HERC2 deficiency activates C-RAF/MKK3/p38 signalling pathway altering the cellular response to oxidative stress

Vol.:(0123456789) 1 3 Cellular and Molecular Life Sciences (2022) 79:548 https://doi.org/10.1007/s00018-022-04586-7 ORIGINAL ARTICLE HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response tooxidative stress JoanSala‑Gaston1 · LeonardoPedrazza1· JuanmaRamirez2· ArturoMartinez‑Martinez1· LettieE.Rawlins3,4· EmmaL.Baple3,4· AndrewH.Crosby3· UgoMayor2,5· FrancescVentura1· JoseLuisRosa1 Received: 27 April 2022 / Revised: 3 October 2022 / Accepted: 3 October 2022 / Published online: 14 October 2022 © The Author(s) 2022 Abstract HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases. Keywords Neurodevelopmental disorder· Angelman· Ubiquitin· MAPK· Cell stress Introduction Hereditary neurodevelopmental disorders arise from alterations in central nervous system development and manifest perinatally or during infancy and childhood. Despite showing wide genetic and clinical heterogeneity, most share some common phenotypic features, such as developmental delay, impaired motor function and intellectual disability. The identification of genes responsible for these disorders has enabled genetic diagnosis, accurate genetic counselling, and better management [1]. The HECT and RCC1-like domain 2 (HERC2) gene encodes an unusually large protein with 4834 amino acid residues. The HERC2 protein is an E3 ubiquitin ligase that functions in ubiquitylation by accepting ubiquitin from ubiquitin-conjugating enzymes (E2) and transferring it to a target protein [2]. Ubiquitylation affects proteins in many ways, variously marking them for proteasome degradation Cellular andMolecular Life Sciences * Jose Luis Rosa [email protected] 1 Department ofPhysiological Sciences, Bellvitge Biomedical Research Institute (IDIBELL), University ofBarcelona (UB), C/ Feixa Llarga s/n, 08907L’HospitaletdeLlobregat, Spain 2 Department ofBiochemistry andMolecular Biology, Faculty ofScience andTechnology, UPV/EHU, Leioa, Bizkaia, Spain 3 RILD Wellcome Wolfson Medical Research Centre, RD&E (Wonford) NHS Foundation Trust, University ofExeter Medical School, Exeter, UK 4 Peninsula Clinical Genetics Service, Royal Devon & Exeter Hospital (Heavitree), Exeter, UK 5 Ikerbasque, Basque Foundation forScience, 48013Bilbao, Spain J.Sala-Gaston et al. 1 3 548 Page 2 of 20 or, affecting their activity, localisation or interactions with other proteins. Therefore, ubiquitin ligases are key regulators of many cellular processes, with their dysregulation being common in numerous cancers and neurodegenerative diseases [3]. For example, HERC2 mutations are associated with breast, skin (melanoma), gastric, colorectal, and haematological (leukaemia) cancers [4]. The underlying molecular mechanism could be that HERC2 regulates BRCA1, XPA, USP20 or RPA2 protein ubiquitylation, involved in regulating DNA repair and genomic stability [5–9]. HERC2 also regulates p53 transcriptional program by promoting p53 tetramerisation and subsequent activation, independent of its ubiquitin ligase activity [10–12]. Besides, HERC2 is essential during embryonic development and plays an important role in regulating motor coordination [13]. Moreover, it is highly expressed in the nervous system and has been linked with hereditary neurodegenerative disorders [14]. Biallelic HERC2 variants associated with HERC2 Angelman-like syndrome include missense and frameshift mutations with a premature stop codon that result in a loss of function. These cases are associated with a complete loss or markedly reduced levels of HERC2 protein [15–19]. The condition was first described in Amish/ Mennonite communities, associated with homozygosity for a HERC2 (c.1781C > T, p.Pro594Leu) founder gene variant at increased frequency in the population (autosomal recessive mental retardation type 38; OMIM # 615516) [15, 16]. Proteomic studies of peripheral blood-derived lymphoblasts from individuals with this condition suggest derangements of multiple cellular pathways probably involving disparate pathogenic mechanisms [20]. Despite these efforts, the molecular mechanisms underlying HERC2-related disorders remain elusive, impeding efforts to find potential treatments for these rare diseases. Further investigation of their molecular basis could reveal not only the underlying pathology but also potential therapeutic targets. In this study, we analysed intracellular signalling pathways in skin fibroblasts from individuals with the pathological variant HERC2 Pro594Leu (HERC2 P594L). They displayed altered mitogen-activated protein kinase (MAPK) signalling that affected the oxidative stress response, with increases in C-RAF protein levels and MAPK p38 activation. These effects were reproduced in other human and mouse cells with HERC2 protein knockdown. Furthermore, we showed that HERC2 regulates C-RAF ubiquitylation and that HERC2 deficiency triggers MKK3/p38 pathway activation in a RAF-dependent manner. In line with this, cells with the HERC2 P594L variant had increased resistance to H2O2-induced oxidative stress, dependent on the activities of RAF and p38. Finally, we discuss both the implications of these findings for neurodevelopmental disorders caused by HERC2 variants and the potential therapeutic use of RAF inhibitors. Materials andmethods Human cell sample, cell lines andculture conditions Samples of human skin fibroblasts were obtained with approved informed