The autophagy protein RUBCNL/PACER represses RIPK1 kinase-dependent apoptosis and necroptosis

ABSTRACT

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A₁; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a

Introduction

Mesenchymal stem cells (MSCs) have been the subject of numerous inflammatory disease-related studies due to their immunoregulatory properties, which suggest their application in a wide range of clinical settings. However, MSCs suffer from poor survival after being transplanted, limiting their therapeutic efficacy [1–4], partly due to their exposure to a hostile and proinflammatory microenvironment in the host tissue [5,6]. This proinflammatory environment can include significantly elevated levels of TNF (tumor necrosis factor), IFNG/IFNγ (interferon gamma), and interleukins (IL1, IL6, IL17). Macroautophagy, hereafter referred to as simply autophagy, is a homeostatic cellular process that catabolizes cytoplasmatic materials, such as organelles, proteins, and lipids [7]. Autophagy can be upregulated in response to environmental stressors, such as starvation, hypoxia, oxidative stress, inflammation, and others [8]. MSCs are likely exposed to multiple of those conditions after transplantation, and autophagy could impact the survival rate of cells exposed to stress [4,9]. In this context, activated autophagy constitutes a stress adaptation pathway that promotes cellular homeostasis and cell survival [8].

RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like) belongs to a putative protein family called the RUBCN/Rubicon family, consisting of 5 members, which are highly homologous in their C-terminal region containing a RUBCN homolog/RH domain [10–16]. Recently, RUBCNL has been shown to positively regulate autophagy [12,17,18]. RUBCNL was found to stimulate autophagosome maturation and autolysosome formation promoting hepatic autophagy and liver homeostasis [12,17]. Furthermore, RUBCNL is directly phosphorylated by the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) at S157 under nutrient-rich conditions, preventing autophagosome maturation. Conversely, the dephosphorylation of RUBCNL promotes its acetylation and autolysosome formation [17]. Moreover, we have identified RUBCNL as a regulator of proteostasis associated with amyotrophic lateral sclerosis/ALS pathogenesis. RUBCNL deficiency led to impaired autophagy and accumulation of ALS-associated protein aggregates, which correlated with the induction of neuronal cell death [17]. However, it remained unclear if the role of RUBCNL in cell death is limited to proteostasis unbalance conditions or if RUBCNL has a role in cell death independent of the control it exerts over autophagy. Furthermore, recently, we demonstrated that RUBCNL overexpression in MSCs improves their autophagic capacity in response to inflammatory stimulus, as well as their immunosuppressive ability in vivo [19].

Depending on the cellular context, TNF, can either promote inflammation directly by activating the canonical pathway of NFKB (nuclear factor of kappa light polypeptide gene enhancer in B cells) or indirectly by inducing regulated cell death (RCD)-dependent or not on RIPK1 (receptor (TNFRSF)-interacting serine-threonine kinase 1). TNF binds to TNFRSF1A/TNFR1 (tumor necrosis factor receptor superfamily, member 1a), and it induces the formation of a protein complex called complex I. TRADD (TNFRSF1A-associated via death domain) and RIPK1 form the core of complex I. The assembly of complex I is essential for initiating downstream signaling events that regulate cell death. Both apoptosis or necroptosis are triggered by complex II, which originates from dissociation of complex I from the TNFRSF1A, and subsequent recruitment of FADD (Fas associated via death domain) and CASP8 (caspase 8) [20]. Apoptosis is a form of RCD characterized by cell morphological changes (cell shrinkage, chromatin condensation, and cellular fragmentation) triggered by the activation of caspases and is involved in normal development and tissue homeostasis [21]. Necroptosis is a caspase-independent, lytic form of RCD characterized by organelle swelling and plasma membrane rupture, allowing the leakage of intracellular components [22]. Necroptosis mediates plasma membrane permeabilization through phosphorylation of MLKL (mixed lineage kinase domain-like) by forming the necrosome that contains RIPK3 (receptor-interacting serine-threonine kinase 3) in complex with RIPK1, FADD, and CASP8. Downstream of RIPK1 kinase activity, RIPK3 kinase phosphorylates MLKL [22]. It is currently unknown if MSCs die by apoptosis or necroptosis after TNFRSF1A activation, yet it comprises a critical point for improving their survival and therapeutic efficacy.

Although some reports indicate a crosstalk between autophagy and apoptosis/necroptosis, the mechanistic details have remained poorly understood so far. Some studies link the autophagy machinery with these two cell death types of stress responses [23–25]. Goodall and colleagues showed that the autophagy machinery facilitates necrosome assembly due to the recruitment of RIPK1 mediated by the autophagy receptor SQSTM1/p62. This was later corroborated in neurons where SQSTM1 promotes RIPK1 recruitment, stimulates self-oligomerization, and activates necroptosis [26]. Huyghe and colleagues showed that ATG9A (autophagy related 9A) promotes degradation of the complex II, protecting against TNF-induced cell death and mitigating embryonic lethality and inflammatory skin disease in mouse models [24]. Also, deficiency of ATG14/BAKOR (autophagy related 14) in intestinal epithelium leads to spontaneous villus loss and cell death, with sensitivity to TNF-induced apoptosis [25]. Consequently, autophagy-related factors play a crucial role in cell death by functioning as a scaffold for the death complex.

Here, we found using proteomics analyses that RUBCNL has a suggested function in cell death. First, we characterized which TNF-induced RCD pathway is active in MSCs. Then, we determined the role of RUBCNL in TNF-induced RCD in MSCs with loss and gain of function approaches. We found that RUBCNL protects from TNF-induced apoptosis and necroptosis independent of its role in autophagy by regulating the availability of RIPK1 to bind TNFRSF1A affecting the formation of complex I and the execution of the cell death program. These results allow us to propose that RUBCNL has both active anti-apoptotic and anti-necroptotic roles under proinflammatory conditions side-by-side with its reported role in autophagy and hence could constitute a multi-purpose target to improve the therapeutic efficacy of MSCs.

Results

RUBCNL negatively regulates TNF-induced cell death in MSCs

To better understand the role RUBCNL plays in MSCs, the murine MSC proteome was analyzed with mass spectrometry under RUBCNL loss of function. MSCs were transfected with siRNA targeting Rubcnl mRNA (siRubcnl) or nonsense siRNA (siCtrl). Samples were collected 48 h post-transfection for mass spec analysis. One hundred seventy-four differential expressed proteins (DEPs) of siRubcnl versus siCtrl passing the p-value cutoff of 0.05 were identified Figure 1(A-C), Fig. S1A. The EnrichR tool was used to determine the main pathways and GO terms enriched in the dataset. Besides expected terms and pathways, such as protein processing and vesicle-mediated transport, membrane trafficking, or endocytosis, respectively, the negative regulation of apoptosis and apoptosis as a pathway were suggested Figures 1(D,E). In total, 12 DEPs of siRubcnl overlapped with apoptosis, some very highly differentially expressed, such as Retsat or Gsn Figure 1(F,G), Fig. S1A. To confirm these results, we generated stably Rubcnl-deficient MSCs with a small hairpin RNA (shRNA) construct delivered by lentivirus, as previously described (Fig. S1B) [18,19], and assessed the impact on cell death. Cells were stimulated with oxidative stress agent (H₂O₂); ER-stress-inducing agents that block N-glycosylation (tunicamycin [Tm]) or inhibit the ER-calcium pump ATP2A/SERCA (thapsigargin [Tg]); the ferroptosis-inducing agent that blocks GPX4 (ML162); etoposide (ETO) that induces intrinsic apoptosis or with human TNF that activates TNFRSF1A triggering either survival and inflammation or apoptosis or necroptosis Figure 1(H,I). The effect of RUBCNL depletion in cell death triggered by these compounds was evaluated by kinetic cell death analysis measuring nuclear SytoxGreen (SG) fluorescence in dead cells, as previously described [27] Figure 1(I), Fig. S1C-G. The depletion of Rubcnl did not affect the induction of cell death triggered by H₂O₂, Tm, ML162, or ETO Figure 1(I), Fig. S1C-G. However, RUBCNL loss of function increased the susceptibility of MSCs to TNF-induced cell death Figure 1(I), Fig. S1G. Next, we stably overexpressed Flag-tagged human RUBCNL (RUBCNL-Flag) in murine MSCs and assessed its impact on cell death (Fig. S2A and S2B). Conversely to the results obtained under Rubcnl knockdown, RUBCNL-Flag overexpression significantly protected against TNF-triggered cell death but not when other stimuli were used (Fig. S2C-H). Together, these results indicate RUBCNL as a negative regulator of cell death triggered by TNFRSF1A activation.

