Molecular Mechanisms of Protein Sorting by the Type II Secretion System

II 型分泌系统蛋白质分选的分子机制

• 批准号: 10239182
• 负责人: Maria B Sandkvist
• 金额: $ 39.39万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239182
• 关键词: Acinetobacter baumannii; Acute Diarrhea; Biochemical; Biological Models; C-terminal; Caspase; Cell Wall; Cell surface; Cells; Cellular biology; Charge; Chimera organism; Chitinase; Cholera; Cholera Toxin; Cholera Toxin Protomer B; Cholera Vaccine; Cleaved cell; Collaborations; Data; Destinations; Diabetes Mellitus; Diarrhea; Disease; Disease model; Engineering; Environment; Enzymes; Ethanolamines; Eukaryota; Extracellular Matrix Proteins; Extracellular Space; Family; Future; GPI Membrane Anchors; Glycine; Gram-Negative Bacteria; Health; Human; Hydrophobicity; Individual; Infection; Knowledge; Lipase; Lipids; Lipoproteins; Literature; Malignant Neoplasms; Mediating; Medical; Medicine; Membrane; Membrane Proteins; Microbial Biofilms; Modification; Molecular; Molecular Conformation; Multiprotein Complexes; Mus; Mutagenesis; Organism; Parasites; Parkinson Disease; Pathway interactions; Peptide Hydrolases; Phosphatidylethanolamine; Process; Production; Protein Sortings; Proteins; Public Health; Role; Serine Protease; Signal Transduction; Sorting - Cell Movement; Specificity; Structure; Support System; Surface; Symptoms; System; TRIM Motif; Testing; Tissues; Toxin; Trypsin; Type II Secretion System Pathway; Vesicle; Vibrio; Vibrio cholerae; Virulence; Virulence Factors; cell envelope; cytotoxicity; design; diarrheal disease; experimental study; extracellular; human disease; human pathogen; immunogenic; immunogenicity; insight; member; mouse model; novel; nuclease; pathogen; periplasm; rhomboid; sortase; suckling; vaccine development

Cholera, an acute diarrheal disease, remains a global burden to human health. The key factor chiefly responsible for this devastating disease is cholera toxin, an AB5 toxin that is produced and secreted by Vibrio cholerae. Its extracellular secretion is dependent on the type II secretion system (T2SS), which is also responsible for the outer membrane translocation of proteases, lipases, nucleases and chitinases. Common to many Gram-negative pathogens, it uniquely transports these factors from the periplasmic compartment to the extracellular environment in their fully folded conformations. Despite a dramatic increase in structural knowledge of the T2SS and its individual components in recent years, the mechanism by which T2S substrates are recognized and sorted for outer membrane translocation remains to be determined. A lack of significant sequence and structural similarity between these proteins complicates the identification of a common secretion signal. Other confounding factors include the findings that the T2SS supports the extracellular transport of both soluble proteins and lipoproteins and that there are differences in the final destination among T2S substrates. Some T2S substrates such as cholera toxin are released to the extracellular space once transported through the outer membrane; however, others remain surface associated or may reattach to the bacterial cell surface following extracellular release. The trypsin-like protease VesB is an example of a T2S substrate that is primarily retained on the cell surface. VesB belongs to a unique class of extracellular enzymes that have a C-terminal extension consisting of two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM domain). Rhombosortase, a newly discovered member of the intramembrane rhomboid protease family, cleaves off the GlyGly-CTERM domain during transit of VesB through the cell envelope, and the posttranslationally modified VesB is localized to the cell surface. The experiments described in this proposal are designed to test the hypothesis that the T2S system, in collaboration with rhombosortase, mediates the maturation and surface localization of GlyGly-CTERM containing proteins. Specifically, this proposal will determine the mechanism of surface anchoring of proteins produced with GlyGly-CTERM extensions, decipher how GlyGly-CTERM proteins are differentially recognized and secreted by the T2SS, and assess the T2SS/rhombosortase system for surface localization of GlyGly-CTERM-tagged heterologous proteins. The findings will facilitate understanding of the function and specificity of rhombosortase as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the T2S/rhombosortase system for preventative use.

霍乱是一种急性腹泻病，仍然是全球人类健康的负担。主要负责的关键因素 造成这种毁灭性疾病的罪魁祸首是霍乱毒素，这是一种由霍乱弧菌产生和分泌的 AB5 毒素。它是 细胞外分泌依赖于 II 型分泌系统 (T2SS)，该系统也负责 蛋白酶、脂肪酶、核酸酶和几丁质酶的外膜易位。许多革兰氏阴性菌共有 病原体，它独特地将这些因子从周质室运输到细胞外 处于完全折叠构象的环境。尽管 T2SS 的结构知识显着增加 近年来，T2S底物的识别机制和其各个组成部分 外膜易位的分类仍有待确定。缺乏重要的顺序和结构 这些蛋白质之间的相似性使得共同分泌信号的识别变得复杂。其他混杂因素 因素包括 T2SS 支持可溶性蛋白质和细胞外转运的发现 脂蛋白，并且 T2S 底物之间的最终目的地存在差异。一些T2S底物 例如霍乱毒素通过外膜转运后释放到细胞外空间； 然而，其他细菌仍保持表面相关或可能在细胞外细胞后重新附着到细菌细胞表面。 发布。胰蛋白酶样蛋白酶 VesB 是主要保留在细胞上的 T2S 底物的一个例子 表面。 VesB 属于一类独特的胞外酶，其 C 末端延伸由以下组成： 两个突出的甘氨酸和一个疏水性螺旋，后面是带正电荷的残基（GlyGly-CTERM 领域）。菱形排序酶是膜内菱形蛋白酶家族的新发现成员，可裂解 在 VesB 通过细胞膜的过程中，GlyGly-CTERM 结构域会被关闭，并且翻译后 修饰的 VesB 定位于细胞表面。 本提案中描述的实验旨在测试 T2S 系统的假设 与菱形排序酶合作，介导含有 GlyGly-CTERM 的成熟和表面定位 蛋白质。具体来说，该提案将确定用以下物质产生的蛋白质的表面锚定机制： GlyGly-CTERM 扩展，破译 GlyGly-CTERM 蛋白如何被差异性识别和分泌 T2SS，并评估 T2SS/rhombosortase 系统对 GlyGly-CTERM 标记的表面定位 异源蛋白质。这些发现将有助于理解菱形排序酶的功能和特异性 以及更广泛的医学相关菱形蛋白酶类别，并可能确定操纵 用于预防性用途的 T2S/菱形排序酶系统。