consent as previously described elsewhere [16]. U2OS, HEK 293T, H1299, RAW 264.7, mouse embryonic fibroblasts (MEFs) and human skin fibroblasts were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2mM l-glutamine, 100 U/ mL penicillin, and 0.1mg/mL streptomycin sulphate. Mouse primary osteoblasts were cultured in Minimum Essential Medium α with 10% FBS, 2mM l-glutamine, 1mM pyruvate, 100 U/ml penicillin, and 0.1mg/ml streptomycin with 50μg/ml ascorbic acid and 4mM β-glycerophosphate. All cells were maintained in a humidified incubator at 37°C and 5% CO2 atmosphere. Cell treatments andinduction ofcellular stress Cells were treated with one of three inhibitors, as indicated: 1µM LY3009120 (Selleckchem), 1µM Sorafenib (Santa Cruz Biotechnology) or 10µM SB203580 (Selleckchem). Different cellular stress types were induced using different stressors: oxidative stress by 500µM or 50µM hydrogen peroxide (H2O2) (Panreac), depending on the experiment; saline stress by 100mM NaCl. Plasmid andsiRNAs transfections Plasmid transfection was performed using the Lipofectamine LTX method (15338; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Myc-tagged fragments of HERC2 (F1, F2, F3, F4, F5 and F5CT) were kindly provided by Dr. Ohta [21]. Green fluorescent protein (GFP) and C-RAF fusion constructs (CR1, CR2, CR3 and full-length) were generated, sub-cloned and tested elsewhere [22]. Plasmids expressing HERC2 full-length protein pcDNA5 FRT/TO SF-HERC2 (ShB-R) (Addgene plasmid # 55613; http:// n2t. net/ addge ne: 55613; RRID:Addgene_55613) and pcDNA5 FRT/TO SF-HERC2 C4762S (ShB-R) (Addgene plasmid # 55614; http:// n2t. net/ addge ne: 55614; RRID:Addgene_55614) were a gift from David Chan [23]. His-Ubiquitin constructs were kindly provided by Dr. Erazo [24]. The plasmid expressing a biotinylatable version of ubiquitin had been previously described elsewhere [25]. For gene interference, siRNAs were transfected using the calcium phosphate method described elsewhere [10]. Custom double-stranded siRNA oligonucleotides were HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 3 of 20 548 obtained from GeneCust (Boynes, France). The forward sequences were as follows: negative control (NC) = 5′-UUC UCC GAA CGU GUC ACG UTT; HERC2 (H2.2) = GAC UGU AGC CAG AUU GAA ATT; HERC2 (H2.4) = GGA AAG CAC UGG AUU CGU UTT; HERC1 = CGG CAU GGA UGA ACA AAU UTT; MKK3 = GGA AGA AGG AUC UAC GGA UTT; C-RAF = UAG UUC AGC AGU UUG GCU ATT; A-RAF = AAC AAC AUC UUC CUA CAU GAG TT; B-RAF = AAA GAA UUG GAU CUG GAU CAU TT; p53 = GAC UCC AGU GGU AAU CUA CTT. Lentiviral particle production andtarget cell infection Lentiviral vectors were produced in HEK 293 T cells. Cells were transfected with 7μg pMD2.G, 7μg psPAX2 (VSVG) and 7μg of either empty pLKO.1-Puro or pLKO.1‐ shHERC2 by the calcium phosphate method. Media containing lentiviral particles were collected, filtered using polyvinyl difluoride filters (Millex-HV filter 0.45μm, Millipore SLHV033RB) and stored in aliquots at − 80°C. Target cells were seeded at a confluence of 50–60% in a 6-well plate before adding 300 μL of the medium containing the lentiviral vectors to each well. Fresh medium, supplemented with 5μg/mL polybrene, was added to make a total volume of 1mL. Media with lentiviral vectors were removed the next day and after 24h, 5μg/mL puromycin was added for selection. MISSION shRNA clone of mouse HERC2 (TRCN0000039444) was purchased from SigmaAldrich. The plasmid vector pLKO.1—TRC control was a gift from David Root (Addgene plasmid #10879; http:// n2t. net/ addge ne: 10879; RRID:Addgene_10879) [26], and the VSV-G envelope expressing plasmid pMD2.G (Addgene plasmid #12259; http:// n2t. net/ addge ne: 12259; RRID:Addgene_12259) and the lentivirus packaging plasmid psPAX2 (Addgene plasmid #12260; http:// n2t. net/ addg e ne: 12260; RRID:Addgene_12260) were a gift from Didier Trono. Protein extraction, PAGE, andimmunoblotting For protein extraction, cells were washed twice in ice-cold phosphate-buffer saline after discarding the media. Cell lysis was performed by scrapping after adding of NP40 lysis buffer (50mM Tris–HCl, pH 7.5, 150mM NaCl, 50mM NaF, 0.5% NP40) containing protease and phosphatase inhibitors as previously described [27]. Lysates were maintained on ice under agitation for 20min, and then centrifuged at 13,000×g at 4°C for 10min. Supernatants were collected before analysis using the Tris–Acetate PAGE system [28]. Band intensities were detected using a gel documentation system (LAS-3000, Fujifilm) and quantified with ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej. nih. gov/ ij/). We used the following antibodies: anti-HERC2 monoclonal (BD Biosciences #612366); anti-C-RAF (BD Biosciences #610151); anti-Clathrin Heavy Chain (TD.1) (Santa Cruz Biotechnology #sc12734); anti-P-ERK1/2 (Sigma-Aldrich #M 8159); anti-p44/42 MAPK (ERK1/2) (Cell signalling #9102); anti-phospho-p38 (Cell signalling #9211); anti-p38 (Santa Cruz Biotechnology #sc-535); antiHERC1 (410) [10]; anti-P-MKK3 (Cell signalling #9231); anti-MKK3 (Proteintech #13898–1-AP); anti-A-RAF (A-5) (Santa Cruz #sc-166771); anti-B-RAF (F-7) (Santa Cruz Biotechnology #sc-5284); anti-HERC2 polyclonal (bvg3) [10]; anti-c-myc (clone 9E10) (Roche #1 667 149); antiGFP (Abcam #ab13970); anti-Flag M2 (Sigma-Aldrich #F 3165); anti-p-HSP27 (Enzo Life Sciences #ADI-SPA-523); anti-HSP27 (Santa Cruz Biotechnology #sc-1049); antiNRF2 (Cell signalling #12721); anti-ubiquitylated proteins (clone