Allosteric regulation of the sensory protease DegS

描述（由申请人提供）：细胞已经进化出相互作用的蛋白质的复杂网络，这些蛋白质调节基因表达以响应损伤、外部压力或化学信号。受调节的膜内蛋白水解（RIP）是一种高度保守的监视和反应机制。Ce胁迫反应途径是在大肠杆菌和许多其他革兰氏阴性细菌中发现的RIP系统。在热休克条件下，外膜孔蛋白（OMP）错误折叠并积累在周质中。新暴露的错误折叠的外膜蛋白的C-末端与E. coliDegS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，启动导致应激反应基因转录的级联事件。这项提案的超越目标是扩大我们对DegS变构和调节机制的理解。将构建具有活性PDZ结构域和/或蛋白酶结构域的不同组合的不对称DegS三聚体并进行生物化学表征，以询问DegS活化和调节中对称性的重要性。关键的突变蛋白质将被结晶，它们的结构将被解析，以帮助解释生化数据。将检查DegS单体内和之间的变构通信的作用。将采用结构分析和基于序列的统计方法来鉴定稳定活性或非活性DegS的残基，并且将构建和表征适当的突变体以理解DegS内变构的进化。最后，将开发荧光测定法，以允许严格测定DegS活化的动力学和热力学，作为活化肽、底物和环境条件的函数。初步结果表明，DegS标记的溶剂化显色荧光团改变荧光在一个OMP-肽依赖性的方式，设置这些研究的阶段。在许多细菌中，Ce相关基因的表达是致病所必需的。由于DegS作为细菌应激反应的分子看门人，因此它是抗生素抑制的有吸引力的靶标。因此，了解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，包括与半胱天冬酶非依赖性细胞凋亡和癌症预防相关的人类同源物HtrA 2/Omi。 公共卫生相关性：许多细菌的致病性和抗生素抗性取决于它们检测和响应细胞外应激的能力。因此，了解反应系统的分子调控机制对人类健康具有重要意义。本提案中描述的研究将拓宽和加深我们对DegS感觉蛋白酶用于促进细菌在恶劣条件下存活的变构机制的理解。

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.