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

Autophagy. 2024 Jan;20(1):76-93. doi: 10.1080/15548627.2023.2249392. Epub 2023 Aug 30.

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-1β: 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.

Keywords: AIM2; USP50; pristimerin; selective autophagy; tendinopathy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / genetics
  • DNA-Binding Proteins / metabolism
  • Deubiquitinating Enzymes / metabolism
  • Humans
  • Indomethacin / pharmacology
  • Inflammasomes* / metabolism
  • Inflammation
  • Interleukin-1beta / metabolism
  • Macroautophagy
  • NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
  • Sequestosome-1 Protein / metabolism
  • Tendinopathy*
  • Ubiquitin / metabolism

Substances

  • Inflammasomes
  • Sequestosome-1 Protein
  • celastrol methyl ester
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Ubiquitin
  • Indomethacin
  • Deubiquitinating Enzymes
  • Interleukin-1beta
  • AIM2 protein, human
  • DNA-Binding Proteins
  • PYCARD protein, human

Grants and funding

This work was supported by grants from National Natural Science Foundation of China (82171741), Guangdong Basic and Applied Basic Research Foundation (2019B1515120033, 2021A1515012140 and 2023A1515010421), Guangdong Zhujiang Youth Scholar funding, the Start-up Fund for High-level Talents of Southern Medical University to X. Yu, and the China Postdoctoral Science Foundation (2022M721504) to H. Jiang.