FK2; Biomol); and peroxidase-conjugated secondary antibodies (Invitrogen). Confocal microscopy We seeded U2OS cells on glass coverslips and performed fixation by incubating cells at room temperature for 20min in 4% paraformaldehyde. Then, cells were permeabilised for 20min with 0.05% saponin in phosphate-buffered saline containing 0.5% bovine serum albumin. The primary antibody, anti-phospho-p38 (Cell signalling #9211) (1:200), was incubated at 37°C for 1h. After washing, Alexa-Fluor 488 secondary antibody (Invitrogen) (1:500) was incubated at 37°C for 45min. Actin filaments were stained by incubation with phalloidin-Alexa 647 (BioProbes) (100ng/mL) for 20min at room temperature. Nuclei were stained with DAPI (Sigma-Aldrich) (1μg/mL). All images were acquired using a confocal laser scanning microscope (LSM 880 spectral, Carl Zeiss Microscopy GmbH, Jena, Germany). Immunoprecipitation andpull‑downs For immunoprecipitation, cells were lysed with CHAPS buffer (10mM Tris–HCl, pH 7.5, 100mM NaCl, 0.3% CHAPS) containing protease and phosphatase inhibitors as described above. Cell lysates (input) were incubated with pre-immune serum or anti-HERC2 polyclonal antibody (bvg3) for 2h at 4°C with gentle rotation and immunoprecipitated with protein A-Sepharose (GE Healthcare) for 1h at 4°C. Beads were pelleted by centrifugation at 2500×g, washed five times with CHAPS buffer, and analysed by electrophoresis and immunoblot. J.Sala-Gaston et al. 1 3 548 Page 4 of 20 For the GFP pull-downs, supernatants were incubated with 2 μL GFP-TrapA (ChromoTek) for 2h at 4°C. Pellets were washed five times with CHAPS buffer and analysed by electrophoresis and immunoblot. For ubiquitome proteomic experiments, biotin-pulldowns were performed in triplicates as previously described [25], in order to compare proteins more ubiquitinated in Flag-HERC2 WT-overexpressing cells, relative to FlagHERC2 C4762S-overexpressing cells. Ubiquitylation assay HEK 293T cells were transfected with the indicated plasmids for 48h. Before the ubiquitylation assay, the cells were treated for 4h with 10µM of the proteasome inhibitor MG132 (Sigma-Aldrich/Merck #C2211). Then, cells were lysed with denaturing buffer #1 (6M guanidinium-HCl, 10mM Tris, 100mM Na2HPO4–NaH2PO4 buffer, pH 8) and the cells extracts were incubated with the nickel beads (Ni2+-NTA agarose; Qiagen) for 2h at 4°C under rotation. Beads were successively washed as follows: twice with 1ml of denaturing buffer #1 plus 10mM 2-mercaptoethanol; three times with 1ml of buffer #2 (8M urea, 10mM Tris, 10mM 2-mercaptoethanol, 100mM Na2HPO4–NaH2PO4 buffer, pH 8); twice with 1ml of buffer #3 (8M urea, 10mM Tris, 100mM Na2HPO4–NaH2PO4 buffer, pH 6.3) containing 0.2% Triton X-100; once with 1ml of buffer #3 containing 0.1% Triton X-100 and 0.5M NaCl; and three times with 1ml of buffer #3. Finally, proteins were eluted by incubating the beads with 200mM imidazole in 5% SDS, 0.15M Tris–HCl, pH 6.7, 30% (vol/vol) glycerol, 0.72M 2-mercaptoethanol for 1h at 37°C with mixing. The samples were analysed by immunoblotting as indicated above. Reverse transcription andquantitative PCR Total RNA was isolated from U2OS cells using the TRIsure reagent according to the manufacturer’s protocol (Bioline). Total RNA (2μg) was reverse-transcribed using the highcapacity cDNA Reverse Transcription kit (Applied Biosystems). PCR amplification reactions were performed with the ABI Prism 7900 HT Fast Real-Time PCR System. Applied Biosystems’ TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify the gene expressions of the following: GUSB (Hs00939627_m1), NFE2L2 (Hs00975960_m1), SOD1 (Hs00533490_m1), SOD2 (Hs00167309_m1), GPX1 (Hs00829989_Gh), and the housekeeping gene GAPDH (Hs99999905_m1), which was used to normalise. MTT assay forcell viability andcell proliferation Using 96-well plates, U2OS cells and human skin fibroblasts were seeded to final concentration of 10,000 cells/well or 15,000 cells/well, respectively. After incubation at 37°C for 24h in the cell incubator, we initiated treatments, as indicated and performed the MTT assay (M5655; Sigma/ Merck) according to manufacturer’s instructions. Briefly, we added MTT at a final concentration of 0.5mg/mL to each well, incubated the cells for 4h in a humidified incubator, then discarded the media and solubilised the formazan crystals with isopropanol. Finally, absorbance at a wavelength of 570nm was determined using a 96-well plate spectrophotometer. MitoSox staining To evaluate mitochondrial reactive oxygen species (ROS), human skin fibroblasts were seeded in a µ-Slide 8 wellchambered coverslip at a concentration of 15,000 cells/well. The next day, cells were stained with 1µg/mL of Hoechst 33,342 (H3570, ThermoFisher, USA) for 30min at 37°C and with 2µM MitoSOX Red (Invitrogen) for 15min at 37°C. Cells were examined in a Zeiss LSM 880 laser scanning confocal spectral microscope equipped with an incubation control system (37°C, 5% CO2). Fluorescence intensity per cell was measured, quantified and expressed as arbitrary units (a.u). Images were analysed using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej. nih. gov/ ij/). Mitotracker staining For mitochondria staining, human skin fibroblasts were seeded in a µ-Slide 8 well-chambered coverslip at a concentration of 15,000 cells/well. The next day, cells were stained with 1µg/mL of Hoechst 33,342 (H3570, ThermoFisher, USA) and 50nM Mitotracker Red CMXRos (M7512, ThermoFisher, USA) for 