Allosteric regulation of the sensory protease DegS

描述(申请人提供)：细胞进化出错综复杂的相互作用蛋白质网络，调节基因表达以应对损伤、外部压力或化学信号。调节膜内蛋白分解(RIP)是一种高度保守的监测和反应机制。Ce包膜应激反应通路是在大肠杆菌和许多其他革兰氏阴性菌中发现的一种RIP系统。在热激条件下，外膜孔蛋白(OMP)错误折叠并聚集在周质中。新暴露的错误折叠的OMPS的C-末端与大肠杆菌DEGS的PDZ结构域结合，并通过变构激活蛋白酶活性。激活的PDZ-蛋白水解酶作为CE反应网络的分子开关，启动了一系列导致应激反应基因转录的事件。这项建议的过分目标是扩大我们对DEGS变构和监管机制的理解。将构建具有不同活性PDZ结构域和/或蛋白酶域组合的不对称DEGS三聚体，并对其进行生化表征，以询问对称性在DEGS激活和调节中的重要性。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。我们将研究DEGS单体内部和之间的变构通讯的作用。结构分析和基于序列的统计方法将被用来识别稳定活性或非活性DEGS的残基，并将构建合适的突变体并对其进行表征，以了解DEGS中变构的进化。最后，将开发一种荧光分析，以允许作为激活肽、底物和环境条件的函数的DEGS激活的动力学和热力学的严格测定。初步结果表明，用溶剂变色荧光团标记的DEGS以OMP多肽依赖的方式改变荧光，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力所必需的。由于DEGS作为包膜应激反应的分子守门人，它是抗生素抑制的一个有吸引力的靶点。因此，了解这种调节酶内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蛋白水解酶的范例，包括与caspase非依赖性细胞凋亡和癌症预防有关的人类同系物HtrA2/Omi。 公共卫生相关性：许多细菌的致病性和抗生素耐药性取决于它们检测和响应细胞外压力的能力。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。这项建议中描述的研究将拓宽和加深我们对DEGS感觉蛋白水解酶用来促进细菌在不适宜居住的条件下生存的变构机制的理解。

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.