Figure 1.

Proteomics and bioinformatic analysis suggest a role for RUBCNL in cell death. (A-G) Proteomic and bioinformatic analyses of MSCs treated with siCtrl or siRubcnl. The experiment was performed in triplicates and detected by mass spectrometry. Global changes in protein levels are displayed in (A). Differentially up- or downregulated proteins in the siRubcnl condition versus siCtrl are shown in a volcano plot (B). 174 proteins were consistently detected to be differentially expressed passing the p-value 0.05. Of those, 88 were upregulated and 86 were downregulated (C). GO term (D) and Pathway (E) analysis showed that a significant number of proteins associated to the process of apoptosis, which were identified in (F) by overlapping analyses performed in (C-E), as well as by displaying levels of differentially detected proteins in (G). (H-I) The effect of RUBCNL loss of function on different cell death modalities was interrogated using different stimuli. MSCs were stably transduced with lentiviral expression vectors for shRubcnl or shCtrl. Cells described were treated with indicated compounds (50 μM H₂O₂, 10 μM Tm, 10 μg/ml Tg, 10 μM ML162, 5 μM ETO, and 10 ng/mL TNF) and cell death was measured in function of time by SG-positivity. The results are presented as mean±S.E.M. of three independent experiments. Statistical significance was determined by two-way ANOVA. Significance between samples is indicated as follows: n.s., p > 0.05; ****, p ≤ 0.0001.

TNF induces RIPK1-dependent apoptosis but not necroptosis in MSCs

Our results suggested that RUBCNL expression specifically confers protection against TNF-induced RCD. However, it was unclear which cell death modality (apoptosis or necroptosis) MSCs die from when TNFRSF1A is activated. To our knowledge, no prior detailed studies defined TNF-induced cell death pathways in MSCs. Previously, Liu et al. showed that IFNG enhances apoptosis of bone marrow-derived MSCs when the TNF concentration reaches at least 50 ng/ml [28]. However, we observed that lower and single TNF doses, from 0.25 to 25 ng/ml, significantly induced cell death in a dose-dependent manner after 21 h (Figure 2A). The cell death induced by TNF was completely blocked by necrostatin 1s (Nec1s), a pharmacological inhibitor of RIPK1 (Figure 2A). Also, we determined the effect of 10 ng/ml TNF on kinetic cell death and CASP3 activity, observing 15% of cell death (Figure 2B) and a 35% increment of CASP3 activity (Figure 2C) after 21 h of TNF treatment. Additionally, we observed that TNF induced the cleavage of PARP-1 (Figure 2D,E and Fig. S3), and apoptotic DNA fragmentation (TUNEL assay) (Figure 2D,F). In all cases, Nec1s completely blocked cell death, CASP3 activation, the cleavage of PARP-1 and the generation of TUNEL⁺ cells Figure 2(A-F) and Fig. S3. To validate the participation of RIPK1 in TNF-induced apoptosis in MSCs, we tested the effect of three other RIPK1 inhibitors (Nec1, GSK963, and GSK157) [29], as well as one RIPK3 (receptor-interacting serine-threonine kinase 3) inhibitor (GSK840), on cell death (Figure 2G) and caspase 3 activity (Figure 2H). All RIPK1 inhibitors tested completely blocked TNF-induced apoptosis. The RIPK3 inhibitor GSK840 partially, but significatively, reduced TNF-induced cell death but not CASP3 activity in MSCs (Figure 2G,H). Since the catalytic kinase activities of RIPK1 and RIPK3 are critical for TNF-induced apoptosis and necroptosis, we decided to target RIPK1 and RIPK3 expression through siRNAs. We also targeted the expression of MLKL, the executor of necroptosis Figure 2(I,J). As expected, due to its role as a scaffold protein in the TNFRSF1A-NFKB pathway, targeting RIPK1 increased the susceptibility of MSCs to TNF-induced cell death (Figure 2K). However, knocking down RIPK3 or MLKL did not result in significant changes in TNF-induced cell death (Figure 2I), indicating necroptosis is not significantly induced upon stimulation of MSCs with TNF. The pan-caspase inhibitor zVAD prevents apoptosis by inhibiting CASP8 and the executor caspases, like CASP3. Conversely, CASP8 inhibits necroptosis by suppressing the function of RIPK1 and RIPK3 to activate MLKL [30,31]. Then, to avoid apoptosis we pre-treated cells with zVAD, and then exposed them to TNF to induce necroptosis, observing a significant reduction in cell death levels when RIPK1, RIPK3, and MLKL were knocked down (Figure 2K). In summary, these results show that a single and low dose of TNF induces RIPK1 kinase-dependent apoptosis in MSCs, but not necroptosis. However, when the catalytic activity of caspases is blocked by zVAD, MSCs die by necroptosis.

Figure 2.

TNF induces RIPK1-dependent apoptosis in MSCs. (A) WT MSCs were pre-treated or not for 20 min with 10 μM Nec-1s, and then MSCs were exposed to indicated concentrations of TNF by 21 h, and cell death was measured by SytoxGreen (SG) positivity. (B-C) WT MSCs were pre-treated or not for 20 min with 10 μM Nec1s, and then cells were exposed to 10 ng/ml TNF, and TNF-mediated cell death (B) and caspase activity (C) were measured in function of time, respectively, by SG-positivity and DEVD-AMC fluorescence. (D-F) WT MSCs were pre-treated or not for 20 min with 10 μM Nec1s, and then stimulated with 10 ng/ml TNF for 48 h. Then, cPARP⁺ and TUNEL⁺ cells were observed by IF and confocal microscopy. (D) Representative images show: DAPI in cyan, and TUNEL⁺ in magenta, cPARP⁺ in green, and F-actins in yellow. Scale bar: 20 µm. (E) Quantification of cPARP⁺ cells (%). (F) Quantification of TUNEL⁺ cells (%). (G and H) WT MSCs were pre-treated for 20 min with 10 μM Nec1, 10 μM Nec1s, 10 μM GSK963, 1 μM GSK157 and 10 GSK840, and then cells were exposed to 10 ng/ml TNF for 21 h, and TNF-mediated cell death (G) and CASP3 activity (H) were measured, respectively, by SG positivity and DEVD-AMC fluorescence. (I) Model of signaling following TNFRSF1A activation. The TNF signaling pathway begins with the activation of its transmembrane receptor TNFRSF1A. (I.i) RIPK1 has a scaffold protein role in the TNFR1-NFKB pathway, positively impacting cell survival. (I.ii) RIPK1 enzymatic activity in the presence of CASP8 activity induces apoptosis. (I.iii) RIPK1 enzymatic activity positively regulates necroptosis with RIPK3 and MLKL. (J) WT MSCs were transfected with siRnaNAs targeting Ripk1, Ripk3, Mlkl mRNA, or the control (siCtrl). After 48 h, total protein extracts were generated and probed as indicated. (K) Cells described in (J) were pre-treated for 20 min with 50 μM zVAD or DMSO (control), and then stimulated with 10 ng/ml TNF for 21 h. TNF-mediated cell death was measured by SG-positivity. The results are presented as mean±S.E.M. of three or four independent experiments. Statistical significance was determined by two-way ANOVA. Significance between samples is indicated as follows: n.s., p > 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001.