30min at 37°C. Images were taken using a Zeiss LSM 880 laser scanning confocal spectral microscope equipped with an incubation control system (37°C, 5% CO2). Fragmented mitochondrial percentage was calculated by counting spherical non-contiguous mitochondrial particles and dividing by the number of total structures comprised in the mitochondrial network. Images were analysed using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej. nih. gov/ ij/). HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 5 of 20 548 Statistical analysis The results indicate the means and standard error of the mean (± SEM) of, at least, three independent experiments. Individual data points are plotted as single dots. Significance was calculated by Student t-test and indicated as follows: *, **, or *** for p values of ≤ 0.05, ≤ 0.01, or ≤ 0.001, respectively. Figures were created, and statistical analysis was performed, using GraphPad Prism version 8.4.3 for Windows (GraphPad Software, San Diego, California USA), www. graph pad. com. Results Human HERC2 Pro594Leu cells display MAPK pathway alteration Several recessive mutations affecting the HERC2 gene cause developmental delay with Angelman-like features [14, 19]. Knowing how pathologic HERC2 variants affect intracellular signalling could reveal the underlying pathology and identify possible therapies. Therefore, we studied cells from an individual with the mutant HERC2 P594L variant described in most cases. Since HERC1 had previously been reported to regulate the ERK and p38 MAPK signalling pathways [22, 29], we wondered if HERC2 also had a modulatory role. As expected, cells with the HERC2 P594L mutation showed almost undetectable HERC2 protein levels (Fig.1A–C). Interestingly, although they showed higher protein levels of C-RAF (Fig.1A), this did not correlate with the canonical activation of the ERK signalling pathway, assessed by ERK phosphorylation (Fig.1B). An increment in p38 phosphorylation was also detected while total p38 protein levels remained stable (Fig.1C). In order to provide more evidence that these changes in MAPK signalling pathways are a general hallmark of disease in patients with biallelic HERC2 mutations, we analysed samples of two more individuals carrying the mutant HERC2 P594L variant. Consistently, patients with the HERC2 P594L mutation (P1, P2 and P3) showed lower HERC2 protein levels than the wild-type controls (C1, C2 and C3). In addition, C-RAF protein levels and p38 phosphorylation were upregulated in all three patients, but no changes were detected in ERK activation (Fig.1D). These results showed that cells with the HERC2 P594L mutation exhibit altered MAPK signalling pathway activation, as reflected by higher C-RAF and phospho-p38 protein levels. HERC2 regulates C‑RAF protein levels To delve deeper into the molecular mechanisms involved in the altered MAPK signalling pathway in HERC2 P594L cells, we considered human cells with low levels of HERC2 protein shared this alteration. In knockdown experiments performed in human U2OS cells, cells were transfected with either a negative control (NC) small-interfering RNA (siRNA), an siRNA against HERC2, or a positive control siRNA against HERC1. The positive control was chosen because previous work had shown that HERC1 controls ERK and p38 signalling pathways modulating C-RAF protein levels [22, 29]. HERC2 knockdown mimicked the effect observed in HERC2 P594L cells, with depletion of HERC2 correlating with increased C-RAF protein levels. As expected, this was also observed after HERC1 silencing (Fig.2A). HERC2 depletion modified neither A-RAF nor B-RAF protein levels (Fig.2B, C). These data indicated that RAF regulation by HERC2 is specific for the C-RAF isoform. Next, we analysed the RAF MAPK signalling pathway, in which canonical RAF activation triggers ERK phosphorylation [29]. We noted that C-RAF upregulation observed after HERC1 depletion correlated with increased phosphorylated ERK levels, while total ERK protein levels remained stable. However, we detected no changes in ERK phosphorylation in the HERC2-depleted cells (Fig.2D). These results suggested that C-RAF upregulation caused by HERC2 depletion was not signalled through the canonical MEK/ERK pathway. HERC2 regulates p38 phosphorylation Given that HERC1 regulates the MKK3/p38 axis through a RAF-dependent mechanism [29], we decided to study if this mechanism was the same for HERC2. We analysed levels of p38 phosphorylation in U2OS cells transfected with a negative control siRNA, an siRNA against HERC2, and a positive control siRNA against HERC1. We observed the induction of p38 phosphorylation in HERC2-depleted cells, though with total p38 protein levels remaining stable and higher C-RAF protein levels(Fig.3A). Analogous behaviour was detected in HERC1-depleted cells(Fig.3A). The same results for p38 phosphorylation were obtained when silencing HERC2 with siRNAs containing different RNA sequences (HERC2 H2.2 and HERC2 H2.4) (Fig.3B). The phosphorylation of p38 is associated with its activation and nuclear translocation. To check this, we analysed p38 subcellular localisation. Immunofluorescence J.Sala-Gaston et al. 1 3 548 Page 6 of 20 experiments showed increased p38 nuclear localisation in HERC2-depleted cells (Fig.3C). This was quantified assessing the nucleus:cytoplasm ratio, which was higher in HERC2-depleted cells compared with control cells (Fig.3D). After HERC2 silencing, p38 activation, was