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

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.

Mettl3-mediated m6A modification plays a role in lipid metabolism disorders and progressive liver damage in mice by regulating lipid metabolism-related gene expression.

AIMS: N6-methyladenosine (m6A), the most abundant and conserved epigenetic modification of mRNA, participates in various physiological and pathological processes. However, the roles of m6A modification in liver lipid metabolism have yet to be understood entirely. We aimed to investigate the roles of the m6A "writer" protein methyltransferase-like 3 (Mettl3) in liver lipid metabolism and the underlying mechanisms. MAIN METHODS: We assessed the expression of Mettl3 in liver tissues of diabetes (db/db) mice, obese (ob/ob) mice, high saturated fat-, cholesterol-, and fructose-induced non-alcoholic fatty liver disease (NAFLD) mice, and alcohol abuse and alcoholism (NIAAA) mice by quantitative reverse-transcriptase PCR (qRT-PCR). Hepatocyte-specific Mettl3 knockout mice were used to evaluate the effects of Mettl3 deficiency in mouse liver. The molecular mechanisms underlying the roles of Mettl3 deletion in liver lipid metabolism were explored by multi-omics joint analysis of public data from the Gene Expression Omnibus database and further validated by qRT-PCR and Western blot. KEY FINDINGS: Significantly decreased Mettl3 expression was associated with NAFLD progression. Hepatocyte-specific knockout of Mettl3 resulted in significant lipid accumulation in the liver, increased serum total cholesterol levels, and progressive liver damage in mice. Mechanistically, loss of Mettl3 significantly downregulated the expression levels of multiple m6A-modified mRNAs related to lipid metabolism, including Adh7, Cpt1a, and Cyp7a1, further promoting lipid metabolism disorders and liver injury in mice. SIGNIFICANCE: In summary, our findings demonstrate that the expression alteration of genes related to lipid metabolism by Mettl3-mediated m6A modification contributes to the development of NAFLD.