RUBCNL deficiency sensitizes to RIPK1 kinase-dependent apoptosis and necroptosis

To determine the effect of RUBCNL deficiency in RIPK1-dependent apoptosis induced by TNF, we transfected MSCs with siRNA targeting Rubcnl (siRubcnl) or siCtrl (Figure 3A). After 48 h of transfection, a single TNF dose was applied, leading to the appearance of classical morphological changes resembling apoptosis, including cell shrinkage and detachment (Figure 3B). This phenotype was confirmed through real-time quantification of SG-positive cells and CASP3 activity Figure 3(C,D) respectively. Interestingly, the transient depletion of RUBCNL increased the susceptibility of MSCs to TNF-induced apoptosis, which was completely prevented by Nec1s Figure 3(C,D). With our proteomic data we could exclude that RUBCNL depletion per se indirectly affects the expression of TNF receptor signal adaptors, since no significant changes in their protein levels were detected (Fig. S4A). The repressive effect of RUBCNL on RIPK1-dependent apoptosis was also confirmed by determining cleaved CASP3 (cCASP3), and cPARP by western blot (Fig. S4B). Given that we identified a new negative regulator of TNF-induced death, we decided to further evaluate the contribution of RUBCNL to the different cellular death outcomes of TNFRSF1A engagement in MSCs. Binding of TNF to TNFRSF1A can trigger RIPK1 kinase-dependent apoptosis when it is combined with MAP3K7/TAK1 inhibitor (5Z)-7-oxozeaenol (MAP3K7-inh.) and RIPK1 kinase-dependent necroptosis in the presence of zVAD [32,33]. We tested the effect of TNF in combination with MAP3K7-inh., or zVAD, in control MSCs or MSCs lacking RUBCNL, similar as shown in Figure 3(C). The effect RUBCNL loss of function in response to TNF was also observed in TNF- and MAP3K7-inh.-induced apoptosis (Fig. S4C). Furthermore, we found that siRNA-mediated repression of Rubcnl in MSCs sensitizes to TNF+zVAD-induced cell death, which was associated with increased phosphorylation of MLKL Figure 3(E,F), indicative of increased necroptosis induction. In line with this, we found that this susceptibility was not cell type specific, as siRNA-mediated repression of Rubcnl in mouse embryonic fibroblast (MEF) cells (Fig. S4D) also sensitized them to TNF+zVAD-induced necroptosis (Figure 3G). To exclude any off-target effect of the Rubcnl-targeting siRNA, we confirmed our results by using lentivirus to stably express shRNA targeting Rubcnl mRNA (Fig. S4E-H) [18,19]. As shown in Fig. S4E-H, stable depletion of Rubcnl sensitized MSCs to RIPK1 kinase-dependent apoptosis and necroptosis, similar as observed for siRNA-mediated repression of Rubcnl (Figure 3A-F). Altogether, these results show that the presence of RUBCNL restrains RIPK1-dependent apoptosis and necroptosis triggered by the activation of TNFRSF1A.

Figure 3.

RUBCNL deficiency sensitizes to apoptosis and necroptosis. (A) MSCs were transfected with siRnas targeting Rubcnl mRNA (siRubcnl) or the siRNA control (siCtrl). After 48 h, total protein extracts were generated and probed as indicated. (B) MSCs described in (A) were treated with 10 ng/ml TNF by 21 h, and cell morphology was visualized by phase contrast microscopy. (C-D) Cells were pre-treated for 20 min with 10 μM Nec1s, and then cells were exposed to 10 ng/ml TNF. TNF-mediated cell death (C) and CASP3 activity (D) were measured in function of time, respectively, by SG positivity and DEVD-AMC fluorescence. (E-F) Cells were pre-treated for 20 min with 50 μM zVAD presence or absence of Nec1s and stimulated with 10 ng/ml TNF, and cell death was measured in function of time, by SG-positivity fluorescence, and protein extracts were generated and probed as indicated. (G) MEFs were transfected with siRNAs targeting Rubcnl mRNA (siRubcnl) or the siRNA control (siCtrl). After 48 h, total cells were exposed to 10 ng/ml TNF+zVAD and cell death was measured in function of time, by SG. The results are presented as mean±S.E.M. of four independent experiments. Statistical significance was determined by two-way ANOVA. Significance between samples is indicated as follows: ****, p ≤ 0.0001.

RUBCNL overexpression represses RIPK1 kinase-dependent apoptosis and necroptosis

In our primary screening, we determined that human RUBCNL-Flag overexpression led to a significant protection against TNF but not against other stimuli (Fig. S2). Also, we and others demonstrated that RUBCNL promotes autophagosome formation and autophagosome maturation [12,17,18]. We confirmed the positive effect of RUBCNL-Flag overexpression (Fig. S2B) on the autophagy process in MSCs (Figure 4A). This effect was not affected upon TNFRSF1A activation, suggesting that RUBCNL-Flag overexpression could stimulate autophagy and independently repress TNF-induced cell death in MSCs. To test this hypothesis, we determined the effect of stable overexpression of RUBCNL-Flag in the different cell death modalities triggered by TNF in MSCs. We used Nec1s as a positive control to monitor RIPK1 kinase dependency in cell death. Strikingly, we found that RUBCNL-Flag overexpression represses RIPK1-dependent apoptosis triggered by a single dose of TNF (Figure 4B) or by the combination of TNF+MAP3K7-inh (Figure 4C). Furthermore, RUBCNL-Flag overexpression also completely inhibited RIPK1 kinase-dependent necroptosis induced by the combination of TNF+zVAD, as well as Nec1s (Figure 4D). We confirmed that necroptosis-repression mediated by RUBCNL was not cell type and construct specific, as RUBCNL with a C-terminal V5-tag also protected L929 cells from TNF-induced necroptosis (Figure 4E,F). Taken together, our results confirm the positive effect of RUBCNL in autophagy in MSCs and reveal a new activity of RUBCNL in promoting MSCs survival in response to TNF, independent if TNFRSF1A activation induces apoptosis or necroptosis.

Figure 4.

RUBCNL overexpression protects from TNF-induced cell death in MSCs. (A) MSCs were stably transduced with lentiviral expression vectors for RUBCNL-Flag or empty vector (Mock) (see also Figure S2B). Total protein extracts were generated and probed as indicated. (B) Cells described in (A), were pre-treated for 20 min with 10 μM Nec1s, and then cells were exposed to 10 ng/ml TNF and cell death was measured by SG-positivity in function of the time. (C-D) Cells described in (A) were pre-treated with MAP3K7-inh. (C) or zVAD (D), in presence or absence of Nec1s and stimulated with 10 ng/ml TNF, and cell death was measured in function of time by SG-positive fluorescence. (E) L929 cells were transfected with expression vectors for RUBCNL-V5 or empty vector (Mock) by 48 h. Total protein extracts were generated and probed as indicated. (F) Cells described in (E) were pre-treated for 20 min with 10 μM Nec1s and 50 μM zVAD, and then cells were exposed to 10 ng/ml TNF and cell death was measured by SG-positivity in function of the time. The results are presented as mean±S.E.M. of three or four independent experiments. Statistical significance was determined by two-way ANOVA. Significance between samples is indicated as follows: n.s., p > 0.05; ****, p ≤ 0.0001.