replicated in other human cells, such as the p53-lacking human non-small lung carcinoma cell line (H1299) and the non-tumorigenic human kidney 293T cell line (HEK 293T) (Fig.3E). In addition, the same results were obtained in mouse cells and Fig. 1 Patient-derived cells with a homozygous mutation in human HERC2 gene show MAPK pathway alterations. A–C We analysed lysates of human skin fibroblasts from an individual with the wild-type HERC2 (HERC2 WT) and the p.Pro594Leu mutant HERC2 variant (HERC2 P594L) by immunoblot, using the indicated antibodies. C-RAF (A), phospho-ERK (P-ERK) (B) or phospho-p38 (P-p38) (C) levels were quantified and normalised based on clathrin heavy chain (CHC), ERK or p38 protein levels, respectively. The results are expressed relative to the control condition. Plots represent mean ± standard error of the mean. Representative results are shown for experiments repeated at least three times and the data points of each experimental repetition are plotted as single dots. (D) We analysed lysates of human skin fibroblasts from three different control individuals with the wild-type HERC2 (C1, C2 and C3) and three different patients with the HERC2 P594L mutant variant (P1, P2 and P3) by immunoblot. Levels of HERC2 and C-RAF proteins were quantified as in A. Levels of P-ERK and P-p38 were quantified as in B, C, respectively. The results are expressed relative to the control condition. Plots represent mean ± standard error of the mean. Representative results are shown for experiments repeated at least three times and the data points of each of the individuals analysed are plotted as single dots. Significance levels: ns = non-significance; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 7 of 20 548 when using a different HERC2 silencing method. RAW 264.7 macrophage cell line, primary mouse osteoblasts and MEFs were infected with lentiviral particles carrying either an empty vector as a control (plKO) or a short hairpin RNA (shRNA) against HERC2. All HERC2 knockdown cells presented higher phospho-p38 protein levels compared to controls, while total p38 protein levels remained constant (Fig.3F). In conjunction, these results demonstrated that HERC2 participates in regulating p38 signalling. HERC2 regulates theMKK3/p38 pathway throughcrosstalk mediated byC‑RAF MAPK kinase (MAPKK or MKK) mediates p38 activation through phosphorylation. MKK3 is the dominant isoform Fig. 2 HERC2 regulates C-RAF protein levels. A U2OS cells were transfected with an siRNA negative control (NC), an siRNA against HERC2, or an siRNA against HERC1. The indicated protein levels were analysed by immunoblot. Levels of C-RAF proteins were quantified, normalised based on clathrin heavy chain (CHC) protein levels (loading control), and expressed relative to the control condition. B, C U2OS cells were transfected with a NC siRNA or an siRNA against HERC2. Levels of A-RAF (B) or B-RAF (C) were analysed by immunoblot, quantified, normalised based on CHC protein levels, and expressed relative to the control condition. (D) U2OS cells were transfected as in A, and the indicated protein levels were analysed by immunoblot. Phospho-ERK (P-ERK) levels were quantified, normalised based on ERK protein levels and expressed relative to the control condition. Plots represent the mean ± standard error of the mean. Representative results are shown from experiments repeated at least three times and the individual data points are plotted as single dots. Significance levels: ns = non-significance; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 J.Sala-Gaston et al. 1 3 548 Page 8 of 20 in human U2OS cell lines [29], and its activation has been analysed by measuring its phosphorylation at Ser189 [30]. Thus, we analysed MKK3 activation and its total protein expression in HERC2-depleted U2OS cells, revealing that neither MKK3 phosphorylation at Ser189 nor total MKK3 protein levels were altered compared with control cells (Fig.4A). To confirm whether p38 phosphorylation triggered by HERC2 depletion depends on MKK3, we cotransfected U2OS with an MKK3 siRNA and either the negative control or the HERC2 siRNA. This revealed that MKK3 knockdown significantly abolished the increment in p38 phosphorylation after HERC2 depletion (Fig.4B). HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 9 of 20 548 These data suggested that MKK3 activation caused the increase in phospho-p38 independent of phosphorylation at Ser189. Given the finding that HERC2 regulates C-RAF and p38 activation, we used two specific RAF kinase inhibitors to identify a potential crosstalk mechanism between the two pathways. LY300912 was used to inhibit all RAF isoforms, and Sorafenib was used to inhibit only B-RAF and C-RAF. In absence of the inhibitors, cells showed an increase in p38 phosphorylation after HERC2 depletion; remarkably, however, this increase was clearly abrogated after incubation with LY3009120 or Sorafenib inhibitors for 1h (Fig.4C). Since RAF isoforms interact by forming different heterodimers [31], sometimes all isoforms must be depleted to rescue the regulatory effects mediated by RAF proteins. Therefore, we co-transfected U2OS cells with siRNAs against C-RAF or all three RAF isoforms (A-RAF, B-RAF and C-RAF) along with either the negative control siRNA or the siRNA against HERC2 to achieve knockdown (Fig.4D). Although silencing C-RAF alone was insufficient to reduce p38 phosphorylation significantly, silencing all three