UXT attenuates the CGAS-STING1 signaling by targeting STING1 for autophagic degradation

Abstract

STING1 (stimulator of interferon response cGAMP interactor 1), the pivotal adaptor protein of CGAS (cyclic GMP-AMP synthase)-STING1 signaling, is critical for type I IFN production of innate immunity. However, excessive or prolonged activation of STING1 is associated with autoinflammatory and autoimmune diseases. Thus, preventing STING1 from over-activation is important to maintain immune homeostasis. Here, we reported that UXT (ubiquitously expressed prefoldin like chaperone), a small chaperone-like protein, was essential to prevent the excessive activation of STING1-mediated type I IFN signaling through autophagic degradation of STING1 via SQSTM1 (sequestosome 1). Upon DNA mimics or cyclic GMP-AMP (cGAMP) stimulation, UXT specifically interacted with STING1 and promoted STING1 degradation through selective macroautophagy/autophagy. Moreover, UXT was required for more efficient autophagic degradation of STING1 by facilitating the interaction of SQSTM1 and STING1. The in vivo role of UXT in attenuating the CGAS-STING1 signaling was further confirmed in the mouse model of DNA-virus infection and the TMPD (2,6,10,14-tetramethylpentadecane)-induced murine lupus model. Intriguingly, the expression of UXT was consistently impaired and exhibited a remarkable inverse correlation with type I IFN signature in the leukocytes and PBMCs (peripheral blood mononuclear cells) of several large SLE (systemic lupus erythematosus) cohorts. Importantly, the replenishment of UXT effectively suppressed the production of IFNs and ISGs in the PBMCs of SLE patients. Taken together, our study reveals a novel regulatory role of UXT in autophagic degradation of STING1 to maintain immune homeostasis. UXT might be a potential therapeutic target for alleviating aberrant type I IFNs in autoimmune diseasesAbbreviations: 3-MA: 3-methyladenine; BMDMs: bone marrow-derived macrophages; cGAMP: cyclic GMP-AMP; CGAS: cyclic gmp-amp synthase; cKO: conditional knockout; CXCL10: C-X-C motif chemokine ligand 10; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HSV-1: herpes simplex virus type 1; HTDNA: herring testes DNA; IFIT1: interferon induced protein with tetratricopeptide repeats 1; IFNA4: interferon alpha 4; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISD: interferon stimulatory DNA; ISGs: IFN-stimulated genes; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; RNA-seq: RNA sequencing; PBMCs: peripheral blood mononuclear cells; RSAD2: radical S-adenosyl methionine domain containing 2; SLE: systemic lupus erythematosus; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TMPD: 2,6,10,14-tetramethylpentadecane; UXT: ubiquitously expressed prefoldin like chaperone.

Keywords: Autophagic degradation; SLE; SQSTM1; STING1; UXT.

Figure 1.

UXT is a novel regulator of the CGAS-STING1 signaling pathway. (A) MEF cells were transfected with the indicated siRNAs. Cell lysates were collected for western blot analysis of UXT and GAPDH. (B) The heatmap of expression changes of differentially expressed ISGs between negative control (NC) or Uxt siRNA-transfected MEFs stimulated with cGAMP (1 μg per well) for 3 h. For each gene, the expression level was normalized (Z-score) across samples. (C) Significantly enriched biological processes of the upregulated DE genes (FDR<0.01). (D) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Ifnb, Ifna4, and Cxcl10 mRNAs was measured by real-time PCR. (E) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Isg15, Ifit1, and Rsad2 mRNAs was measured by real-time PCR. (F) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with HTDNA (2 μg per well) for 3 h. Then, the induction of Ifnb, Ifna4, and Cxcl10 mRNAs was measured by real-time PCR. (G) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with HTDNA (2 μg per well) for 3 h. Then, the induction of Isg15, Ifit1, and Rsad2 mRNAs was measured by real-time PCR. (H) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with HTDNA for 0, 3, or 6 h. Then, cell lysates were collected for western blot analysis of UXT, IRF3, IRF3 phosphorylation, and ACTB. (I) MEFs transfected with negative control (NC) or Uxt siRNAs were stimulated with cGAMP for 0, 3, or 6 h. Then, cell lysates were collected for western blot analysis of UXT, IRF3, IRF3 phosphorylation, and ACTB. Graphs show the mean ± SEM, and the data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Two-tailed t-test).

Figure 2.