RUBCNL represses RIPK1 cytotoxicity independent of its role in autophagy

Here we established that RUBCNL promotes cellular survival upon TNFRSF1A activation, while previously we and others have shown that RUBCNL promotes autophagy [12,17,18]. These data suggest that RUBCNL acts in two cellular pathways whose connection is not fully understood. To determine the role of autophagy in RUBCNL-regulated TNF-induced cell death, we downregulated the endogenous autophagy machinery by targeting Atg5 expression with siRNAs (siAtg5) (Fig. S5A). Depletion of ATG5 impairs basal autophagy flux observed in the presence of BAF+CQ (Fig. S5B). To determine if basal autophagy flux has a role in RUBCNL-mediated protection in TNF-induced cell death, we targeted ATG5 expression in MSCs overexpressing RUBCNL-Flag and the control Mock (Figure 5A and Fig. S2B). Interestingly, the protection against TNF-induced apoptosis and TNF+zVAD-induced necroptosis generated by RUBCNL-Flag overexpression is not affected by the downregulation of ATG5 Figures 5(B,C) respectively. Then, we tested the effect of Atg5 knockdown together with RUBCNL loss of function. For this purpose, MSCs were co-transfected with siAtg5 and siRubcnl. The respective knockdown efficiencies were evaluated by qPCR (Figure 5D). Under these experimental conditions we found that even under simultaneous siAtg5 and siRubcnl knockdown the depletion of RUBCNL conserved its capability to sensitize to TNF-induced apoptosis and necroptosis (Figure 5D-F). Together, these results show that the loss of function of the early/mid-stage of autophagy does not affect the protection RUBCNL provides from apoptosis and necroptosis upon TNFRSF1A activation.

Figure 5.

Autophagy counteracts TNF-induced cell death, but it is dispensable for RIPK1-dependent apoptosis and necroptosis repression by RUBCNL upon TNFR1 activation. (A) MSCs stably transduced with lentiviral expression vectors for RUBCNL-Flag or empty vector (Mock) (Fig. S2B) were transfected with siRnas targeting Atg5 mRNA (siAtg5) or the control (siCtrl). Total protein extracts were generated and probed as indicated. (B and C) Cells described in (A), were treated with TNF (B) or TNF+zVAD (C). Cell death was measured by SG-positivity in function of time. (D-F) WT MSCs were transfected with siCtrl (2×) (40 nM), siCtrl (20 nM) + siAtg5 (20 nM), siCtrl (20 nM) + siRubcnl (20 nM), and siAtg5 (20 nM) + siRubcnl (20 nM). (D) Efficient knockdown of Rubcnl and Atg5 was assessed at 48 h by qPCR. Foldchange of Rubcnl or Atg5 mRNA levels was calculated using Actin mRNA levels as a reference. Cells were additionally treated with TNF (E) or TNF+zVAD (F) and cell death was measured at indicated times. (G) Illustration of autophagy competent or incompetent V5-tagged RUBCNL mutants. (H and I) Cells described in (Fig. S5C-F) were stimulated with TNF (H) or TNF+zVAD (I), and cell death rate was measured by SG-positivity on the time. The results are presented as mean±S.E.M. of three or four independent experiments. Statistical significance was determined by one-way ANOVA or two-way ANOVA. Significance between samples is indicated as follows: n.s., p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Previously, Cheng et al. showed that autophagy-regulation mediated by RUBCNL depends on its interaction with UVRAG. The mutation of amino acids VEKEN into AAAAA (RUBCNL[5A]) generated the loss of RUBCNL binding to UVRAG [12]. Also, Cheng et al. identified Ser157 of RUBCNL to be phosphorylated by MTORC1, disrupting the association of RUBCNL with STX17 and the HOPS complex, abolishing RUBCNL-mediated autophagosome maturation. Conversely, Ser157 dephosphorylation of RUBCNL induced by nutrient-deprived conditions promotes KAT5/TIP60-mediated RUBCNL acetylation. Two lysine (K) residues, K483 and K573, were identified as targets of KAT5/TIP60, whose acetylation enhances autophagy flux [17]. We generated constructs expressing wild-type human RUBCNL (WT RUBCNL), as well as the above-mentioned RUBCNL mutants, all with a C-terminal V5-tag (Figure 5G). VEKEN residues were mutated into AAAAA (RUBCNL[5A]), and Ser157 residue was replaced by aspartate (RUBCNL^S157D), to mimic phosphorylation. Thus, Ser157 was mutated into alanine (RUBCNL^S157A), a non-phosphorylatable amino acid. Finally, K483 and K573 were replaced by arginines (RUBCNL[2K]), abolishing RUBCNL acetylation. We transfected these vectors in MSCs and tested RUBCNL construct expression by western blot (Fig. S5C). First, we assessed the effect of these constructs on autophagic flux under basal conditions in presence of lysosome inhibitors (BAF+CQ). We detected the enhancement of basal autophagy flux generated by WT RUBCNL expression (Fig. S5D). In contrast, RUBCNL[5A] expression failed to stimulate autophagy since the LC3-II levels were similar to the Mock (Fig. S5D). RUBCNL^S157A stimulated autophagy, whereas RUBCNL^S157D (Fig. S5E), and RUBCNL[2K] (Fig. S5F) had no effect. Additionally, we confirmed the effect of Rubcnl knockdown on autophagy flux induced by starvation in MSCs (Fig. S5G). To determine if RUBCNL-mediated protection against apoptosis and necroptosis is dependent on its function in autophagy we tested the effect of WT RUBCNL and RUBCNL mutants expressed in MSCs exposed to TNF or TNF+zVAD (Figure 5H,I), and the cell death rate was measured. As previously observed, WT RUBCNL significantly protected MSCs against both RIPK1 kinase-dependent apoptosis and necroptosis induced by TNF or TNF+zVAD, respectively. Interestingly, RUBCNL[5A], RUBCNL^S157D, RUBCNL^S157A, and RUBCNL[2K] also conferred significant protection to both TNF-induced RCD, similar to WT RUBCNL. To complement these results, we also tested the effect of MTOR inhibitor and autophagy inducer rapamycin (Rapa) on TNF-induced cell death in MSCs. We found that the pharmacologic autophagy induction with rapamycin reduces TNF-induced cell death, suggesting that autophagy has a role in the protection from TNF-induced cell death (Fig. S5H). Furthermore, rapamycin also rescued from TNF-induced cell death under RUBCNL depletion, reducing the levels of TNF-induced cell death in similar proportions in siCtrl and siRubcnl treated cells (Fig. S5I). In summary, these data suggest that RUBCNL can inhibit RIPK1-dependent apoptosis and necroptosis mechanistically independent of its autophagy enhancer function.

RUBCNL forms a dynamic complex with RIPK1

To interrogate the role that RUBCNL plays in TNFRSF1A signaling, MSCs stably transduced with lentiviral constructs to target Rubcnl mRNA (shRubcnl or shCtrl (Fig. S1B), were stimulated for 10 min with TNF-Flag and subjected to immunoprecipitation using anti-Flag antibody to pull down TNF-Flag-TNFRSF1A-RIPK1 complex and analyzed by western blot (Figure 6A). We found that the loss of Rubcnl expression significantly increases the amount of RIPK1 in complex I in response to TNF-Flag treatment (Figure 6A,B), suggesting that the absence of RUBCNL facilitates the recruitment of RIPK1 by complex I, which could explain the anti-apoptotic and anti-necroptotic effect of RUBCNL. We then investigated if RUBCNL and RIPK1 form a complex with each other in vitro by using proximity ligation assay (PLA). MSCs were transiently transfected with empty vector (Mock) or a vector expressing human V5-tagged RUBCNL (RUBCNL-V5). Interaction of RUBCNL with endogenous Ripk1 was detected by confocal microscopy (Figure 6C and Fig. S6). Under steady-state conditions, RUBCNL-V5 and RIPK1 were found to interact, whereas treatment with TNF abolished this interaction in a time-dependent manner until no interaction was observed at 120 min (Figure 6C,D). Accordingly, co-immunoprecipitation using V5-tagged RUBCNL in HEK293T cells showed that RIPK1 is in complex with RUBCNL-V5, and again, this interaction is lost when cells were treated with TNF for 120 min (Figure 6E). In summary, RUBCNL appears to regulate the availability of RIPK1 to bind TNFRSF1A affecting the formation of complex I and the execution of the cell death program. Hence the presence of RUBCNL negatively regulates RIPK1 kinase-dependent cell death signaling by tethering RIPK1 (Figure 6F).

