The MM cell line JJN-3 and INA-6 were kind gifts from Dr. Jennifer Ball (University of Birmingham, UK) and Dr. Martin Gramatzki (University of Erlangen-Nuremberg, Erlangen, Germany), respectively. OH-2 and IH-1 were established in our laboratory (16, 17). The cells were cultured in RPMI-1640 medium with fetal calf serum or human serum as previously described (18). For T-cell isolation and activation, human PBMCs were isolated from buffy coat using Lymphoprep (Stem Cell Technologies, Vancouver, Canada) according to the manufacturer’s protocol. PBMCs were incubated overnight in full medium (RPMI medium with 10% heat-inactivated fetal calf serum (FCS) with gentamicin) before T cells were isolated with Dynabeads Untouched Human T Cells Kit (Thermo Fisher Scientific, Waltham, MA, USA). T cells were cultured in full medium supplemented with IL-2 at 30 U/ml (PeproTech/Thermo Fisher Scientific, waltham, MA, USA). To increase the number of T cells and to increase IL-32 expression, T cells were activated with Dynabeads™ Human T-Activator CD3/CD28 (Thermo Fisher) with a 1:1 cell:bead ratio for 72 h before bead removal. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

For cycloheximide (CHX) protein degradation chase assays, CHX was used at a final concentration of 5 or 7 µg/ml, while proteasomal and autophagic inhibitors MG132 and bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA) was used at a concentration of 20 μM and 90 nM, respectively. Carfilzomib and bortezomib (Selleck Chemicals, Houston, TX, USA), were both used at a final concentration of 50 nM. DUB inhibitors and the concentrations used are found in Supplementary Table S1.

Using buffer R (Amaxa Nucleofector Kit R, Lonza, Basel, Switzerland) and program T-001 on Nucleofector device (Amaxa Biosystems, Cologne, Germany), 2 µM ADO ON-TARGETplus siRNA (L-015855-01-0005) and ON-TARGETplus Non-targeting Pool (D-001810-10-05) siRNA from Dharmacon were transfected into JJN-3 cells by electroporation. The cells were seeded into 12-well plates 24 h after siRNA transfection and cultured overnight in normoxic (20% O₂, 5% CO₂) or hypoxic conditions (2% O₂, 5% CO₂) before 5 µg/ml CHX was added and harvested at a timepoint of 0 min/h. Plates were then placed back in the normoxic incubator (reoxygenation and normoxia) and the hypoxic incubator (hypoxia), and the cells were harvested at the indicated time points.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and complementary DNA (cDNA) was synthesized from total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using StepOne Real-Time PCR System and TaqMan Gene Expression Assays (Thermo Fisher Scientific) with standard settings (2′ 50°C, 10′ 95°C, 40 cycles at 95°C for 15 s, 1′ 60°C). The comparative Ct method was used to estimate relative changes in IL-32 gene expression using β-actin and GAPDH as housekeeping genes. Probes were as follows: human IL-32 (Hs00992441_m1) and housekeeping gene GAPDH (Hs99999905_m1) housekeeping gene β-actin (Hs0160665_g1) both from Thermo Fisher Scientific.

Cells were lysed in lysis buffer (50 mM Tris–HCl, 1% NP40, 150 mM NaCl, 10% glycerol, 1 mM Na₃VO₄, 50 mM NaF, and a complete protease inhibitor (Roche Diagnostics, Mannheim, Germany). Lysates were denatured in 1× NuPage LDS sample buffer supplemented with 0.1 mM DTT for 10 min at 70°C before they were separated on 4%–12% Bis‐Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen, Camarillo, CA, USA). Membranes were blocked using 5% bovine serum albumin (Sigma-Aldrich) in Tris‐buffered saline with 0.01% Tween, followed by overnight incubation with the primary antibodies. Detection was performed using horseradish peroxidase (HRP)-conjugated antibodies (DAKO, Glostrup, Denmark) and developed with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL, USA). Images were obtained with LI‐COR Odyssey Fc and analyzed using Image Studio Software (LI‐COR, Lincoln, NE, USA). The following antibodies were used: anti-IL-32 (#AF3040, R&D Systems, Minneapolis, MN, USA), anti-β-actin (#4967, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (# ab8245, Abcam, Cambridge, UK), anti-HA (#H6908, Sigma-Aldrich), anti-ubiquitin (# 13-1600, Thermo Fisher), and anti-ADO (ab134102, Abcam).

JJN-3 cells were seeded in poly-l-lysine coated 96-well glass-bottom plates and treated with carfilzomib for 4 h at a concentration of 100 nM at 37°C before being fixed with 4% PFA for 10 min at RT. After quenching for 10 min with 50 mM NH₄Cl, cells were washed with PBS and then permeabilized using 0.15% triton X-100 in PBS. Cells were washed with 0.02% Tween before blocking with 20% human serum (HS) in PBS. Anti-IL-32 (R&D) antibody were diluted to 2 μg/ml in 1% HS and 0.02% Tween and was left on cells overnight at 4°C. The next day, secondary antibody chicken anti-goat IgG (H + L) antibody Alexa Fluor 647 (2 μg/ml) in 1% HS, 0.02% Tween was added for 30 min before leaving cells in Hoechst (Thermo Fisher Scientific) in PBS (2 μg/ml) for imaging.

