REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

正确的蛋白质运输必须发生在所有原核和真核细胞的细胞膜上。所有系统中分泌蛋白的统一特征是对信号肽的需求。这项工作的长期目标是确定蛋白质分泌所必需的信号肽的物理性质，并描述信号肽与蛋白质运输装置组分之间的相互作用，这些组分用于指导蛋白质到达最终目的地。以大肠杆菌为模型系统，本研究的具体目的是：(1)确定模型前蛋白的特征，包括信号肽的调节因子，这些因子赋予了SecB依赖性；(2)根据信号肽和前蛋白的物理性质确定SecA的底物特异性；(3)确定信号肽结合所需的SecA的性质；(4)确定SecYEG如何调节信号肽的相互作用；(5)确定信号肽与分泌机制组分之间的相互作用如何整合以实现整体运输。在这些研究中，我们将以碱性磷酸酶为原型，用模型序列重新设计其信号肽或成熟区域，以放大某些性状，以测试疏水性，构象和电荷的作用。具有这些序列的突变体在体内被评估在野生型和Sec缺陷寄主菌株中完成不同分泌过程的程度。代表将用于体外分析，以建立具有特定性质的信号肽与Sec机制之间的直接相互作用。生化分析和与相应合成信号肽的直接结合研究旨在建立与我们在体内观察功能相同的体外结合层次。了解信号肽如何增强细菌的正确区隔化，将有助于理解其他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，运输其他蛋白质的载体设计以及运输抑制剂的开发。

The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. The unifying feature among secreted proteins in all systems is the requirement for a signal peptide. The long-term goals of this work are to identify the physical properties of signal peptides which are necessary for protein secretion, and to delineate the interactions between the signal peptide and components of the protein transport apparatus which serve to direct a protein to its final destination. Using Escherichia coli as a model system, the specific aims of the proposed research are to: (1)determine the characteristics of model preproteins, including the modulatory factors of signal peptides, which confer SecB dependence; (2)determine the substrate specificity of SecA with regard to the physical properties of signal peptides and preproteins; (3)determine the properties of SecA required for signal peptide binding; (4) determine how SecYEG modulates signal peptide interactions; (5)determine how interactions between the signal peptide and components of the secretion machinery are integrated to achieve transport overall. For these studies we will use alkaline phosphatase as a prototype, redesigning its signal peptide or mature regions with model sequences designed to amplify certain traits to test the roles of hydrophobicity, conformation, and charge. Mutants with these sequences are evaluated in vivo for the extent to which different steps of the secretion process are accomplished in wild type and Sec- deficient host strains. Representatives will be used for in vitro analyses to establish direct interactions between signal peptides with particular properties and the Sec machinery. Biochemical analyses and direct binding studies with the corresponding synthetic signal peptides are designed with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. Knowledge of how signal peptides enhance correct compartmentalization in bacteria will be useful in understanding secretion in other normal and diseased cells. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents, the design of vehicles to transport other proteins, and the development of transport inhibitors.