期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：43
页码：13237-13242
DOI：10.1073/pnas.1507579112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceInvading pathogens and other danger-associated signals are recognized by the innate immune system. Subsequently, the eukaryotic protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)] assembles to long filaments, which might serve to amplify the signal and activate an inflammatory response. We have determined the structure of the mouse ASC filament at atomic resolution. The pyrin domain of ASC forms the helical filament core, and the CARD, thus far elusive to experimental observation, is flexibly unfolded on the filament periphery. The integration of data from two structural methods, cryo-electron microscopy and solid-state NMR spectroscopy, opens perspectives for structural studies of inflammasomes and related molecular assemblies. Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.
关键词：inflammation ; protein structure ; protein filament ; ASC speck ; innate immune response