JJN3 cells were incubated overnight in hypoxic (2% O₂, 5% CO₂) conditions. Before harvest, the cells were stimulated for 4 h with 20 µM MG-132 in normoxic conditions. Approximately 10 million JJN-3 cells were lysed in lysis buffer (1% NP40, 10% glycerol, 150 mM NaCl, 50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 100 mM n-ethylmaleimide, 1 mM Na₃VO₄, 50 mM NaF, and a complete protease inhibitor (Roche Diagnostics, Mannheim, Germany)). Ubiquitinated proteins were pulled down with tandem ubiquitin binding entities (TUBEs) conjugated to magnetic beads with the kit UM411M: UbiTest Magnetic TUBE Elution Kit (LifeSensors) according to the manufacturer’s protocol.

HA-Ubiquitin (19) was a gift from Edward Yeh (Addgene plasmid # 18712 (12); http://n2t.net/addgene:18712; RRID : Addgene_18712). Five micrograms of HA-ubiquitin plasmid was transfected into JJN-3 cells by electroporation using buffer R (Amaxa Nucleofector Kit R, Lonza) and program T-001 on the Nucleofector device (Amaxa Biosystems, Cologne, Germany). Cells were cultured overnight in hypoxic conditions (2% O₂, 5% CO₂) and then harvested for IL-32 immunoprecipitation.

IL-32 antibody (R&D) was conjugated to M-270 beads according to the manufacturer’s instructions, using Dynabeads Antibody Coupling Kit (Thermo Fisher) and 10 µg of resuspended IL-32 antibody/mg of beads. JJN-3 KO and WT cells were incubated in hypoxia for 24 h before lysis with 4× pellet volume RIPA buffer (1% CHAPS, 50 mM Tris, 150 mM NaCl, 1 mM Na₃VO₄, 50 mM NaF, and a complete protease inhibitor (Roche Diagnostics)) for 1 h at 4°C on rotation. Lysate was incubated with IL-32 antibody-conjugated beads for 1 h at 4°C. After washing four times with PBS, beads were resuspended in 1× NuPage LDS sample buffer supplemented with 0.1 mM DTT and heated for 10 min at 70°C before beads were removed and the sample proceeded to immunoblotting.

We previously identified interaction partners of IL-32 in MM cells by IL-32 pulldown of cell lysates, followed by mass spectrometry (13). Two of the identified binding partners were the proteasomal alpha ring-subunit PSMA3 and the E3 ubiquitin ligase WWP2, which may suggest that IL-32 is ubiquitinylated and degraded by the proteasome. Indeed, when we treated the MM cell line JJN-3 with the proteasome inhibitor MG132, we observed an accumulation of IL-32 protein that was evident after 2 and 4 h of treatment (Figure 1A), while IL-32 mRNA did not increase (Figure 1B). IL-32 protein was similarly stabilized by MG132 treatment in three other myeloma cell lines: INA-6, IH-1, and OH-2 (Supplementary Figures S1A, B). Treatment with carfilzomib and bortezomib, proteasomal inhibitors that are used in the treatment of MM, had a similar effect: accumulation of IL-32 protein (Supplementary Figure S1C) with no change in mRNA expression (Supplementary Figure 1D). Treatment with bafilomycin A also led to some accumulation of IL-32 protein (Supplementary Figures S1C, D), indicating that IL-32 may also be partially degraded through an autophagosomal pathway. Confocal imaging of carfilzomib-treated JJN-3 cells showed that IL-32 was localized at distinct puncta in the cytosol in untreated cells while abundantly present in the cytosol after 4 h with proteasome inhibitor treatment (Figure 1C).

Proteins are targeted for proteasomal degradation by poly-ubiquitination executed by E1, E2, and E3 ligases (20). To see if IL-32 is ubiquitinated, we transfected JJN3 IL-32 CRISPR/Cas9 KO (13) and WT cells with a HA-ubiquitin plasmid and performed a pulldown of IL-32. Staining for the HA tag on the Western blot showed a high molecular weight smear on the WT pulldown samples with no smear on the KO pulldown sample (Figure 1D), which supports that IL-32 is poly-ubiquitinated. To demonstrate the endogenous ubiquitination of IL-32, we purified the polyubiquitinylated proteome of JJN-3 IL-32 KO and WT cells using magnetic bead-coupled TUBEs. IL-32 was detected upon TUBE-pulldown in WT cells but not in KO cells, supporting that it is polyubiquitinylated (Figure 1E). When treating the TUBE-pulldown sample with DUBs, only a ~27-kDa IL-32 band appeared in the pulldown samples, validating TUBE pulldown of ubiquitinated IL-32. IL-32 also accumulated when the E1 ubiquitin-activating enzyme was inhibited using the small molecule E1 inhibitor TAK234 (21), further supporting that IL-32 is degraded in a ubiquitin-dependent manner (Figure 1F).

