A BRISC-SHMT complex deubiquitinates IFNAR1 and regulates interferon responses

Abstract

Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here, we identify serine hydroxymethyltransferase (SHMT) as a previously unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub-mediated internalization and lysosomal degradation. BRISC-deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a mechanism of DUB regulation and suggest a therapeutic use of BRISC inhibitors for treating pathophysiological processes driven by elevated IFN responses.

Figure 1. Identification of SHMT Species as a 5^th Component of the BRISC Complex

(A) Schematic comparison of the nuclear RAP80 complex that directs BRCC36-mediated DUB activity at K63-Ub chains formed at DNA damage sites with the cytoplasmic BRISC complex whose substrates and targeting components remain unknown. (B) Proteins co-purified with either Flag-HA- Abraxas (eAbraxas) or KIAA0157 (eKIAA0157) were separated by SDS-PAGE and stained using Coomassie Brilliant Blue. (C) Tabular display of the number of tryptic peptides from each of the indicated proteins that co-purified with eKIAA0157. (D) Flag immunoprecipitation (IP) reveals association of SHMT2 with KIAA0157 but not Abraxas. (E) KIAA0157 was detected by immunoblot following IP of endogenous SHMT1 or SHMT2. (F) 293T cells transiently expressing wild and mutant Flag tagged versions of KIAA0157 were lysed and used for Flag-IP. Co- IPed proteins were detected using different antibodies as indicated. (G) Immunoblot displaying similar amounts of SHMT2 from IP of either Flag-HA-KIAA0157 or endogenous SHMT2 from HeLa and 293T cells. SHMT catalytic activity was assayed from these immunoprecipitates (IPs). (H) SHMT activity was observed in IPs of SHMT, but not KIAA0157. Similar results were observed in both cell lines.

Figure 2. Interferon Stimulation Enhances BRISC-SHMT2 interaction and Association with IFNAR1

(A) 293T cells were treated with IFNα for the indicated times. IFNAR1 was IPed and then SHMT2 and IFNAR1 were detected by immunoblotting using the indicated antibodies. WCL: Whole Cell Lysates. (B) Cells expressing wildtype (WT) and Δ215–222 mutant KIAA0157 were treated with or without IFNα for 15 minutes. Flag-IP was performed and co-purified proteins were detected using the indicated antibodies. (C) Proximity ligation assay showing interaction between KIAA0157 or SHMT2 and IFNAR1 (Proximity foci). U2OS cells with stable expression of Flag-HA-KIAA0157 were transfected with the indicated siRNAs and interaction between KIAA0157 or SHMT2 and IFNAR1 was studied using rabbit IFNAR1 and mouse anti-HA antibodies. (D) Quantification of foci from A. Data and error bars here and thereafter represent Mean±S.E.M. from at least three independent experiments that included at least 100 cells per sample. P values were calculated using the Student’s t-test. (E) 293T cells were transfected with shRNAs against GFP or β-Trcp (BTR). Cells were treated with IFNα for indicated times. IFNAR1 was IPed and SHMT2, K63-Ub, pS535 and IFNAR1 were detected by immunoblotting using the indicated antibodies. WCL was used for detection of β-Trcp and β-Actin by immunoblotting. (F) 293T cells were transfected with Flag-IFNAR1 wild type (WT) or Flag-IFNAR1 S535A. Cells were treated with IFNα for indicated times. Flag-IFNAR1 was immunoprecipitated, and SHMT2, K63-Ub, pS535 and Flag-IFNAR1 were detected by immunoblotting using the indicated antibodies.

Figure 3. The BRISC-SHMT Complex Regulates K63-linked Ubiquitination of IFNAR1

(A) 2fTGH cells with stable knockdown of KIAA0157 or SHMT2 were incubated with or without IFNα as indicated. IFNAR1 was IPed from cell lysates and K63-Ub, pS535 and IFNAR1 were detected using the indicated antibodies. (B) 2fTGH cells with stable knockdown of BRCC36 were treated as in B. IFNAR1 was IPed from cell lysates and K63-Ub, pS535 and IFNAR1 were detected using the indicated antibodies. (C) Flag-IFNAR1 was IPed from 293T cells and immunoblot was performed using antibodies specific to K48-Ub chains as indicated. (D) K63-Ub modification and degron phosphorylation of endogenous IFNAR1 in cells expressing wild type KIAA0157 or its Δ215–222 mutant. (E) MEFs from wild type or KIAA0157-null mice were treated with or without mIFNβ for 30 min. IFNAR1 was IPed from cell lysates and K63-Ub, pS526 (corresponding to human pS535) and IFNAR1 were detected using the indicated antibodies.

