Allosteric regulation of the sensory protease DegS

描述（由申请人提供）：细胞已经进化出复杂的相互作用蛋白质网络，这些相互作用蛋白质调节基因表达，以响应损伤，外部压力或化学信号。调节膜内蛋白水解（RIP）是一种高度保守的监测和反应机制。Ce包膜-应激反应途径是在大肠杆菌和许多其他革兰氏阴性菌中发现的RIP系统。在热休克条件下，外膜孔蛋白（OMP）错误折叠并积聚在外周质中。新暴露的错误折叠OMPs的c端与大肠杆菌DegS的PDZ结构域结合，并变构激活蛋白酶活性。激活的pdz蛋白酶作为Ce反应网络的分子开关，启动一系列事件，导致应激反应基因的转录。本提案的超额目标是扩大我们对DegS变构和调节机制的理解。具有活性PDZ结构域和/或蛋白酶结构域不同组合的不对称DegS三聚体将被构建和生物化学表征，以询问对称在DegS激活和调节中的重要性。关键的突变蛋白将被结晶，它们的结构将被解决，以帮助解释生化数据。DegS单体内部和之间的变构通信的作用将被检查。将采用结构分析和基于序列的统计方法来确定稳定活性或非活性deg的残基，并构建和表征合适的突变体，以了解deg内变构的演变。最后，将开发一种荧光测定法，以严格确定DegS激活的动力学和热力学，作为激活肽、底物和环境条件的函数。初步结果表明，用溶剂致变色荧光团标记的DegS以依赖于omp肽的方式改变荧光，为这些研究奠定了基础。在许多细菌中，Ce相关基因的表达是毒性所必需的。由于DegS作为包膜-应激反应的分子看门人，它是抗生素抑制的一个有吸引力的靶点。因此，了解在这种调节蛋白酶中进化的变构机制可能为新的治疗方法奠定基础。最后，了解DegS功能的分子决定因素可能会导致发现适用于其他pdz蛋白酶的范例，包括与caspase非依赖性细胞凋亡和癌症预防相关的人类同源物HtrA2/Omi。

DESCRIPTION (provided by applicant): Cells have evolved intricate networks of interacting proteins that regulate gene expression in response to damage, external stress, or chemical signals. Regulated intramembrane proteolysis (RIP) is a highly conserved surveillance and response mechanism. The Ce envelope-stress response pathway is a RIP system found in Escherichia coli and many other Gram-negative bacteria. Under heat-shock conditions, outer membrane porins (OMP) misfold and accumulate in the periplasm. The newly exposed C-terminus of the misfolded OMPs binds to the PDZ domain of E. coli DegS and allosterically activates protease activity. The activated PDZ-protease serves as a molecular switch for the Ce response network, setting in motion a cascade of events that leads to the transcription of stress-response genes. The overreaching goal of this proposal is to expand our understanding of the mechanism of DegS allostery and regulation. Asymmetric DegS trimers with different combinations of active PDZ domains and/or protease domains will be constructed and characterized biochemically to interrogate the importance of symmetry in DegS activation and regulation. Key mutant proteins will be crystallized and their structures solved to aid in interpreting the biochemical data. The role of allosteric communication within and between DegS monomers will be examined. Structural analysis and sequence based statistical methods will be employed to identify residues that stabilize active or inactive DegS, and appropriate mutants will be constructed and characterized to understand the evolution of allostery within DegS. Finally, a fluorescence assay will be developed to allow rigorous determination of the kinetics and thermodynamics of DegS activation as a function of activating peptide, substrate, and environmental conditions. Preliminary results indicate that DegS labeled with a solvatochromic fluorophore changes fluorescence in an OMP-peptide dependent manner, setting the stage for these studies. In many of bacteria, Ce related gene expression is required for virulence. As DegS serves as the molecular gatekeeper for the envelope-stress response, it is an attractive target for antibiotic inhibition. Thus, understanding the allosteric mechanism that has evolved within this regulatory protease may lay a foundation for novel therapeutics. Finally, understanding the molecular determinants of DegS function may lead to the discovery of paradigms that are applicable to other PDZ-proteases including the human homolog HtrA2/Omi that has been linked to caspase-independent apoptosis and the prevention of cancer. PUBLIC HEALTH RELEVANCE: The pathogenicity and antibiotic resistance of many bacteria depends on their ability to detect and respond to extracellular stress. Therefore, understanding the molecular control mechanisms of the response systems is of importance to human health. The research described in this proposal will broaden and deepen our understanding of the allosteric mechanisms used by the DegS sensory protease to facilitate bacterial survival under inhospitable conditions.