USP5 attenuates NLRP3 inflammasome activation by promoting autophagic degradation of NLRP3

The NLRP3 (NLR family pyrin domain containing 3) inflammasome is involved in diverse inflammatory diseases, thus strict control of its activation is necessary to prevent excessive inflammation. Protein ubiquitination has been reported to regulate the assembly, protein expression and activation of the NLRP3 inflammasome. Until now, several deubiquitinases (DUBs) have been reported to affect the degradation of NLRP3 through the proteasome pathway. However, there is no research on DUBs regulating NLRP3 degradation through macroautophagy/autophagy. Here, we demonstrated the pivotal function of USP5 (ubiquitin specific peptidase 5) in restraining the activation of the NLRP3 inflammasome independent of its deubiquitinating enzyme activity. USP5 selectively promoted K48-linked polyubiquitination of NLRP3 and mediated its degradation through the autophagy-lysosomal pathway by recruiting the E3 ligase MARCHF7/MARCH7. Knockdown of <i>USP5</i> facilitated the two-signal model of lipopolysaccharide and ATP-triggered IL1B/IL-1β production. Simultaneously, USP5 overexpression <i>in vivo</i> reduced IL1B and polymorphonuclear (PMN) infiltration in alum-induced peritonitis. Overall, the data revealed that USP5 is a key scaffold protein recruiting the E3 ligase MARCHF7 to NLRP3, and promoting autophagic degradation of NLRP3. The findings provide new insight into USP5 in the regulation of excessive activation of the NLRP3 inflammasome and inflammatory innate immune response. 3-MA: 3-methyladenine; AIM2: absent in melanoma 2; ATG5: autophagy related 5; BafA1: bafilomycin A₁; CASP1: caspase 1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CQ: chloroquine; DUBs: deubiquitinases; IL1B/IL-1β: interleukin 1 beta; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MARCHF7/MARCH7: membrane associated RING-CH-type finger 7; NFKB/NF-κB: nuclear factor kappa B; Nig.: nigericin; NLRC4: NLR family CARD domain containing 4; NLRP3: NLR family pyrin domain containing 3; PECs: peritoneal exudate cells; PMN: polymorphonuclear; PMs: peritoneal macrophages; PYCARD/ASC: PYD and CARD domain containing; TLRs: toll like receptors; TNF/TNF-α: tumor necrosis factor; Ub: ubiquitin; USP5: ubiquitin specific peptidase 5; WT: wild type.