isoforms led to a significant decrease in p38 activation in HERC2-depleted cells (Fig.4D). Unlike pharmacological inhibition of RAF, triple knockdown failed to produce a complete abrogation of p38 phosphorylation after HERC2 depletion, which is probably due to the fact that siRNA silencing did not achieve sufficient RAF isoforms knockdown. Altogether these results confirm the existence of a crosstalk between the RAF and p38 signalling pathways regulated by HERC2. HERC2 interacts withC‑RAF To further investigate the mechanism behind C-RAF regulation by HERC2, we analysed whether these two proteins can interact. In immunoprecipitation experiments in U2OS cells with a specific anti-HERC2 antibody (bvg3), endogenous HERC2 and C-RAF immunoprecipitated, while HERC1 did not, indicating that the interaction of HERC2 and C-RAF was independent of HERC1 (Fig.5A). RAF hetero-dimerisation between its isoforms is a well-reported process [31], and consistent with this, A-RAF and B-RAF were also detected in HERC2 immunoprecipitated complexes (Fig.5B, C). The same results were obtained in the human 293T cell line (Fig.5D–F). To identify the region of HERC2 interacting with C-RAF, we co-expressed a GFP-C-RAF fusion protein with a series of Myc-HERC2 fusion proteins in HEK 293T cells (Supplementary Fig.1A), followed by pull-down assays with GFP-binding beads. Constructs F4, F5, and F5CT coimmunoprecipitated with GFP-C-RAF, indicating that the HERC2 and C-RAF protein interaction occurs mainly in the carboxyl-terminus of HERC2 polypeptide chain. F5CT construct, which contains the HECT domain holding the ubiquitin ligase activity, showed the highest affinity with C-RAF, suggesting that this is the most relevant interaction site (Supplementary Fig.1A). HEK 293T cells were then co-transfected with a Flag-HERC2 full-length fusion protein along with GFP (as a negative control) or the GFPC-RAF fusion constructs (CR1, CR2, CR3 or full-length) to map the C-RAF region involved. In the GFP pull-down, Flag-HERC2 was coimmunoprecipitated with CR1, CR3, and the full-length constructs (Supplementary Fig.1B). To characterise this interaction further, we co-expressed the F4 Myc-HERC2 construct with GFP-C-RAF fusion constructs and performed a GFP pull-down, which showed preferential co-immunoprecipitation of the F4 construct with CR3 (Supplementary Fig.1C). In parallel, the same experiment was done but with the F5CT Myc-HERC2 construct instead of F4, and this revealed co-immunoprecipitation of F5CT with CR1 and CR3 (Supplementary Fig.1D). In conjunction, pull-down experiments confirmed the interaction between HERC2 and C-RAF, and indicated the possible domains involved. The HERC2 HECT domain, contained in the F5CT construct, showed the highest affinity for C-RAF and its catalytic domain (CR3), suggesting that the HECT and CR3 domains could be the most relevant at the physiological level. Subsequent structural studies should confirm this relevance. HERC2 regulates C‑RAF ubiquitylation Having shown that the ubiquitin E3 ligase HERC2 interacts with C-RAF and regulates its protein levels, we wanted Fig. 3 HERC2 regulates p38 phosphorylation. A U2OS cells were transfected with an siRNA negative control (NC), an siRNA against HERC2, or an siRNA against HERC1. The indicated protein levels were analysed by immunoblot. Levels of phospho-p38 (P-p38) were quantified, normalised based on total p38 protein levels, and expressed relative to the control condition. B U2OS cells were transfected with an siRNA negative control (NC) and two different siRNA sequences against HERC2: H2.2 or H2.4. The indicated protein levels were analysed by immunoblot and phospho-p38 levels were quantified and represented as in (A). C U2OS cells transfected with NC or HERC2 siRNA were analysed by immunoblot against the indicated proteins and by confocal microscopy. Fixed cells were stained for phospho-p38 (green), F-actin with phalloidin (red), and nuclei with DAPI (blue) and analysed by immunofluorescence. D Fluorescence intensity in the nucleus and cytoplasm per cell was measured and quantified. The ratio nucleus/cytoplasm was calculated. Each data point represent mean of a different field. E HEK 293T and H1299 cells were transfected with a NC or HERC2 siRNA. The indicated protein levels were analysed by immunoblot. F A RAW 264.7 mouse macrophage cell line, mouse primary osteoblasts and mouse embryonic fibroblasts (MEFs) were infected with lentiviral particles carrying either the empty plKO vector as a negative control (plKO) or an shRNA against HERC2. The indicated protein levels were analysed by immunoblot. Plots represent mean ± standard error of the mean. Representative results are shown from experiments repeated at least three times and the individual data points are plotted as single dots. Significance levels: ns = non-significance; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 ◂ J.Sala-Gaston et al. 1 3 548 Page 16 of 20 HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 17 of 20 548 HERC2-depleted cells, confirmed that p38 acts upstream of NRF2 activation. Still, given the variety of p38 substrates we cannot discard that other transcription factors, apart from NRF2, could also be involved in the regulation of the studied antioxidant genes. The transcription factor ATF-2 is another important mediator of p38 in the induction of SOD2 expression upon H2O2-induced oxidative stress in MEFs [45]. This possible cooperation between