UXT represses the CGAS-STING1 signaling responses. (A) MEF cells were transfected with the indicated plasmids. Cell lysates were collected for western blot analysis of UXT and GAPDH. (B) The heatmap of expression changes of differentially expressed ISGs between control vectors (Vec) or UXT expression plasmids (UXT) transfected MEFs stimulated with cGAMP (1 μg per well) for 3 h. For each gene, the expression level was normalized (Z-score) across samples. (C) Significantly enriched biological processes of the downregulated DE genes (FDR<0.01). (D) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Ifnb, Ifna4, and Cxcl10 mRNAs was measured by real-time PCR. (E) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Isg15, Ifit1, and Rsad2 mRNAs was measured by real-time PCR. (F) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA (2 μg per well) for 3 h. Then, the induction of Ifnb, Ifna4, and Cxcl10 mRNAs was measured by real-time PCR. (G) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA (2 μg per well) for 3 h. Then, the induction of Isg15, Ifit1, and Rsad2 mRNAs was measured by real-time PCR. (H) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA for 0, 3,6 h, respectively. Then, cell lysates were collected for western blot analysis of UXT, IRF3, IRF3 phosphorylation and ACTB. (I) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with cGAMP for 0, 3,6 h, respectively. Then, cell lysates were collected for western blot analysis of UXT, IRF3, IRF3 phosphorylation and ACTB. Graphs show the mean ± SEM, and the data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Two-tailed t-test).

Figure 3.

UXT deficiency enhances the CGAS-STING1 signaling response. (A) Control and uxt cKO BMDMs were isolated from control and uxt cKO mice. Cell lysates were collected for western blot analysis of UXT and GAPDH. (B) The heatmap of expression changes of differentially expressed ISGs between control and uxt cKO BMDMs stimulated with cGAMP (1 μg per well) for 3 h. For each gene, the expression level was normalized (Z-score) across samples. (C) Significantly enriched biological processes of the upregulated DE genes (FDR<0.01). (D) Control and uxt cKO BMDMs were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Ifnb, Ifna4, and Cxcl10 mRNAs was measured by real-time PCR. (E) Control and uxt cKO BMDMs were stimulated with cGAMP (1 μg per well) for 3 h. Then, the induction of Isg15, Ifit1, and Rsad2 mRNAs was measured by real-time PCR. (F) Control and uxt cKO BMDMs were stimulated with cGAMP for 0, 3, 6 h, respectively. Then, cell lysates were collected for western blot analysis of UXT, IRF3, IRF3 phosphorylation and ACTB. (G) Control and uxt cKO mice were injected intravenously with HSV-1 (2 × 10⁷ pfu per mouse). The survival rates were monitored for 15 days after infection. (H) uxt cKO mice and control littermates were injected intravenously with HSV-1 (1 × 10⁷ pfu per mouse) for 12 h. Then, the serum was collected, and the concentration of IFNB was measured by ELISA. (I) Hematoxylin and eosin staining of lung sections isolated from control and uxt cKO mice infected with HSV-1 by intravenous injection for 16 h (scale bar: 100 μm). (J) Control and uxt cKO mice were injected intravenously with HSV-1 (1 × 10⁷ pfu per mouse) for 9 h. The relative induction of Ifna4, Ifnb mRNA in lungs or spleens from control and uxt cKO mice was measured by real-time PCR. Graphs show the mean ± sem., and the data shown are representative of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 (Two-tailed t-test).

Figure 4.

UXT regulates the CGAS-STING1 signaling at the STING1 level. (A) Luciferase activity in HEK293 cells transfected with an IFNB luciferase reporter, together with an empty vector or increasing amount of plasmid expressing UXT, activated by CGAS, STING1, TBK1 or IRF3 5D. The firefly- and renilla luciferase signals were detected with Dual Glo® luciferase assay (Promega). (B) Luciferase activity in HEK293 cells transfected with an IFNB luciferase reporter, together with negative control (NC) or Uxt siRNAs, activated by CGAS, STING1, TBK1 or IRF3 5D. The firefly- and renilla luciferase signals were detected with Dual Glo® luciferase assay (Promega). (C) HEK293T cells were transfected with the indicated plasmids. Then, cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (D) MEFs were stimulated with HTDNA for 3 h, and the cell lysates were immunoprecipitated with an anti-UXT antibody or normal IgG, and then immunoblotted with the indicated antibodies. (E) MEFs were stimulated with cGAMP for 3 h, and the cell lysates were immunoprecipitated with an anti-UXT antibody or normal IgG, and then immunoblotted with the indicated antibodies. (F) MEFs were stimulated with HTDNA for 3 h, then PLA analysis was applied to detect the interaction between UXT and STING1. Scale bars: 20 μm. Graphs show the mean ± SEM, and the data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Two-tailed t-test).

