Deubiquitination of proteasome subunits by OTULIN regulates type I IFN production

Abstract

OTULIN is a linear deubiquitinase that negatively regulates the nuclear factor κB (NF-κB) signaling pathway. Patients with OTULIN deficiency, termed as otulipenia or OTULIN-related autoinflammatory syndrome, present with early onset severe systemic inflammation due to increased NF-κB activation. We aimed to investigate additional disease mechanisms of OTULIN deficiency. Our study found a remarkable activation of type I interferon (IFN-I) signaling in whole blood, peripheral blood mononuclear cells, monocytes, and serum from patients with OTULIN deficiency. We observed similar immunologic findings in OTULIN-deficient cell lines generated by CRISPR. Mechanistically, we identified proteasome subunits as substrates of OTULIN deubiquitinase activity and demonstrated proteasome dysregulation in OTULIN-deficient cells as the cause of IFN-I activation. These results reveal an important role of linear ubiquitination in the regulation of proteasome function and suggest a link in the pathogenesis of proteasome-associated autoinflammatory syndromes and OTULIN deficiency.

Fig. 1.. Patients with otulipenia display overactivation of IFN-I signaling.

(A) NanoString analysis of IFN-I signaling in whole blood samples from two patients, four healthy controls, and a type I interferonopathy patient control (PC) with deoxyribonuclease 2 deficiency. (B) Cytokine levels in whole blood samples. The whole blood samples from P1 and his unaffected sibling were stimulated with poly(I:C) (20 μg/ml) or LPS (1 μg/ml) for 22 hours. (C) Cytokine levels in the supernatant of cultured PBMCs from three patients and one healthy control. (D) Cytokine levels in the supernatant of purified monocytes from P1 and three healthy controls. Cells used to detect IP10 and monokine induced by IFN-γ (MIG) were at basal level. Cells used to detect monocyte chemoattractant protein 1 (MCP1) were stimulated with LPS (1 μg/ml) for 48 hours. P1 was sampled twice. (E) Serum cytokine levels from three patients and seven healthy controls. (F) Intracellular cytokine staining of IFN-β and RANTES (CCL5) in T cells and B cells of P3 and two healthy controls. (G) Phosphorylation of STAT1 and p65 of patient P3 T cells compared to two healthy controls at basal level and after IL-6 (100 ng/ml) stimulation for 6 hours as determined by flow cytometry analysis. The patients carry homozygous disease-associated variants in OTULIN: P1 (Leu²⁷²Pro), P2 (Tyr²⁴⁴Cys), and P3 (Gly¹⁷⁴Aspfs*2).

Fig. 2.. OTULIN deficiency causes proteasome dysfunction.

(A) The Lys⁴⁸ ubiquitination (Ub) levels in patients’ and one healthy control’s fibroblasts. (B) Proteasome chymotryptic, tryptic, and caspase-like activity in fibroblasts from three patients and two healthy controls. (C) Proteasome chymotryptic, tryptic, and caspase-like activity in PBMCs from two patients and six healthy controls. P3 was sampled twice. (D to F) Native gel analysis of the proteasome assembly in fibroblasts from three patients and three healthy controls. The assembly of different parts of the proteasome was illustrated using antibodies of different subunits correspondingly. CP, core particle; RP, regulatory particle. (G) Native gel analysis of the proteasome assembly in PBMCs from the patient P3 and four healthy controls. (H) Western blots analysis of linear ubiquitination levels on purified proteasome from patient P1’s and one healthy control’s fibroblasts. Data in (B) and (C) are shown as means ± SEM from four repeated technical analyses. P values were determined by unpaired two-tailed t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. Proteasome subunits as substrates of OTULIN deubiquitinase activity.

(A) Identification of proteasome subunits that are regulated by OTULIN-mediated deubiquitination of linear ubiquitin using immunoprecipitation (IP). LUBAC complex consists of the catalytic subunit HOIP and two accessory proteins: HOIL-1 and Sharpin. WCL, whole-cell lysate. Ub-KO, ubiquitin mutant with all lysines mutated to arginines, which only forms linear polyubiquitin chains. (B) Schematic illustration of the different OTULIN deletion constructs that are used in experiments shown in (C). (C) Immunoprecipitation of 293T cells transfected with the indicated OTULIN constructs and various proteasome subunits provides evidence that the interaction between OTULIN and proteasome subunits relies on the OTU domain.

Fig. 4.. Graphical abstract of the pathogenic mechanism of otulipenia.

OTULIN deficiency leads to the excessive linear ubiquitination on proteasome subunits, especially on the immunoproteasome subunit, which disrupts immunoproteasome assembly and function. The defected proteasome will cause accumulation of unfolded or ubiquitinated protein, leading to a strong IFN-I response. Clinically, patients with otulipenia usually suffer from severe multiorgan systemic inflammation.