into conserved mechanisms of C-terminal membrane anchoring in higher organisms.

摘要 霍乱仍然是一个全球负担，表现为影响贫困和 不稳定地区。包括霍乱弧菌在内的细菌利用II型分泌系统（T2 SS）进行 分泌多种效应蛋白和毒素，以适应环境变化，特别是霍乱 霍乱症状的病原体。虽然包括霍乱毒素在内的多种T2 SS底物是 完全分泌后，一个亚群保留在细胞表面。最近发现的一组不同的底物， 包括霍乱弧菌在内的一些革兰氏阴性细菌含有一个同源的C-末端结构域，称为GlyGly- Cterm，将其乘客蛋白定位于细胞表面。这个新识别的域由 菱形分选酶，菱形蛋白酶的一个亚家族，在T2 SS转运之前。目前还不清楚是什么序列 GlyGly-Cterm是表面定位所需的，除了菱形分选酶之外， 负责GlyGly-Cterm蛋白成熟，重要的是，为什么霍乱弧菌有这种独特的表面- 保留系统模型蛋白，来自霍乱弧菌的VesB，将用于探测 GlyGly-Cterm结构域，采用荧光显微镜，细胞分级分离和活性测定。额外 已经鉴定了编码可能参与GlyGly-Cterm蛋白质加工的推定酶的基因 在有序的霍乱弧菌转座子文库筛选中。这将通过生成干净的基因来跟进 缺失和评估VesB表面定位。定量和空间蛋白质组学以及酶 将使用测定来表征这些基因产物的作用。GlyGly-Cterm的重要性将是 通过表达具有和不具有该延伸的GlyGly-Cterm蛋白来探测。目前表征的细胞表面 相关的细菌酶几乎完全通过N-末端脂化保留，而GlyGly-Cterm 系统代表了一种新的C-末端细胞表面锚定机制。含有A的酶 GlyGly-Cterm参与多种功能，有助于霍乱弧菌的环境持久性， 发病机制，包括营养物质的获取，生物膜的形成，并可能维持膜 完整GlyGly-Cterm蛋白质的重要性通过菱形分选酶的破坏来证明， 导致细胞生长受损、生物膜形成减少和对膜应力敏感。表征 这一系统有可能解释为什么一些细菌已经进化出这种独特的细胞表面保留 系统，并可能提供深入了解保守的机制，C-末端膜锚定在更高的 有机体