High Throughput Screening for Tat Transport Inhibitors

Tat 转运抑制剂的高通量筛选

• 批准号: 7617460
• 负责人: SIEGFRIED M MUSSER
• 金额: $ 14.65万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617460
• 关键词: Animals; Antibiotics; Arginine; Automobile Driving; Bacteria; Biological Assay; Biotechnology; Cell membrane; Complex; Culture Media; Cytoplasm; Cytoplasmic Protein; Defect; Development; Endoplasmic Reticulum; Follow-Up Studies; Future; Genome; Growth; Growth Inhibitors; Health; Human; Industry; Ions; Membrane; Membrane Proteins; Methods; Microbial Physiology; Numbers; Organism; Oxidation-Reduction; Pathway interactions; Pharmacologic Substance; Plants; Protein Export Pathway; Protein Import; Protein Secretion; Protein translocation; Proteins; Proton-Motive Force; Public Health; Recombinant Proteins; Site; System; Toxic effect; Twin Multiple Birth; Virulence Factors; electric field; high throughput screening; in vitro Assay; inhibitor/antagonist; interest; periplasm; polypeptide; protein expression; protein structure; protein transport; secretory protein; small molecule; stem; therapeutic protein; translocase; transport inhibitor

DESCRIPTION (provided by applicant): The bacterial twin-arginine translocation (Tat) system exports proteins across the cytoplasmic membrane. Unlike most well-studied protein translocation systems, which transport "linearized," or unfolded, polypeptides across a membrane, the Tat system translocates fully folded and assembled proteins and protein complexes. The Tat system transports many proteins that must assemble complex metallo-redox centers before transport. In some cases, the quaternary contacts between distinct subunits must be established before an assembled protein complex can be transported. Since the bacterial cytoplasmic membrane supports ion gradients, a major unresolved question is how large protein complexes > 100 kDa can be transported across this membrane by the Tat machinery without collapsing the proton motive force used to make ATP. Due to its ability to transport large protein structures that must be fully folded before export, the Tat machinery is potentially important for the biotechnology industry as a system to bacterially express protein therapeutics that require a cytoplasm for maturation. Products could be recovered directly from the growth medium. Though the Tat transport system is not required for growth in all organisms that encode it, it is responsible for the export of a number of bacterial virulence factors, and the absence of a functional Tat system often leads to growth defects. Considering that the Tat system is found in many bacteria, but not found in animals, including humans, the Tat system is likely to be an excellent target for antibiotic development. Currently, the mechanism of Tat translocation is poorly understood. Three membrane proteins, TatA, TatB and TatC comprise the membrane translocase, forming numerous oligomeric complexes within the membrane. The transmembrane electric field is essential for driving efficient transport, presumably through a gated-pore. The common method to characterize protein translocation systems involves trapping a cargo protein during transport, that is, to form translocation intermediates. The Tat machinery has thus far resisted this approach. The Specific Aim of this proposal is to develop a high-throughput screening assay that will be used to search for candidate inhibitors of Tat transport. Positive hits from the primary screen will be validated using secondary screens and in vitro assays. Bona fide Tat transport inhibitors will be used to assist with mechanistic studies of Tat transport, and will be evaluated for pharmaceutical potential. PUBLIC HEALTH RELEVANCE: RELEVANCE: This proposal seeks inhibitors of the bacterial twin-arginine translocation (Tat) system, protein secretion machinery that is responsible for the export of a number of bacterial virulence factors, and that contributes to efficient bacterial growth. These inhibitors will be used to assist with future mechanistic studies of Tat transport, and will be evaluated for their possible pharmaceutical potential. Understanding the mechanism of Tat transport is essential for utilizing this unique system for the bacterial expression of protein therapeutics that requires a cytoplasm for maturation.

描述（由申请人提供）：细菌双精氨酸易位（TAT）系统在整个细胞质膜上导出蛋白质。与最研究的蛋白质易位系统不同，该系统在膜上传输“线性化或展开的多肽，TAT系统都会易位完全折叠和组装的蛋白质和蛋白质复合物。 TAT系统运输了许多蛋白质，这些蛋白质必须在运输前组装复杂的金属核中心。在某些情况下，必须在可以运输组装的蛋白质复合物之前建立不同亚基之间的第四纪接触。由于细菌细胞质膜支持离子梯度，因此一个主要的未解决的问题是，大型蛋白质复合物可以通过TAT机械在该膜上运输> 100 kDa，而不会折叠用于制造ATP的质子动力。由于其运输大型蛋白质结构必须在出口之前完全折叠的能力，因此TAT机械对于生物技术行业来说可能很重要，作为一种细菌表达需要细胞质成熟的细菌表达蛋白质疗法的系统。产品可以直接从生长培养基中回收。尽管所有编码的生物的生长并不需要TAT传输系统，但它是导致多种细菌毒力因子出口的原因，并且缺乏功能性TAT系统通常会导致生长缺陷。考虑到在许多细菌中发现了TAT系统，但在包括人类在内的动物中未发现，TAT系统可能是抗生素发育的绝佳靶标。目前，TAT易位的机制知之甚少。三种膜蛋白，TATA，TATB和TATC包括膜易位酶，形成膜内的许多低聚物复合物。跨膜电场对于通过门控孔进行有效运输至关重要。表征蛋白质易位系统的常见方法涉及在运输过程中捕获货物蛋白，即形成易位中间体。到目前为止，TAT机械已经抵抗了这种方法。该提案的具体目的是开发高通量筛选测定法，该测定法将用于寻找TAT运输的候选抑制剂。主要屏幕的正命中将使用二级筛选和体外测定进行验证。真正的TAT转运抑制剂将用于协助TAT运输的机械研究，并将评估药物潜力。 公共卫生相关性：相关性：该提案寻求细菌双精氨酸易位（TAT）系统，蛋白质分泌机制的抑制剂，该机制负责出口多种细菌毒力因子，并有助于有效的细菌生长。这些抑制剂将用于协助对TAT运输的未来机械研究，并将评估其可能的药物潜力。了解TAT转运的机制对于利用这种独特的系统来用于需要细胞质成熟的蛋白质疗法的细菌表达。