High Throughput Screening for Tat Transport Inhibitors

描述（由申请人提供）：细菌双精氨酸易位（Tat）系统通过细胞质膜输出蛋白质。与大多数研究充分的蛋白质易位系统不同，Tat系统易位的是完全折叠和组装的蛋白质和蛋白质复合物，这些蛋白质易位系统是通过膜运输“线性化”或未折叠的多肽。Tat系统运输许多蛋白质，这些蛋白质必须在运输前组装复杂的金属氧化还原中心。在某些情况下，不同亚基之间的四级接触必须在组装的蛋白质复合物运输之前建立。由于细菌细胞质膜支持离子梯度，一个主要的未解决的问题是，如何在不破坏用于制造ATP的质子动力的情况下，通过Tat机制将bbb100 kDa大的蛋白质复合物运输到细胞膜上。由于它能够运输在出口前必须完全折叠的大型蛋白质结构，Tat机器对于生物技术行业作为一种需要细胞质成熟的细菌表达蛋白质疗法的系统具有潜在的重要意义。产品可直接从培养基中回收。虽然Tat转运系统并不是所有编码该系统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，而缺乏功能性Tat系统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系统很可能成为抗生素开发的绝佳靶点。目前，Tat易位的机制尚不清楚。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传输是必不可少的，可能是通过门控孔。表征蛋白质易位系统的常用方法涉及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，泰国政府一直抵制这种做法。本提案的具体目的是开发一种高通量筛选试验，用于寻找Tat转运的候选抑制剂。主筛选的阳性结果将通过二次筛选和体外试验进行验证。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。

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.