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