Figure 6.

Treatment with TNF reduces the interaction between RUBCNL and RIPK1 in MSCs. (A) MSC treated with shCtrl or shRubcnl (Fig. S1B) were stimulated with FLAG-HsTNF. Complex I was immunoprecipitated, and TNFRSF1A-bound RIPK1 was analyzed by immunoblotting. (B) Levels of RIPK1 in complex with TNFRSF1A were quantified in 3 independent experiments. (C and D) Detection of an interaction between RUBCNL and RIPK1 by PLA in vitro. Representative images of MSCs were transfected with Mock or V5-tagged RUBCNL and then treated with TNF for 120 minutes. Complex formation between RUBCNL-V5 and endogenous RIPK1 was observed by confocal microscopy. (C) Representative images are shown for PLA test. DAPI is depicted in cyan, and the RUBCNL-RIPK1 complex in magenta. Scale bar 20 µm. (D) Quantification of the amount of RUBCNL-RIPK1 complex spots per nucleus radius in each sample. Complex formation was observed under non-treated conditions or TNF treatment for 10, 30, 60, and 120 min. (E) HEK293T cells were transfected with empty vector (Mock) or a vector for RUBCNL-V5 and treated or not with TNF (10 ng for 120 min). Forty-eight h post-transfection, a co-immunoprecipitation was performed using the V5 tag on RUBCNL. RIPK1 and V5-tagged RUBCNL were detected by western blot. Representative blots of 3 independent experiments are shown. (F) Model of a possible mode of action of RUBCNL on RIPK1-dependent cell death signaling. Statistical significance was determined by one-way ANOVA. Significance between samples is indicated as follows: n.s., p > 0.05; ****, p ≤ 0.0001.

Discussion

TNF indirectly promotes inflammation by triggering cell death, which may depend on RIPK1 enzymatic activity [34]. Protective mechanisms, also called cell death checkpoints, normally repress TNF cytotoxicity. Consequently, death by TNF usually requires inactivation of one of these protective brakes. In this study, we show that, in contrast to most cell types, MSCs succumb by RIPK1 kinase-dependent apoptosis upon sensing of TNF alone. Caspase inhibition by zVAD does not protect MSCs from death, but switches the TNF response to necroptosis, which can be efficiently repressed by pharmacological inhibition of RIPK1, or downregulation of RIPK1, RIPK3 or MLKL. Nevertheless, RIPK1 is known to act as a scaffold protein for the TNF/NFKB pathway, sensitizing cells to TNF-dependent death when downregulated [35]. Necroptosis can be executed in MSCs when caspases are inactivated after TNFRSF1A activation, suggesting a possible role in the control of the inflammatory response mediated by MSCs [36]. Also, RIPK1 has been identified as a key point in determining cellular fate for development and tissue homeostasis [37]. For instance, recently, it was shown that RIPK1 expression was required for both MSC proliferation and survival in a TP53/p53-BBC3/PUMA axis-dependent manner [38]. Our results reveal a key function for RIPK1 in cell death promotion in MSCs and, thereby, an active role in their inflammatory response. Interestingly, our results suggest that RIPK1 inhibitors (Nec1, Nec1s, GSK963, and GSK157) could be effective pharmacological alternatives for the control of cell death in MSC therapy. However, more detailed studies of the effect of RIPK1 inhibitors are required since, under certain conditions, the cell death process could contribute to the therapeutic efficacy of MSCs [39].

An active role for autophagy in MSC differentiation has been proposed previously [40,41], however, the role of autophagy in the immunosuppressive function of MSC remained elusive. Recently, Bergmann et al. described that RUBCNL overexpression enhances the effectiveness of MSCs in ameliorating the symptoms of DSS-induced colitis in mice [19]. Interestingly, RIPK1 is involved in cell death and inflammation in DSS-induced colitis [42], a widely used experimental model of inflammatory bowel disease. RIPK1 inhibitors have been proposed as drug candidates for inflammatory diseases, given that RIPK1 is a critical driver of inflammation of various pathways downstream of TNFRSF1A [43,44]. BECN1, an interactor of RUBCNL and a core component of the autophagy machinery, has also been described to regulate the therapeutic capacity of MSCs in a model of encephalitis [45–47]. Interestingly, BECN1 was shown to interact with phosphorylated MLKL in HT-29 cells, abrogating necrosome formation and necroptosis activation upon TNF treatment by preventing MLKL oligomerization [48].

Here, we identify a previously unknown relationship between RUBCNL and RCD. We provide evidence that RUBCNL represses apoptosis and necroptosis triggered by TNF in MSCs but not upon other stimuli related to ferroptosis, oxidative stress, DNA damage, or ER stress. Furthermore, we demonstrate that necroptosis repression mediated by RUBCNL is not cell type-specific, given that RUBCNL also protects L929 and MEFs cells from necroptosis triggered by TNF and TNF+zVAD, respectively. Conversely, RUBCN, which was reported to function antagonistically to RUBCNL by repressing autophagy, is required for UV-induced immunosuppression in vivo but it does not affect cell death of MEFs exposed to UV [47]. Another component of the autophagy machinery, the autophagy receptor SQSTM1, promotes necrosome assembly by functioning as a scaffold for the death complex [23,26]. It is unknown if the scaffolding role of autophagy machinery components in cell death is restricted to SQSTM1. Interestingly, the downregulation of UVRAG impairs autophagosome-lysosome fusion, resulting in SQSTM1 accumulation and facilitating necroptotic cell death. Consistently, AAV-mediated UVRAG overexpression reduced MLKL phosphorylation induced by TNF in both in vitro and murine model of Alzheimer disease [26]. Recently, another autophagy protein, ATG9A, was shown to promote degradation of the cytotoxic complex II through an LC3-independent lysosomal targeting pathway, protecting against TNF-induced apoptosis in vitro and in mouse models of inflammatory skin disease [24]. Also, deficiency of ATG14 in the intestinal epithelium results in spontaneous cell death and villus loss, with sensitivity to TNF-induced apoptosis [25]. Therefore, similar to RUBCNL, UVRAG, ATG9A, and ATG14 also suppress TNF-cytotoxicity. Conversely, results reported for RUBCN in MLKL-mediated necroptosis are contradictory, with opposing effects in acute kidney injury and kidney ischemia-reperfusion injury [49,50]. The involvement of multiple regulatory autophagy proteins, such as RUBCNL, SQSTM1, UVRAG, BECN1, RUBCN, ATG14, and ATG9A in the regulation of cell death may indicate close ties between both pathways.

Given that RUBCNL promotes autophagy [12,17–19] and represses apoptosis and necroptosis, we wonder about the mechanics of the bidirectional relationship between both phenomena. Interestingly, the MTOR inhibitor rapamycin also enhances the immunosuppressive function of MSC mediated by TGFB secretion [51], suggesting that autophagy induction affects MSC survival and their capacity for immunoregulation. Here we show that the protection against TNF-induced apoptosis and TNF+zVAD-induced necroptosis generated by RUBCNL expression is not affected by the downregulation of ATG5, indicating that autophagy is dispensable for RUBCNL-mediated protection from cell death upon TNFRSF1A activation. Moreover, taking advantage of previously reported post-translational regulation of RUBCNL, we tested the effect of RUBCNL mutants defective in their effect on autophagy regulation [12,17]. Remarkably, RUBCNL represses RIPK1-kinase-dependent apoptosis and necroptosis even when its ability to stimulate autophagy is lost, suggesting that these two functions exist side-by side within separate domains in the RUBCNL protein. Furthermore, we found that RUBCNL can negatively regulate complex I formation by limiting RIPK1 availability through binding, hence increased levels of RUBCNL can impair the execution of cell death and promote survival by holding RIPK1 in check upon TNFRSF1A activation. It remains to be investigated in detail what happens with RIPK1 after forming a complex with RUBCNL, one option could be that RUBCNL is involved in the steady-state turnover of RIPK1 possibly through autophagy mediated degradation. Moreover, it remains to be investigated which domain of RIPK1 and RUBCNL on the other side is responsible for their interaction and if this interaction is direct or through another protein.

