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.

NLR家族pyrin结构域包含3（NLRP3, NLR family pyrin domain containing 3）炎性小体参与多种炎症性疾病，因此严格调控其激活对于预防过度炎症反应至关重要。已有研究证实，蛋白质泛素化可调控NLRP3炎性小体的组装、蛋白表达与激活过程。截至目前，已有数种去泛素化酶（deubiquitinases, DUBs）被报道可通过蛋白酶体通路调控NLRP3的降解，但尚未有研究探讨去泛素化酶通过巨自噬/自噬通路调控NLRP3降解的相关机制。
本研究证实，泛素特异性蛋白酶5（USP5, ubiquitin specific peptidase 5）可在不依赖其自身去泛素化酶活性的情况下，发挥负向调控NLRP3炎性小体激活的关键作用。USP5可选择性促进NLRP3的K48位连接的多聚泛素链修饰，并通过招募E3泛素连接酶MARCHF7/MARCH7（membrane associated RING-CH-type finger 7），通过自噬-溶酶体通路介导NLRP3的降解。敲低USP5可增强脂多糖（lipopolysaccharide, LPS）与ATP诱导的双信号模型中白细胞介素1β（IL1B/IL-1β, interleukin 1 beta）的产生水平。同时，在体内过表达USP5可缓解明矾诱导的腹膜炎模型中IL1β的表达及多形核白细胞（polymorphonuclear, PMN）的浸润程度。
综上，本研究数据表明，USP5作为关键支架蛋白，可招募E3泛素连接酶MARCHF7至NLRP3，进而促进NLRP3的自噬性降解。本研究结果为USP5调控NLRP3炎性小体过度激活以及先天性炎症免疫应答提供了全新的研究视角。
3-MA：3-甲基腺嘌呤（3-methyladenine）；AIM2：黑色素瘤缺失蛋白2（absent in melanoma 2）；ATG5：自噬相关蛋白5（autophagy related 5）；BafA1：巴弗洛霉素A₁（bafilomycin A₁）；CASP1：半胱氨酸天冬氨酸蛋白酶1（caspase 1）；CHX：环己酰亚胺（cycloheximide）；Co-IP：免疫共沉淀（co-immunoprecipitation）；CQ：氯喹（chloroquine）；DUBs：去泛素化酶（deubiquitinases）；IL1B/IL-1β：白细胞介素1β（interleukin 1 beta）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；LPS：脂多糖（lipopolysaccharide）；MARCHF7/MARCH7：膜相关RING-CH型指蛋白7（membrane associated RING-CH-type finger 7）；NFKB/NF-κB：核因子κB（nuclear factor kappa B）；Nig.：尼日利亚菌素（nigericin）；NLRC4：NLR家族CARD结构域包含4（NLR family CARD domain containing 4）；NLRP3：NLR家族pyrin结构域包含3（NLR family pyrin domain containing 3）；PECs：腹腔渗出细胞（peritoneal exudate cells）；PMN：多形核白细胞（polymorphonuclear）；PMs：腹腔巨噬细胞（peritoneal macrophages）；PYCARD/ASC：PYD和CARD结构域包含蛋白（PYD and CARD domain containing）；TLRs：Toll样受体（toll like receptors）；TNF/TNF-α：肿瘤坏死因子α（tumor necrosis factor）；Ub：泛素（ubiquitin）；USP5：泛素特异性蛋白酶5（ubiquitin specific peptidase 5）；WT：野生型（wild type）