Pristimerin suppresses AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy.

ABSTRACT

Macroautophagy/autophagy plays an important role in regulating cellular homeostasis and influences the pathogenesis of degenerative diseases. Tendinopathy is characterized by tendon degeneration and inflammation. However, little is known about the role of selective autophagy in tendinopathy. Here, we find that pristimerin (PM), a quinone methide triterpenoid, is more effective in treating tendinopathy than the first-line drug indomethacin. PM inhibits the AIM2 inflammasome and alleviates inflammation during tendinopathy by promoting the autophagic degradation of AIM2 through a PYCARD/ASC-dependent manner. A mechanistic study shows that PM enhances the K63-linked ubiquitin chains of PYCARD/ASC at K158/161, which serves as a recognition signal for SQSTM1/p62-mediated autophagic degradation of the AIM2-PYCARD/ASC complex. We further identify that PM binds the Cys53 site of deubiquitinase USP50 through the Michael-acceptor and blocks the binding of USP50 to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains of PYCARD/ASC. Finally, PM treatment in vivo generates an effect comparable to inflammasome deficiency in alleviating tendinopathy. Taken together, these findings demonstrate that PM alleviates the progression of tendinopathy by modulating AIM2-PYCARD/ASC stability via SQSTM1/p62-mediated selective autophagic degradation, thus providing a promising autophagy-based therapeutic for tendinopathy.Abbreviations 3-MA 3-methyladenine; AIM2 absent in melanoma 2; AT Achilles tenotomy; ATP adenosine triphosphate; BMDMs bone marrow-derived macrophages; CHX cycloheximide; Col3a1 collagen, type III, alpha 1; CQ chloroquine; Cys cysteine; DARTS drug affinity responsive target stability; DTT dithiothreitol; DUB deubiquitinase; gDNA genomic DNA; GSH glutathione; His histidine; IL1B/IL-1ß interleukin 1 beta; IND indomethacin; IP immunoprecipitation; LPS lipopolysaccharide; MMP mitochondrial membrane potential; NLRP3 NLR family, pyrin domain containing 3; PM pristimerin; PYCARD/ASC PYD and CARD domain containing; SN supernatants; SOX9 SRY (sex determining region Y)-box 9; SQSTM1 sequestosome 1; Tgfb transforming growth factor, beta; TIMP3 tissue inhibitor of metalloproteinase 3; TNMD tenomodulin; TRAF6 TNF receptor-associated factor 6; Ub ubiquitin; USP50 ubiquitin specific peptidase 50; WCL whole cell lysates.