Autophagy plays conflicting roles in various diseases, where the difference between a beneficial or detrimental effect seems to depend on the physiological state of the tissue affected which is defined by the timepoint during disease progression. In recent years the roles of autophagy in cell death have become more and more recognized, yet the mechanisms underlying these roles have remained elusive. Our results advocate RUBCNL as a dual regulator of autophagy and apoptosis/necroptosis, whose titration could function as a dynamic switch between different cell survival/death modalities making RUBCNL an attractive therapeutic target for diverse diseases, including cancers or neurodegenerative diseases, as well as for improving the therapeutic efficacy of MSCs.

Materials and methods

Antibodies and reagents

Antibodies and reagents were purchased from the following companies: anti-Flag M2 (Sigma-Aldrich, F3165), TUBB/β-tubulin (Cell Signaling Technology, 2146), anti-CASP3 (Cell Signaling Technology, 9662), anti-cCASP3 (Asp175, 5A1E; Cell Signaling Technology, 9664), anti-cPARP (Asp214; Cell Signaling Technology, 9544S), anti-RIPK1 (Cell Signaling Technology, 3493), anti-RIPK1 (Becton Dickinson Transduction Laboratory 610,458), anti-RIPK3 (Abcam, Cambridge, UK 56,164), anti-phospho-MLKL (Ser358, D6H3V; Cell Signaling Technology 91,689), anti-RUBCNL/Pacer [20,21] (Abmart, custom antibody), anti-LC3B (Cell Signaling Technology, 2575), anti-SQSTM1/p62 (Abcam, ab56416), anti-ATG5 (Cell Signaling Technology, 2630), anti-V5 (D3H8Q; Cell Signaling Technology 13,202), anti-V5 (Cell Signaling Technology 132,025), ACTB/β-actin (Cell Signaling Technology, 4967), cycloheximide (CHX; Sigma-Aldrich, C-7698), 9-epimer-11,12-dihydro-(5Z)-7-oxozeanol ((5Z)-7-oxozeaenol or MAP3K7-inh.) (AnalytiCon Discovery GmbH, NP-0009245), zVAD-fmk (Bachem, N-1510), GSK2656157/GSK157 (ApexBio, B2175), GSK840 and GSK963 were provided by GlaxoSmithKline, Nec1 (Calbiochem 480,065), Nec1s (Laboratory of Medicinal Chemistry, University of Antwerp, Belgium), H₂O₂ (Sigma-Aldrich, 7722-84-1), tunicamycin (Sigma-Aldrich, T7765), ML162 (Sigma-Aldrich, SML2561), etoposide (Sigma-Aldrich, E1383), thapsigargin (Sigma-Aldrich, T-9033), bafilomycin A₁ (BAF; Cell Signaling Technology, 54645S), chloroquine (Sigma-Aldrich, C6628), recombinant human TNF and FLAG-tagged human TNF (TNF-flag) was produced and purified to at least 99% homogeneity in our laboratory and have a specific biological activity of 6.8 × 10⁷ IU/mg and 2.3 × 10⁹ IU/mg, respectively (Inflammation Research Center, Ghent University).

Cell culture

Murine bone marrow MSCs were obtained from GIBCO (S1502–100) and Cyagen (MUB0100). MSCs were cultured at 37°C with 5% CO₂ in complete alpha-modified Eagle’s medium (αMEM; Gibco 12,571–063) containing 5% heat-inactivated fetal bovine serum (FBS; Gibco, 10437028, 100 U/ml penicillin and 100 μg/ml streptomycin (Pen-strep; Gibco, DW101203–031-1B). Cells were used between passages 6 and 12. The human embryonic kidney 293 (HEK293T) cells (Sigma 12,022,001) were grown at 37°C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Gibco 12,800,017) supplemented with 5% FBS (Gibco 10,437,028) and 1% Pen-strep.

Lentivirus stable transduction

HEK293T cell line was used for lentiviral particle production using a Lenti-ORF clones kit (Origene, TR30022) according to the manufacturer’s protocol. For RUBCNL gain-of-function experiments, lentiviral particles were produced in HEK293T cells. Briefly, HEK293T cells were seeded at 2.5 × 10⁶ in a 10 cm dish in 10 ml complete DMEM growth media (without antibiotic) and incubated overnight. Then the cells were transfected with 5 μg of either empty vector or human Flag-tagged RUBCNL plus 6 μg of packaging plasmids from Lenti-ORF clones kit (Origene, TR30022). The medium was replaced 12 h post-transfection. The viral supernatant was collected at 24 h and 48 h and filtered through a 0.45-μm filter to remove cellular debris. High titer lentiviral stocks were produced (10⁶–10⁷ TU/ml). Murine MSCs were transduced at passage 9 with lentivirus according to Lenti-ORF clones following the manufacturer’s instruction, generating MSC transduced with empty vector (Mock) or with human Flag-tagged RUBCNL (RUBCNL-Flag). Both constructs carry a puromycin resistance gene; hence MSCs resistant to puromycin (10 μg/ml) were selected over 3 passages and used up to passage 18. We generated stable MSC with reduced levels of Rubcnl mRNA lentiviral expression of shRNA targeting endogenous Rubcnl mRNA (shRubcnl). As a control, a scramble shRNA construct (shCtrl) was employed. Both shRubcnl and shCtrl constructs were purchased from Origene (TR30021, TL516617A-D). We screened five different constructs and selected the two most efficient constructs after real-time PCR analysis [21].

Transient transfection and overexpression

WT human RUBCNL and human mutant RUBCNL (RUBCNL[5A], RUBCNL^S157D, RUBCNL^S157A, RUBCNL[2K]) were cloned into pLV lentiviral vector and V5-tagged (Vectorbuilder). The constructs were transfected using TransIT-x2 dynamic delivery system (Mirus Bio, MR.MIR6000) according to the manufacturer’s instructions.

siRNA transfection

For RUBCNL loss-of-function experiments, MSC were seeded at a concentration of 2 × 10⁵ cells/well in six-well plates and transfected 24 h later with ON-TARGET plus smart-pool siRNAs targeting mouse Rubcnl (siRubcnl), ON-TARGET plus smart-pool siRNAs targeting mouse Atg5 (siAtg5) and ON-TARGETplus non-targeting siRNAs as a control (siCtrl) (All of them from Dharmacon) using Dharmafect Transfection Reagents (Dharmacon, T-2001-01). Briefly, 4 μl Dharmafect was used to obtain a final 20 nM siRNA/well concentration. In the experiments involving double knockdown, the final concentration of each siRNA was set at 20 nM, resulting in a total concentration of 40 nM siRNA/well. Control samples were transfected with 40 nM siCtrl/well. After 48 h of transfection, the cells were used in cell biology experiments.