NRF2 and ATF-2, or some other transcription factor targeted by p38, should be studied further. Overall, our findings may have both physiological and clinical repercussions. Physiologically, we revealed a pro-survival function of p38 that is regulated by HERC2. HERC2 potentially fine-tunes the cellular response to oxidative stress by controlling protein levels of C-RAF and, therefore, C-RAF/MKK3/p38 signalling to regulate antioxidant gene expression. Clinically, these findings may be relevant to cancer, as well as individuals with HERC2 Angelman-like syndrome due to biallelic HERC2 gene variants [16, 18]. Several HERC2 mutations have been associated with a wide number of tumours [4]. In renal cancer, higher HERC2 gene expression correlates with better patient prognosis [46], supporting the hypothesis that HERC2 may act as a tumour suppressor [4, 46]. We previously demonstrated that HERC2, and NEURL4, regulate the transcriptional activity of the tumour suppressor p53, facilitating its oligomerisation. HERC2 knockdown accordingly increases cell proliferation due to the impaired capability to arrest the cell cycle through p53 [10–12]. Equally, although the precise mechanism remains elusive, it is well established that the production of reactive oxygen species in tumour cells increases due to the higher metabolic rate, with the resulting excess being countered by an increased antioxidant cellular response [47]. Supporting this, in mice, oncogenic alleles of Kras, Braf and Myc, associated with increased Nfe2l2 expression. This appear to stably enhance NRF2 antioxidant program and lower intracellular reactive oxygen species [48]. Furthermore, in p53 mutated cancer cells, the NRF2dependent antioxidant response was selectively modulated to enhance cancer cell survival [49]. Our data reveal a new mechanism by which HERC2 deficiency may contribute to tumour malignancy by impairing p53 transcriptional activity, and also by boosting the cellular antioxidant response making cancer cells more resistant to oxidative stress (Fig.8). In this context, combination treatments with drugs causing non-genotoxic activation of p53 oligomerisation and FDA-approved RAF inhibitors, such as Sorafenib, represent potential therapeutic candidates for tumours associated with HERC2 deficiency. Finally, a previous proteomic analysis of human HERC2 mutants (including the p.Pro594Leu variant studied here) has already identified an enrichment of the NRF2-mediated oxidative stress response in HERC2 mutants compared to control group [20]. In addition, protein–protein interaction networks containing signal transduction proteins and MAPKs were found to be differentially expressed in HERC2 mutants [20]. Our results add to these observations and, importantly, provide a possible molecular mechanism explanation. It would be interesting for further research to study the implication of a chronic activation of the C-RAF/MKK3/ p38 signalling pathway in neuronal cells with HERC2 deficiency. Alterations in p38 MAPK signalling in neurons have been linked to neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) [50]. Therefore, we cannot discount the possibility that alterations in this pathway could be associated with clinical outcomes in HERC2 Angelman-like syndrome. Consistent with this, previous studies have shown that SOD1 overexpression, in which gene variants are associated with ALS and whose mRNA levels we found to be increased following HERC2 depletion, is associated with defects in the cerebellar architecture [51, 52]. In addition, while excessive ROS elicit oxidative stress, their persistent depletion, as observed in HERC2-deficient cells (Supplementary Fig.4A), leads to an opposite condition called reductive stress. Persistent activation of antioxidant signalling can cause reductive stress and lead to pathology. In HERC2 P594L cells, the overactivation of NRF2 signalling could be one of the causes. In fact, NRF2 sustained activation has Fig. 7 HERC2 deficiency alters cellular resistance to H2O2-induced oxidative stress. A Human skin fibroblasts derived from an HERC2 wild-type individual (HERC2 WT) and an individual with the p.Pro594Leu HERC2 mutant variant (HERC2 P594L) were treated with 500µM H2O2 to induce oxidative stress for the indicated time points and protein levels were analysed by immunoblot. Phosphop38 (P-p38) levels were quantified, normalised based on total p38 protein levels and expressed relative to the non-treated control condition (HERC2 WT, t = 0). Plots represent mean ± standard error of the mean of 4 independent experiments (n = 4). B Human skin fibroblasts were treated as in (A) and images were acquired by optical microscopy after the indicated treatment times, with representative images shown from experiments repeated three times (n = 3). C, D HERC2 WT and HERC2 P594L human skin fibroblasts were treated with 500µM H2O2 for 6h (C) or with 50µM H2O2 for 24h (D) as indicated. Cells were treated 1µM LY3009120, 1µM Sorafenib or 10µM SB203580 in the specified conditions 1 h before adding H2O2. An MTT assay was performed. Data are presented as a percentage relative to the control and untreated condition. E U2OS were transfected with an siRNA negative control (NC) or an siRNA against HERC2. A p53 siRNA was added when indicated (siRNA p53). Subsequently, cells were plated in a 96-well plate and allowed to grow for 48h to evaluate cell proliferation and an MTT assay was performed. Data are