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）系统跨细胞质膜输出蛋白质。与大多数经过充分研究的蛋白质易位系统（将“线性化”或未折叠的多肽跨膜运输）不同，Tat 系统可易位完全折叠和组装的蛋白质和蛋白质复合物。 Tat 系统运输许多蛋白质，这些蛋白质在运输前必须组装复杂的金属氧化还原中心。在某些情况下，在运输组装的蛋白质复合物之前，必须建立不同亚基之间的四级接触。由于细菌细胞质膜支持离子梯度，一个未解决的主要问题是，在不破坏用于制造 ATP 的质子动力的情况下，Tat 机器可以将大于 100 kDa 的大蛋白质复合物转运穿过该膜。由于其能够运输在出口前必须完全折叠的大型蛋白质结构，Tat 机器作为细菌表达需要细胞质成熟的蛋白质治疗剂的系统，对生物技术行业具有潜在的重要意义。产品可以直接从生长培养基中回收。尽管 Tat 转运系统并不是所有编码它的生物体生长所必需的，但它负责许多细菌毒力因子的输出，并且功能性 Tat 系统的缺失通常会导致生长缺陷。考虑到 Tat 系统存在于许多细菌中，但在包括人类在内的动物中没有发现，Tat 系统可能是抗生素开发的绝佳靶标。目前，人们对 Tat 易位的机制知之甚少。三种膜蛋白 TatA、TatB 和 TatC 组成膜转位酶，在膜内形成许多寡聚复合物。跨膜电场对于驱动有效的运输（大概是通过门控孔）至关重要。表征蛋白质易位系统的常用方法包括在运输过程中捕获货物蛋白质，即形成易位中间体。迄今为止，达塔机构一直抵制这种做法。该提案的具体目标是开发一种高通量筛选方法，用于寻找 Tat 转运的候选抑制剂。初级筛选的阳性结果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 转运的机制研究，并将评估其药物潜力。 公共卫生相关性： 相关性：该提案寻求细菌双精氨酸易位 (Tat) 系统的抑制剂，该系统是负责输出许多细菌毒力因子并有助于细菌高效生长的蛋白质分泌机制。这些抑制剂将用于协助未来 Tat 转运的机制研究，并将评估其可能的药物潜力。了解 Tat 转运机制对于利用这种独特的系统进行需要细胞质成熟的蛋白质治疗药物的细菌表达至关重要。