Real-time PCR

Total RNA was extracted with TRIzol (Life Technologies GmbH 10,296,028) and reverse-transcribed with a First-strand cDNA synthesis kit (Thermo Fisher Scientific, K1621). mRNA levels were determined by real-time PCR using SYBR Green (Kappa biosystem, KK4602) and normalized to mRNA levels of Actb/β-actin. Primer sequences were as follows: mouse Actb 5′-AAGATCATTGCTCCTCCTGA-3′ 5′-TACTCCTGCTTGCTGATCCA-3′; mouse Rubcnl 5′–TTCACCCACCAATCAAGAGGGACA-3′; 5’-ACAAGACTCTGCAGATGAGTGGCA-3′. PCR conditions were: 1 cycle at 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min. The final extension step was carried out at 72°C for 10 min. All primers were synthesized by Macrogene (USA, California).

Cell death analysis

Cell death measurements were carried out using a Fluostar Omega fluorescence plate reader (BMG Labtech, Ortenberg, Germany) with temperature and atmosphere-controlled settings, as previously reported [29,32]. In brief, 10 × 10⁴ cells per well were seeded in duplicate in a 96-well adherent plate. The next day, cells were treated with the indicated compounds in the presence of 5 μM SytoxGreen (SG; Invitrogen, S7020) and/or 10 µM Ac-DEVD-AMC (Pepta Nova, 3171-V). SG intensity was measured in function of time at one-hour intervals with an excitation filter of 485 nm, emission filter of 520 nm, and gains set at 1100. We used 20 flashes per well and orbital averaging with a diameter of 5 mm. Cell death was calculated by subtracting the induced SG fluorescence from the background fluorescence and dividing the result by the maximal fluorescence (minus the background fluorescence) obtained by permeabilizing the cells using Triton x-100 at a final concentration of 0.1%. CASP3 activity (cleaved DEVD-AMC) was obtained using an excitation filter of 355 nM and an emission filter of 460 nM with the same time intervals and gains of 1000 with 20 flashes per well and 5 mm orbital averaging.

Autophagy assays

For basal autophagy experiments, MSCs were treated with BAF (25 nM) and CQ (25 µM) for the indicated times. Then, total protein extracts were generated, and LC3-II was detected by western blot.

Immunoprecipitation assays

For immunoprecipitations (IPs) of complex I, 1 × 10⁶ MSC cells stably knocked down for Rubcnl or control cells (shCtrl or shRubcnl) were seeded the day before in a 10-cm² tissue culture. In a 10 min of pretreatment, MSCs were stimulated with 2 μg/ml FLAG-HsTNF for complex I formation. Cells were then washed two times in ice-cold PBS (Calbiochem 524,650) before 15 min of lysis (4ºC) in 1 ml of NP-40 lysis buffer (10% glycerol, 1% NP-40 [Sigma 74,385], 150 mM NaCl and 10 mM Tris-HCl, pH 8 supplemented with phosphatase [Roche 4,906,845,001] and protease inhibitor cocktail tablets [Thermo Scientific^TM, A32955). The cell lysates were cleared by centrifugation at 10,000× g, for 10 min at 4°C, and the supernatants were then incubated overnight with FLAG® M2 Magnetic Beads (Sigma-Aldrich, M8823) for complex I IPs. The next day, beads were resuspended in 50 μl 1× DUB/λPP buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 5 mM dithiothreitol and 1 mM MnCl₂) after the final wash step. Then, 1.0 μg USP2 (R&D systems, E-504) and/or 800 U Lambda protein phosphatase (New England BioLabs, P0753S) was added as indicated. Reactions were incubated for 45 min at 37°C. All enzymatic reactions were quenched by adding Laemmli buffer (1× final) and by boiling 5 min at 95°C. To evaluate the interaction between RUBCNL and RIPK1, HEK293T cells were transiently transfected with a vector for V5-tagged human RUBCNL (RUBCNL-V5). Forty-six h after transfection, cells were stimulated with TNF for 2 h. Forty-eight h after transfection, the protein extracts were prepared in 500 μL lysis buffer (0.1% NP-40, 100 mM KCl, 50 mM Tris, pH 7.5, 150 mM NaCl), plus protease inhibitor cocktail 1 X (Thermo Scientific^TM, A32955). After incubation overnight on ice, total cell extracts were subjected to immunoprecipitation (IP) using the V5-tagged protein purification kit Ver.2 (MBL Int., 3317) according to the manufacturer’s recommendation. Protein complexes were eluted with V5 peptide. The input and IP eluate were separated by SDS-PAGE and assessed by western blot analysis.

Proximity ligation assay (PLA)

Cells (3 × 10⁵ per well) were seeded in 6-well plates with 4–5 5-mm coverslips. After one day, cells were transiently transfected with a vector for RUBCNL-V5 or an empty vector (Mock) as control. Forty-eight h after transfection, cells were stimulated with TNF for the indicated times, and PLA was developed following manufacturer’s instructions (Duolink®; Sigma-Aldrich, DUO92101) using 1:200 anti-V5 (Cell Signaling Technology 132,025) and 1:500 anti-RIPK1 (BD 610,458). We captured images using a Leica SP8 confocal microscope with an immersion objective of 63× magnification. Each channel was scanned sequentially using a 405-nm diode laser for DAPI and a 546 nm laser for Texas Red (PLA). We set the scanning frequency to 200 Hz, and the acquired images had 1024 × 1024× 10 pixels on the x, y, and z axes, respectively. We processed and analyzed images using the IMARIS software. Initially, we rendered channels on 3D surfaces by thresholding fluorescence intensity at a minimum of 10 AU for nuclei (minimum diameter of 10 µm) and PLA signal (minimum diameter of 2 µm). Using a spot selection tool with a 10-µm diameter per spot, we identified nuclei and classified them as positive if they were within a maximum distance of 15 µm from the PLA 3D surface. Within a 15-µm radius of each nucleus, we detected PLA spots with a mean intensity of 10 AU and a diameter of 2 µm (Fig. S7A). We exported the IMARIS data as.csv files with x and y coordinates for each nucleus and PLA spot. To clean and organize the data, custom-made R scripts were utilized to group PLA spots by nucleus based on x and y coordinates within 15 µm of nuclei. According to the frequency distribution of the number of PLA per nucleus (n_pla), we categorized the data into five groups (n_pla_cat): 0 to 1, 2 to 5, 6 to 10, 11 to 20, and > 20 PLA spots per nucleus and quantified the number of PLA spots per 15 µm nucleus radius (Fig. S7B and S7C). We compared the distribution of PLA spots between the untreated (Mock) group and those treated with TNF for 10, 30, 60, and 120 min using statistical analysis. To evaluate statistical differences between the distribution of cells between the categories, we utilized contingency analysis of chi-squared test with pairwise comparison correction for p-values. In addition, we assessed the number of protein interactions per nucleus using one-way ANOVA with Tukey’s multiple comparisons between conditions. We set the significance threshold for statistical significance at a p-value >0.05 for non-significant (n.s.), a p-value 0.05 for *, a p-value 0.01 for **, and a p-value 0.001 for ***.