Figure 5.

UXT promotes autophagic degradation of STING1. (A) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA (2 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (B) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with ISD (2 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (C) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with cGAMP (1 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (D) MEFs transfected negative control (NC) or Uxt siRNAs were stimulated with HTDNA (2 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (E) MEFs transfected negative control (NC) or Uxt siRNAs were stimulated with ISD (2 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (F) MEFs transfected negative control (NC) or Uxt siRNAs were stimulated with cGAMP (1 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (G) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA (2 μg per well) for 0, 3 h respectively, followed by the treatment of mock, MG132 (10 μM), chloroquine (50 μM), or 3-MA (10 mM). Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (H) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with cGAMP (1 μg per well) for 0, 3 h respectively, followed by the treatment of mock, MG132 (10 μM), chloroquine (50 μM), or 3-MA (10 mM). Then, Cell lysates were collected for western blot analysis of STING1 and GAPDH. (I) MEFs transfected negative control (NC) or Uxt siRNAs and control vectors (Vec) or UXT expression plasmids (UXT) were stimulated with HTDNA (2 μg per well) for 0, 3, 6 h respectively. Then, Cell lysates were collected for western blot analysis of LC3-II and GAPDH. (J) Luciferase activity in HEK293 cells transfected with an IFNB luciferase reporter, together with STING1 and an empty vector or UXT expressed plasmids, followed by the treatment of mock, MG132 (10 μM), chloroquine (50 μM), or 3-MA (10 mM). The firefly- and renilla luciferase signals were detected with Dual Glo® luciferase assay (Promega). Graphs show the mean ± SEM, and the data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Two-tailed t-test).

Figure 6.

UXT facilitates the interaction of SQSTM1 and STING1. (A, B, C and D) HEK293T cells were transfected with the indicated plasmids. Then, cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (E) MEFs were treated with chloroquine and then stimulated with HTDNA for 3 h, and the cell lysates were immunoprecipitated with an anti-UXT antibody or normal IgG, and then immunoblotted with the indicated antibodies. (F) MEFs were treated with chloroquine and then stimulated with cGAMP for 3 h, and the cell lysates were immunoprecipitated with an anti-UXT antibody or normal IgG, and then immunoblotted with the indicated antibodies. (G) MEFs were treated with chloroquine and then stimulated with HTDNA for 3 h, and the cell lysates were immunoprecipitated with an anti-UXT antibody or normal IgG, and then immunoblotted with the indicated antibodies. (H) MEFs transfected negative control (NC) or Uxt siRNAs were treated with chloroquine and then stimulated with HTDNA (2 μg per well) for 3 h, and the cell lysates were immunoprecipitated with an anti-SQSTM1 antibody or normal IgG, and then immunoblotted with the indicated antibodies. (I) MEFs transfected negative control (NC) or Uxt siRNAs were treated with chloroquine and then stimulated with cGAMP (2 μg per well) for 3 h, and the cell lysates were immunoprecipitated with an anti-SQSTM1 antibody or normal IgG, and then immunoblotted with the indicated antibodies. (J) MEFs transfected control vectors (Vec) or UXT expression plasmids (UXT) were treated with chloroquine and then stimulated with cGAMP (2 μg per well) for 3 h, and the cell lysates were immunoprecipitated with an anti-SQSTM1 antibody or normal IgG, and then immunoblotted with the indicated antibodies.

Figure 7.

UXT alleviates aberrant type I IFNs in SLE patients. (A) The expression comparison of UXT in the leukocytes and PBMCs between SLE patients and healthy donors across four SLE cohorts. (B) The expression comparison of UXT in T cells, B cells, and monocytes between SLE patients and healthy donors. (C) The pairwise Pearson expression correlation between UXT and each of the 21 IFN signature genes across four SLE cohorts. The p-value was corrected into FDR. (D) The global expression correlation distribution between UXT and 21 ISGs (21 IFNG) across four SLE cohorts. The global expression correlation between UXT and all expressed protein-coding genes (All PCG) was used as background. (E, F, G, H, I, and J) PBMCs were isolated from the blood sample of SLE patients (n = 5). PBMCs were transfected with control vectors (Vec) or UXT expression plasmids (UXT) by electroporation. Then, UXT, IFNB, CXCL10, ISG15, ISG54, and ISG56 mRNA were measured by real-time PCR. Graphs show the mean ± SEM, and the data shown are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Two-tailed t-test).

Figure 8.

The working model of the negative regulation of CGAS-STING1 signaling pathway by UXT.