Redesign of Structural Regions of Alkaline Phosphatase

描述（由申请人提供）：蛋白质的正确转运必须发生在所有原核和真核细胞的膜上。文献充分表明，这些蛋白质的靶向和运输需要几种蛋白质成分，这些蛋白质成分包括细胞运输途径和分泌蛋白质的氨基端信号肽，以指导进入该途径。对于这些组件如何协同工作以实现传输过程知之甚少。该项目的重点是研究信号肽和前蛋白的成熟区如何与分泌机制的组成部分相结合，并探索使这些组成部分接受前蛋白易位的结构方面。以大肠杆菌为模型系统，结合诱变、生化和生物物理策略，拟开展的研究目的是：(1)确定SecA、信号肽和前蛋白的分子识别要求；(2)对SecA膜活性种的结构进行研究；(3)确定secy信号肽/前蛋白相互作用的性质；(4)确定前导肽酶I如何与转座子和新出现的前蛋白结合。这些研究将利用我们已经在体内和体外生成和表征的合成信号肽库、碱性磷酸酶信号肽突变体和截断的前蛋白。纯化的SecA， SecYEG和先导肽酶I将在膜模拟系统中重组或在内膜囊泡中检测，以表征与前蛋白相互作用的位点，相对亲和力，以及这些关键的蛋白质-蛋白质相互作用如何促进整体分泌。了解信号肽如何增强细菌的正确区隔化，有助于理解正常和病变细胞的分泌。发展的原则可以应用于治疗药物的组织特异性靶向和作为传统抗生素替代品的分泌抑制剂的抗菌剂的开发。

DESCRIPTION (provided by applicant): The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. It is well documented that the targeting and transport of these proteins requires several proteinaceous components which comprise the cellular transport pathway and a signal peptide at the amino-terminus of the secreted protein to direct entry into this pathway. Little is known about how these components function in concert to achieve the transport process. The focus of this project is to examine how the signal peptide and the mature region of the preprotein interface with components of the secretion machinery and to probe the structural aspects which render these components receptive to preprotein translocation. Using Escherichia coli as a model system, and a combination of mutagenesis, biochemical, and biophysical strategies, the aims of the proposed research are to: (1) determine the requirements for molecular recognition of SecA, signal peptide and preprotein; (2) examine the structure of the membrane active species of SecA; (3) determine the nature of SecY-signal peptide/preprotein interactions; (4) determine how leader peptidase I interfaces with the translocon and emerging preprotein. These studies will take advantage of the library of synthetic signal peptides, alkaline phosphatase signal peptide mutants, and truncated preproteins that we have generated andcharacterized in vivo and in vitro. Purified SecA, SecYEG, and leader peptidase I will be reconstituted in membrane mimetic systems or examined in inner membrane vesicles to characterize the sites of interaction with preproteins, the relative affinities, and how these critical protein-protein interactions propel secretion overall. Knowledge of how signal peptides enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials which are secretion inhibitors as alternatives to classical antibiotics.