presented relative to the control condition (NC, WT p53). F U2OS cells were transfected with the NC or HERC2 siRNA along with p53 siRNA (NC + p53/HERC2 + p53). Cell viability was assessed by MTT assay (under the same conditions mentioned in D). Plots represent mean ± standard error of the mean. Representative results are shown from experiments repeated at least three times and the individual data points are plotted as single dots. Significance levels: ns = non-significance; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 ◂ J.Sala-Gaston et al. 1 3 548 Page 18 of 20 already been linked to reductive stress [53]. Highlighting the importance of reductive stress on pathology, mutations in key components of the cellular reductive stress response can cause developmental diseases. For instance, FEM1B gainof-function mutation, which cause a persistent activation of the reductive stress response, elicit developmental syndromes with some similarities to the HERC2 Angelman-like syndrome [33, 34]. An example of the damage that reductive stress can exert on cells is that it can induce mitochondrial dysfunction and impact on the correct cell function [54, 55]. Accordingly, we observed an increased number of fragmented mitochondria in HERC2 P594L cells, which is a common feature observed in neurodegeneration [56]. However, more experiments are needed to confirm these hypotheses and to associate these mechanisms with clinical outcomes in HERC2 Angelman-like syndrome. All things considered, the findings in this study identify p38 and RAF inhibitors as potential therapeutic options for individuals who present with such rare disease. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s0001802204586-7. Acknowledgements We would like to thank Dr. Benjamín Torrejón and Dr. Esther Castaño from “Centres Cientifics i Tecnològics de la Universitat de Barcelona” (CCiT-UB), and Ms. Esther Adanero for technical assistance. We also thank Dr. Ohta and Dr. Erazo for kindly providing us with plasmids. This research was supported by the following grants: JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00/MCIN/AEI/10.13039/501100011033); and FV (PDC2021-121776-I00 and PID2020-117278GB-I00 from MCIN/ AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” “NextGenerationEU”/PRTR; and Grant 202038-30 from “La Marató de TV3”). As well, this article is based upon work from COST Action ProteoCure CA20113, supported by COST (European Cooperation in Science and Technology). J.S.-G. and A.M.-M. received FPU Fellowships (FPU17/02413 and FPU18/06325, respectively) from the Spanish Ministry of Universities. Author contributions Conceived and designed the experiments: JSG, UM, FV, and JLR. Performed the experiments: JSG, LP, JR, AMM, and JLR. All the authors analysed the data. The first draft of the manuscript was written by JSG and JLR, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript. Funding Open Access funding provided thanks to the CRUECSIC agreement with Springer Nature. This study was funded by the following grants: JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00/MCIN/AEI/10.13039/501100011033) and FV (PDC2021-121776-I00 and PID2020-117278 GB-I00 from MCIN/ Fig. 8 Working model of HERC2 function in health and disease. In previous studies, we showed that independently of the ubiquitin ligase activity, HERC2 along with NEURL4, facilitates p53 oligomerisation to promote p53 transcriptional program activation. For example, the target gene p21 regulates the cell cycle and promotes cell cycle arrest. Under conditions of HERC2 deficiency or down-regulation, the transcriptional activation of p53 is impaired due to the compromised p53 oligomerisation process [10–13]. Now, with data presented in this study, we complement this working model by adding an important function of HERC2 dependent on its ubiquitin ligase activity. Under normal conditions, HERC2 controls C-RAF protein levels by regulating its ubiquitylation and targeting it to proteasomal degradation. Hence, in HERC2-deficient cells, C-RAF protein levels increase, which activates a crosstalk between the C-RAF and MKK3/ p38 signalling pathways. Once p38 is activated by phosphorylation, it translocates to the nucleus and activates its target transcription factors (TFs). This eventually activates transcription of genes related to the oxidative stress response such as NFE2L2, SOD1, SOD2 and GPX1, which predisposes cells to an enhanced resistance to oxidative stress. The combination of these effects in the p53/p21 and MKK3/p38 pathways may affect both tumorigenesis and neuronal cell homeostasis HERC2 deficiency activates C‑RAF/MKK3/p38 signalling pathway altering thecellular response… 1 3 Page 19 of 20 548 AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” “NextGenerationEU”/PRTR. And Grant 202038-30 from “La Marató de TV3”). As well, this article is based upon work from COST Action ProteoCure CA20113, supported by COST (European Cooperation in Science and Technology). JSG and AMM received FPU Fellowships (FPU17/02413 and FPU18/06325, respectively) from the Spanish Ministry of Universities. Data availability All data analysed during this study to evaluate the conclusions are included within the article or available in supplemental information. Additional related data need to be requested from the corresponding author. Declarations Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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