High Throughput Screening for Tat Transport Inhibitors

描述（由申请方提供）：细菌双精氨酸易位（达特）系统跨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，该系统转运“线性化”或未折叠的多肽穿过膜，达特系统易位完全折叠和组装的蛋白质和蛋白质复合物。达特系统转运许多蛋白质，这些蛋白质在转运前必须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可以被运输之前建立。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大的蛋白质复合物> 100 kDa可以通过达特机制运输通过这个膜，而不崩溃的质子动力用于制造ATP。由于其运输在输出之前必须完全折叠的大蛋白质结构的能力，达特机构作为细菌表达需要细胞质成熟的蛋白质治疗剂的系统对于生物技术工业是潜在重要的。产物可以直接从生长培养基中回收。虽然达特转运系统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，并且功能性达特系统的缺乏通常导致生长缺陷。考虑到达特系统存在于许多细菌中，但在包括人类在内的动物中未发现，因此达特系统可能是抗生素开发的极好靶标。目前，达特易位的机制知之甚少。三种膜蛋白TatA、TatB和TatC构成膜转位酶，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是必不可少的，可能通过门控孔。表征蛋白质易位系统的常用方法涉及在转运过程中捕获货物蛋白，即形成易位中间体。迄今为止，达特机构一直抵制这种做法。本提案的具体目的是开发一种高通量筛选试验，用于寻找达特转运的候选抑制剂。将使用二次筛选和体外试验验证初步筛选的阳性命中。将使用真正的达特转运抑制剂辅助达特转运的机制研究，并评价其药物潜力。 公共卫生相关性：相关性：该提议寻求细菌双精氨酸易位（达特）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。这些抑制剂将用于协助未来的达特运输机制研究，并将评估其可能的药物潜力。了解达特转运的机制对于利用这种独特的系统用于需要细胞质成熟的蛋白质治疗剂的细菌表达是必不可少的。

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.