Figure 4. BRISC-SHMT Regulates Endocytosis, Proteolytic Turnover and Abundance of IFNAR1

(A) Internalization rate of endogenous IFNAR1 in IFN-stimulated 293T cells that had been transfected the indicated siRNAs. (B) 2fTGH cells with stable knockdown of BRCC36 or KIAA0157 were treated with IFNα together with CHX (20 µg/ml) for the indicated times. IFNAR1 was immunoprecipitated and detected using specific antibodies. β-actin, KIAA0157, and BRCC36 were detected from WCL using the indicated antibodies. (C) 2fTGH cells with stable knockdown of SHMT2 were treated with IFNα together with CHX (20 µg/ml) for the indicated times. IFNAR1 was immunoprecipitated and detected using specific antibodies. β-actin and SHMT2 were detected from WCL using the indicated antibodies. (D) MEFs from wild type or KIAA0157 knockout mice were treated with mouse IFNβ together with CHX (20 µg/ml) for the indicated times. mIFNAR1 was immunoprecipitated and detected using specific antibodies. β-actin and MERIT40 were detected from WCL using the indicated antibodies.

Figure 5. BRISC-SHMT Interaction Promotes Responses to IFN

(A) KIAA0157 +/+ or −/− MEFs were treated with IFNβ for the indicated times. STAT1 Tyr701 phosphorylation and total STAT1 levels were detected from cell lysates using specific antibodies. (B) MEFs of each genotype were either continuously exposed to mouse IFNβ for 24h or pulsed with IFN for 2h and then incubated without IFN for total of 24h. Levels of PKR and β-actin were detected from whole cell lysates using the indicated antibodies. (C) 2fTGH cells stably expressing WT KIAA0157 or mutant KIAA0157-DM1 were treated with human FNα and analyzed as done previously in panel B. (D) 2fTGH cells with stable knockdown of SHMT2 or KIAA0157 or BRCC36 were pulse-treated with IFNα for 2 hours and then IFNα was removed and cells harvested 24 hours after initial treatment. Alternatively, cells were treated with IFNα continuously for 24 hours. Whole cell lysates were used for detection of PKR and β-actin as indicated. (E) KIAA0157 +/+, −/− and reconstituted null MEFs were treated with or without mIFNβ (200U/ml) for 48 hours and cells viability was analyzed by Trypan blue exclusion assay. (F) Viral titers were measured in VSV infected KIAA0157+/+ and −/− MEFs from 2 independent experiments (each in triplicates).

Figure 6. BRISC Deficiency Confers Severely Reduced Interferon Signaling in Response to LPS

(A) FACS analysis of cell surface IFNAR1 levels in peripheral blood leukocytes (PBL) isolated from KIAA0157 +/+ and −/− mice (n=3 for each genotype) 3h after LPS treatment. (B) Real time RT-PCR analysis of the indicated cytokine mRNA levels in PBL from KIAA0157 +/+ and −/− mice (n=4 for each genotype) in response to LPS treatment. (C) KIAA0157 +/+ and −/− MEFs were treated with or without 1µg/ml of LPS for 20h and cell lysates were analyzed by immunoblotting as indicated. (D) KIAA0157 +/+ and −/− MEFs were treated with or without 1µg/ml of LPS for indicated periods of time and levels activated STAT1 (pSTAT-Y701) and other proteins were analyzed in WCL using the indicated antibodies. (E) KIAA0157 +/+, −/− and reconstituted −/− MEFs were treated with or without LPS (10ng/ml) and 10ug/ml CHX for 24 hours. Cells viability was analyzed by Trypan blue exclusion assay.

Figure 7. KIAA0157−/− Mice Display Reduced Interferon Signaling, Tissue Damage, and Mortality in Response to LPS in vivo

(A) KIAA0157 +/+ and −/− mice were injected with the indicated amount of LPS for different time periods and lung and liver were harvested for protein analysis by western blot. (B) Wild type and KIAA0157 null mice were injected with 10.5mg/kg LPS for 4–5 days and their lung were harvested, fixed and then stained with hematoxylin–eosin (H&E) (C) Kaplan-Meier survival curves were generated after injecting 10.5mg/kg LPS in wild type and KIAA0157 null mice. (D) Model depicting IFN inducible SHMT-BRISC assembly and de-ubiquitination activity on IFNAR1 and possibly other substrates (X).