USP16 drives psoriasis progression by deubiquitinating and stabilizing NLRP3 in keratinocytes
Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu
Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu
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Research Article Cell biology Dermatology

USP16 drives psoriasis progression by deubiquitinating and stabilizing NLRP3 in keratinocytes

Abstract

Psoriasis is a chronic inflammatory dermatosis characterized by pathological keratinocyte hyperproliferation and dysregulated immune activation. While ubiquitin-specific peptidase 16 (USP16) has been implicated in modulating multiple cellular signaling pathways, its functional role in psoriatic pathogenesis remains poorly understood. Our investigation revealed pronounced upregulation of USP16 expression in psoriatic epidermis compared with normal controls. Keratinocyte-specific USP16 knockdown demonstrated remarkable therapeutic efficacy, significantly ameliorating characteristic psoriatic phenotypes including epidermal hyperplasia and inflammatory infiltration. RNA-seq analysis showed that USP16 has substantial effects on cell cycle transition and keratinocytes proliferation. Through KEGG analysis, it was found that USP16 primarily regulates the NLRP3 signaling pathway, leading to enhanced cell proliferation and inflammation. Mechanically, USP16 directly binds to the NLRP3 protein to eliminate K48 ubiquitination modification, enhancing the stability of the NLRP3 protein, activating inflammasome activity. Further studies showed that the therapeutic effects of reducing USP16 on psoriasis progression were counteracted by an NLRP3 activator and keratinocyte-specific NLRP3 overexpression adenovirus. Collectively, these results shed light on how USP16 promotes NLRP3 signaling in keratinocytes, exacerbating psoriasis development. This positive regulation highlights the potential of USP16 as a therapeutic target for psoriasis.

Authors

Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu

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Figure 5

USP16 interacted and stabilized NLRP3 by inhibiting its K48 ubiquitination.

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USP16 interacted and stabilized NLRP3 by inhibiting its K48 ubiquitinati...
(A) Immunoblotting and quantitative analysis of NLRP3 protein level that Flag-USP16 or Vector were transfected in HaCaT cells. β-Actin was used as a loading control (n = 4). (B) qPCR analysis for NLRP3 mRNA level that si-Scr or si-USP16 were transfected in HaCaT cells treated with M5 for 24 hours (n = 3). (C) Immunoblotting and quantitative analysis of NLRP3 protein levels that Flag-USP16 or Vector were transfected in HaCaT cells treated with cyclohexamide (CHX) for 0 and 4 hours. β-Actin was used as a loading control (n = 3). (D) Immunoblotting analysis of NLRP3 protein level that si-Scr or si-USP16 were transfected in HaCaT cells treated with MG132 or CQ for 6 hours and stimulated M5 for 24 hours. β-Actin was used as a loading control (n = 3). (E) Immunofluorescent staining of USP16 and NLRP3 in HaCaT cells. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. (F) The HaCaT cell lysates were immunoprecipitated with IgG and anti-USP16 antibody and the expression of USP16 and NLRP3 were detected by Western blot. (G) Molecular docking analysis of USP16 binding to NLRP3 (confidence score: 0.8593). (H) Coimmunoprecipitation experiment was carried out to determine the specific binding domain between Flag-USP16 and GST-NLRP3. (I) USP16 amino acid fragment diagram. (J) HEK293T cells were transfected with HA-NLRP3 and several Flag-USP16 mutants. The whole-cell lysates were immunoprecipitated with anti-HA beads and immunoblotted with anti-Flag and anti-HA antibodies. (K) The HEK293T cells were cotransfected with Flag-USP16 and HA-Ub plasmids. The cell lysates were immunoprecipitated by anti-NLRP3 antibody, and then Western blot assay with anti-HA, anti-NLRP3, and anti-Flag antibody. (L) The HEK293T cells were cotransfected with Flag-USP16 and HA-Ub-K48 plasmids. The cell lysates were immunoprecipitated by anti-NLRP3 antibody, and then Western blot assay with anti-HA, anti-NLRP3, and anti-Flag antibody. Data are shown as the mean ± SEM. *P < 0.05; ***P < 0.001. The P value was determined using unpaired, 2-tailed Student’s t test (A–C). All numbers (n) are biologically independent experiments.