ANALYSIS OF EUKARYOTIC PROTEIN KINASES AND PHOSPHO-REGULATORY SYSTEMS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 内质网(ER)未折叠蛋白反应(UPR)中最保守的分支信号是由内质网应激诱导的激酶内切酶IRE1对HAC1/XBP1 mRNA的序列特异性切割而启动的。我们发现，黄酮醇、槲皮素能激活酵母IRE1‘S核糖核酸酶，并能增强参与IRE1核苷酸结合裂解的天然激活配体ADP的激活作用。酶动力学和共晶体结构表明，在IRE1‘S激酶延伸核酸酶(KEN)结构域的二聚体界面上，有一个意想不到的配体结合口袋与之结合。分析性超速离心法和交联法研究支持了在槲皮素的作用机制中增强二聚体形成的优势。这些发现暗示了内源性细胞质配体的存在，这些配体可能与来自内质网管腔的应激信号一起发挥作用，以调节IRE1的活性，并暗示有可能开发出修改来自这个意想不到的位置的UPR信号的药物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.