To look into the kinetics of IL-32 protein turnover, we treated JJN-3 cells with CHX to inhibit protein translation. Treating JJN-3 cells with CHX showed a 40% reduction of IL-32 protein after 30 min, with a further decrease at later time points (Figures 2A, B). In a similar manner, IL-32 protein was quickly reduced after CHX treatment in INA-6, IH-1, and OH-2 cells (Supplementary Figure S2), supporting a high protein turnover in these cells as well. Combining CHX with the proteasome inhibitor MG132 in JJN-3 cells efficiently maintained the steady-state level of IL-32 protein, suggesting that proteasomal degradation is responsible for the short half-life of IL-32 (Figure 2C). By comparing IL-32 protein levels in MG132-treated samples (degradation inhibited) with CHX and MG132 (translation and degradation inhibited), we could assess how protein translation contributes to IL-32 protein turnover (Figures 2C, D). The level of IL-32 protein in cells treated with MG132 alone for 30 min was roughly doubled compared with cells treated with the combination of CHX and MG132, while at 2 h the difference was nearly threefold (Figure 2D), demonstrating that IL-32 mRNA is rapidly and continuously translated. In conclusion, intracellular IL-32 protein in MM cells is maintained at a steady state by a fast and coordinated translation and degradation.

We previously showed that IL-32 is upregulated in hypoxia and that transcriptional activation is regulated by HIF1α (12). ADO is a recently discovered oxygen-sensing cysteine dioxygenase in humans that targets proteins for proteasomal degradation through the N-degron pathway (15). In the same study, IL-32 protein stability was shown to be regulated by ADO in the RKO cancer colon cell line (15). Ubiquitylation targets proteins for degradation in the proteasome through the N-degron pathway (22). Thus, we investigated if ADO regulates IL-32 protein levels in MM cells in response to changing oxygen levels. We treated cells with CHX to assess the degradation of IL-32 independently of translation. ADO was highly expressed in MM cells, and ADO siRNA only partly depleted the ADO protein (Figure 3A). Regardless, in cells with reduced ADO expression, we observed a translation-independent increase in IL-32 protein levels compared to control cells, supporting that IL-32 is regulated by ADO-dependent degradation (Figure 3A). ADO catalytic activity is dependent on oxygen availability. As expected, in hypoxia (2% O₂) in the presence of CHX, there was no difference in IL-32 protein levels between ADO knockdown cells and control cells (Figure 3A). Reactivation of ADO by reoxygenation (20% O₂) of hypoxic ADO knockdown cells and control cells in the presence of CHX demonstrated that IL-32 was more rapidly degraded in control cells (Figure 3B), supporting the hypothesis that ADO is involved in the regulation of IL-32 in MM cells during oxygen fluctuations.

Despite oxygen-dependent degradation of IL-32 by ADO, MM cells express high IL-32 protein levels in normoxia (10, 12). We thus asked how high levels of IL-32 protein are maintained in physiological oxygen levels despite rapid ubiquitylation and proteasomal degradation. Ubiquitylation is a process that can be reversed by DUBs which remove ubiquitin from target proteins (23). Removal of ubiquitin will reduce the degradation of the target protein. Using the broad targeting nonselective DUB inhibitor PR-619, we observed a clear reduction in IL-32 protein after 2 and 4 h (Figure 4A), indicating that DUBs counteract ubiquitin-dependent degradation of IL-32 in MM cells in normal oxygen conditions. In hypoxia, the differences between PR-619 and untreated cells were smaller, indicating that there is less deubiquitination of IL-32 in hypoxia (Figure 4A). This is in line with reduced ADO-dependent IL-32 ubiquitylation in hypoxia. PR-619 treatment increased the amount of high molecular weight ubiquitinylated IL-32 and decreased nonubiquitinylated IL-32 at 27 kDa in JJN-3 cells (Figure 4B), supporting that DUBs remove ubiquitin on IL-32. DUBs are a large and diverse set of about 100 enzymes divided into six families, and PR-619 is a nonspecific, broad-spectrum DUB inhibitor (24). We, therefore, performed a DUB-inhibitor screen to explore which DUBs could remove ubiquitin from IL-32. Using a panel of more specific DUB inhibitors (Supplementary Table S1), we observed a large reduction of IL-32 protein levels with degrasyn (targets USP9x, USP5, USP14, UCH37), EOAI3402143 (targets USP9x/USP24 and USP5), and ML364 (targets USP2) (Figure 4C). Thus, several different DUBs can potentially contribute to the removal of ubiquitin from IL-32.

To investigate if protein regulation of IL-32 is conserved in cells of the lymphocyte lineage, we assessed IL-32 protein level in PBMC-derived primary human T cells isolated from healthy donors. Indeed, we observed protein turnover of IL-32 within a similar time frame in activated primary human T cells as in MM cells. IL-32 protein showed accumulation upon MG-132 treatment after 1 h of stimulation and was depleted by CHX treatment within the same time interval (Figure 4D). Furthermore, IL-32 was reduced by PR-619 treatment, indicating that DUBs actively remove ubiquitin from IL-32, also in T cells. IL-32 was also slightly increased in hypoxic T cells (Figure 4E).