TUNEL assay and immunofluorescence

In this study, we used the One-step TUNEL Apoptosis Kit (Elabscience, E-CK-A424) adapted for Immunofluorescence to detect cell apoptosis. The principle of detection is based on the cleavage of genomic DNA in apoptotic cells into multimers of 180 ~ 200bp fragments. After fixation and permeabilization, the exposed 3’-OH of the fragmented DNA is catalyzed by terminal deoxynucleotidyl transferase (TdT) with fluorescein-labeled dUTP and subsequently detected by flow cytometry. First, cells were treated with vehicle, TNF, and TNF+Nec1s for 48 h. Briefly, cells were fixed using PBS 1X with 4% paraformaldehyde. After fixation, cells were washed three times for 5 min with PBS 1X. Cells were then incubated 60 min at room temperature in blocking solution containing PBS 1X, 3% BSA (Sigma-Aldrich 810,667) and 0.1% Triton X-100 (Sigma, T9284). The blocking solution was removed, and the cells were incubated overnight in the dark at 4°C with the primary antibody solution for cPARP at a dilution of 1:500 (Cell Signaling Technology, 5625, D64E10). Following primary antibody incubation, cells were washed three times with 1X PBS for 5 min each. Cells were then incubated with DAPI, Phalloidin conjugated with Alexa Fluor® 555 (Life Technologies, A34055), and a secondary antibody anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch, 111-545-003) at a dilution of 1:1000 at room temperature for 60 min. After secondary antibody incubation, cells were washed three times for 5 min each. Cells were then incubated in 30–50 µL of Equilibration Buffer for 15 min in the dark at 37°C. Working labeling solution was prepared according to manufacturer instructions with 70 µL of TdT Equilibration Buffer, 20 µL of Labeling Solution, and 10 µL of TdT Enzyme for 100 µL of the solution. For the negative control, 80 µL of Equilibration Buffer and 20 µL of Labeling Solution were used. The cells were then incubated with the working labeling solution for 60 min at 37°C in a dark chamber. The reaction was halted by incubating the cells in Stop Solution for 10 min. Stop solution was discarded, and cells were washed in PBS three times for 5 min each. Finally, covers were mounted in 5 µl of Vecta Shield (Vector, H1000).

Microscopy and image quantification

In our study, confocal imaging was performed using the Leica SP8 confocal microscope, utilizing a 63× objective lens. The required resolution was achieved through necessary digital zooming. Fluorescence intensity for each channel was meticulously calibrated, using a negative control sample for TUNEL and antibody staining until no signal was perceivable. The established parameters were then consistently applied across all images. The Argon laser at 488 Hz was utilized for cParp detection, while the 693 Hz He Laser was used for capturing the TUNEL signal. For rigorous analysis, four focal fields were captured per independent transfection, which served as technical replicates, with each transfection acting as a biological replicate. Employing the Imaris Software (BitPlane), a 3D reconstruction of labeled structures was achieved. In the pursuit of quantifying TUNEL and cParp signals within the nucleus, we rendered a surface of the DAPI stained nuclei. Consequently, this enabled us to mask the TUNEL and cParp signals and render their surfaces. By measuring the mean fluorescence intensity of both signals within the nuclei, cells were categorized as either positive or negative. The threshold for categorizing a cell as positive was determined using the signal from the control as a reference for negative cells. Subsequent computations yielded the percentage of positive cells for each technical replicate, which was then averaged to represent the biological replicate value in the graphical representation. Data representation and statistical analysis were carried out using GraphPad Prisms 9 Software.

Protein extraction for nLC-MS/MS

The cells were lyophilized and resuspended in 8 M urea with 25 mM ammonium bicarbonate pH 8, and they were homogenized using ultrasound for 1 min in a cold bath with pulses of 10 s (on/off) at an amplitude of 50%. Then, they were incubated on ice for 5 min and later centrifuged to remove debris at 21,000 × g for 10 min at 4°C. Samples were immediately quantified using the Qubit Protein Assay reagent (Invitrogen, Q33212). The proteins were subjected to precipitation using 5:1 (v:v) cold acetone 100% and incubated overnight at −20°C. Then they were centrifuged at 15,000 × g for 10 min, the supernatant was discarded, and the pellet was washed 3 times with acetone at 90%, dried in a rotary concentrator at 4°C, and finally was resuspended in 8 M urea with 25 mM of ammonium bicarbonate pH 8. The proteins were quantified with Qubit protein assay, where 100 µg were reduced with 20 mM DTT for 1 h, alkylated with 20 mM iodoacetamide in the dark for 1 h, diluted ten times with 25 mM of ammonium bicarbonate pH 8, and digested with trypsin/LyC (Promega, V5071) in a 1:50 ratio overnight at 37°C. Peptides were cleaned using Pierce C-18 Spin Columns (Thermo Scientific 89,870) using the protocol suggested by the manufacturer. The eluted peptides were dried using a rotary concentrator at 4°C and resuspended in 2% ACN with 0.1% v:v formic acid (MERCK 100,264) and quantified using DirectDetect (MERCK Millipore, C-134681).

Mass spectrometry (nLC-MS/MS)

A nanoElute liquid chromatography system was used (Bruker Daltonics). 200 ng of tryptic peptides were separated within 90 min at a 400 nL/min flow rate on a reversed-phase column Aurora Series CSI (25 cm x 75 µm i.d. C18 1.6 µm) (IonOpticks, Australia) with 50°C. Mobile phases A and B were water and acetonitrile with 0.1 vol% formic acid, respectively. The B percentage was linearly increased from 2 to 17% within 57 min, followed by an increase to 25% B within 21 min and further to 35% within 13 min, followed by a washing step at 85% B and re-equilibration. All samples were analyzed on a hybrid trapped ion mobility spectrometry (Tims) quadrupole time-of-flight mass spectrometer (MS) (timsTOF Pro, Bruker Daltonics) via a CaptiveSpray nano-electrospray ion source. The MS was operated in a data-dependent mode for the ion mobility-enhanced spectral library generation. Set the accumulation and ramp time at 100 ms each and recorded mass spectra from m/z 100–1,700 in positive electrospray mode. The ion mobility was scanned from 0.6 to 1.6 Vs/cm². The overall acquisition cycle of 1.16 s comprised one full TIMS-MS scan and 10 parallel accumulation-serial fragmentation/PASEF MS/MS scans. Tandem mass spectra were extracted by Tims Control version 2.0 software. All MS/MS samples were analyzed using PEAKS Studio X+ (Bioinformatics Solutions, Waterloo, ON, Canada; version 10.5 [2019- [11–20]). PEAKS Studio X+ was set up to search in the Mus musculus Uniprot/SwissProt database (55310 proteins), assuming trypsin as a digestion enzyme. PEAKS was searched with a fragment ion mass tolerance of 0.05 Da. and a parent ion tolerance of 50 PPM. Carbamidomethyl of cysteine was specified as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine, and N-terminal acetylation were specified as variable modifications. FDR estimation was included using a decoy database. FDR < 0.01 and 1 minimal unique peptide per protein were used for identification.

Bioinformatic analyses

The quantification output reports from PEAKS Studio X+ were exported and processed in the R statistical environment [52]. The intensity values for each run are normalized by adjusting the medians. Missing values are imputed for each condition using the missforest algorithm [53]. Significant differential expression of proteins was determined through a Bayes-based t-test [54]. Any associated protein with a p-value <0.05 is considered significant. The exploratory analysis like dimensional reduction and visualization of data were created using R v.3.6.0 with EnhancedVolcano [55], ComplexHeatmap v.2.0.0 [56], Rtsne [57] and base packages. Functional enrichment analysis was performed using the enrichR webserver [58–60]. Differentially abundant proteins were clusters according to Gene Ontology (Molecular Function 2021 and Biological Process 2021), Reactome 2022, and MSigDB Hallmark 2020 databases. A functional category was considered enriched if presented with an adjusted p-value smaller than 0.05.

Statistical analysis

Statistical analysis was performed with GraphPad Prism V8 software (GraphPad, La Jolla, CA, USA). Statistical significance between experimental groups was determined using two-way ANOVA or Student’s test. Significance between samples is indicated in the figures as follows: n.s.=P > 0.05; *=P < 0.05; **=P < 0.01; ***=P < 0.001).

Funding Statement

The work was supported by the Agencia Nacional de Investigación y Desarrollo (ANID): FONDECYT [11180546 and 1230823 to DRR]; FONDECYT [11240328 to SB]; FONDECYT [1150743, 1200459, and 1240176 to UW]; Fonds Wetenschappelijk Onderzoek [Methusalem BOF09/01M00709 and BOF16/MET_V/007 to MB]; Fonds Wetenschappelijk Onderzoek [G017212N, G013715N, G078713N to MB].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data is available upon request to authors.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2367923.