High Throughput Screening for Tat Transport Inhibitors

描述(申请人提供)：细菌双精氨酸转位(TAT)系统通过细胞质膜输出蛋白质。与大多数研究良好的蛋白质转运系统不同，TAT系统转运的是完全折叠和组装的蛋白质和蛋白质复合体，而不是“线性化的”或未折叠的多肽跨膜运输。TAT系统运输许多蛋白质，这些蛋白质在运输之前必须组装复杂的金属氧化还原中心。在某些情况下，在组装的蛋白质复合体可以运输之前，必须建立不同亚基之间的四元联系。由于细菌细胞质膜支持离子梯度，一个尚未解决的主要问题是，TAT机器如何在不破坏用于制造ATP的质子动力的情况下，将大的蛋白质复合体&GT；100 kDa输送过细胞膜。由于其能够运输必须在出口前完全折叠的大型蛋白质结构，TAT机制作为一种细菌表达需要细胞质才能成熟的蛋白质疗法的系统，对生物技术行业具有潜在的重要意义。产品可直接从生长介质中回收。尽管TAT运输系统并不是编码TAT的所有生物体生长所必需的，但它负责许多细菌毒力因子的输出，而缺乏功能齐全的TAT系统往往会导致生长缺陷。考虑到在许多细菌中都发现了TAT系统，但在包括人类在内的动物中没有发现，TAT系统很可能是开发抗生素的一个很好的目标。目前，人们对TAT易位的机制知之甚少。TATA、TatB和TatC三种膜蛋白组成了膜转位酶，在膜内形成了大量的寡聚复合体。跨膜电场对于驱动有效的传输是必不可少的，大概是通过门控孔。描述蛋白质转运系统的常见方法包括在运输过程中捕获货物蛋白质，即形成转运中间产物。到目前为止，TAT机器一直抵制这种方法。这项建议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。一次筛查的阳性结果将使用二次筛查和体外试验进行验证。真正的TAT转运抑制剂将被用于辅助TAT转运的机制研究，并将被评估为药物潜力。 公共卫生相关性：相关性：这项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有效生长的蛋白质分泌机制。这些抑制剂将被用来协助未来对TAT转运的机制研究，并将对其可能的药物潜力进行评估。了解TAT转运的机制对于利用这一独特的系统进行蛋白质疗法的细菌表达至关重要，因为蛋白质疗法需要细胞质才能成熟。

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.