Neddylation inhibition prevents acetaminophen-induced liver damage by enhancing the anabolic cardiolipin pathway

Article Neddylation inhibition prevents acetaminopheninduced liver damage by enhancing the anabolic cardiolipin pathway Graphical abstract Highlights dHepatic NEDDylation levels are increased after drug liver injury dNEDDylation inhibition (MLN4924) reduced necrosis and liver damage in mouse model dTAM41 stability increases after MLN4924 treatment recovering cardiolipin synthesis dA recovery of mitochondrial activity induces regeneration after MLN4924 treatment Authors Cla `udia Gil-Pitarch, Marina Serrano-Macia ´, Jorge Simon, ..., Naroa Goikoetxea-Usandizaga, Irene Gonza ´lez-Recio, Marı ´a L. Martı ´nez-Chantar Correspondence [email protected] (I.G.-R.), [email protected] (M.L.M.-C.) In brief Gil-Pitarch et al. report that neddylation is significantly enriched during druginduced liver injury (DILI), affecting mitochondrial dysfunction. MLN4924, a NEDD8-activating enzyme (NAE-1) inhibitor, decreases necrosis by promoting liver regeneration through the stabilization of TAM41 and mitochondrial recovery. Gil-Pitarch et al., 2024, Cell Reports Medicine 5, 101653 July 16, 2024 ª2024 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.xcrm.2024.101653 ll Article Neddylation inhibition prevents acetaminopheninduced liver damage by enhancing the anabolic cardiolipin pathway Cla `udia Gil-Pitarch, 1,21 Marina Serrano-Macia ´, 1,21 Jorge Simon, 1 Laura Mosca, 2 Carolina Conter, 1 Claudia M. Rejano-Gordillo, 1,3 L. Estefanı ´a Zapata-Pavas, 1 Patricia Pen ˜a-Sanfe ´lix, 1 Mikel Azkargorta, 4 Rube ´n Rodrı ´guez-Agudo, 1 Sofı ´a Lachiondo-Ortega, 1 Maria Mercado-Go ´mez, 1 Teresa C. Delgado, 1 Marina Porcelli, 2 Igor Aurrekoetxea, 5,6 James D. Sutherland, 7 Rosa Barrio, 7 Dimitris Xirodimas, 8 Patricia Aspichueta, 5,6,16 Felix Elortza, 4,16 (Author list continued on next page) SUMMARY Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, wholebody biotinylated NEDD8 ( bio NEDD8) and ubiquitin ( bio UB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment. INTRODUCTION In the Western world, drug-induced liver injury (DILI) is the leading cause of acute liver failure (ALF) and liver transplantation. It has become a major public health problem, affecting 19 out of every 100,000 people worldwide. 1 Importantly, paracetamol, also known as acetaminophen or APAP, is the leading cause of ALF in the United States (46%) and accounts for 40% to 70% of cases in the United Kingdom and Europe. 2 Every year, APAP is responsible for 500 DILI deaths, 100,000 calls to the 1 Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Derio 48160 Bizkaia, Spain 2 Department of Life Sciences, Health and Health Professions, Link University, Via del Casale di San Pio V, 44 00165 Rome, Italy 3 Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, University Institute of Biosanitary Research of Extremadura (INUBE), 06071 Badajoz, Spain 4 Proteomics Platform, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), ProteoRed-ISCIII, CIBERehd, Science and Technology Park of Bizkaia, 48160 Derio, Spain 5 Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain 6 Biobizkaia Health Research Institute, 48903 Barakaldo, Spain 7 Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain 8 CRBM, University Montpellier, CNRS, 34293 Montpellier, France 9 Department of Physiology, School of Medicine-Instituto de Investigaciones Sanitarias, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain 10 Department of Physiology, CIMUS, 15782 University of Santiago de Compostela, Santiago de Compostela, Spain 11 CIBER Fisiopatologia de la Obesidad y Nutricio ´n (CIBERobn), Santiago de Compostela, Spain 12 Galician Agency of Innovation (GAIN), Xunta de Galicia, Santiago de Compostela, Spain 13 Gastroenterology and Hepatology Department, Marque ´s de Valdecilla University Hospital, Clinical and Translational Digestive Research Group, IDIVAL, 39011 Santander, Spain 14 The Liver Unit, Newcastle-upon-Tyne Hospitals NHS Foundation Trust, NE7 7DN Newcastle upon Tyne, UK (Affiliations continued on next page) Cell Reports Medicine 5, 101653, July 16, 2024 ª2024 The Authors. Published by Elsevier Inc. 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS US Poison Center, 50,000 emergency room visits, and 10,000 hospitalizations in the United States. 3 Nowadays, N-acetylcysteine, a scavenger of reactive oxygen species (ROS), is the only approved pharmacological treatment for hepatotoxicity due to APAP overdose. However, due to the narrow therapeutic window and rapid disease progression, the therapeutic efficacy of N-acetylcysteine is still limited. 4 For patients at the advanced stage, liver transplantation is the only way to improve survival outcomes. Therefore, new therapeutic approaches are required in this field. 5 Previous studies have shown that APAP overdose leads to mitochondrial dysfunction via several mechanisms, including inhibition of several mitochondrial respiratory chain complexes by the reactive metabolite N-acetyl-p-benzoquinoneimine and possibly some components of mitochondrial fatty acid oxidation metabolism and mitochondrial DNA depletion. 6 High APAP concentrations had no effect on complex III, IV, or V but decreased the mitochondrial respiratory capacity of complex I and II in human liver. 5 In addition, N-acetyl-p-benzoquinone imine (NAPQI) leads to depletion of glutathione (GSH) in liver cells, which has significant effects on protein function and mitochondrial redox state. Since GSH serves as the primary intracellular antioxidant, its depletion leads to ROS production. In eukaryotes, protein neddylation is an important ubiquitinlike post-translational modification (PTM). 7 To date, neddylation has been widely used to tag conserved neuronal precursor cellexpressed developmentally downregulated protein 8 (NEDD8) onto substrates to modulate activity and cellular localization. 6 The cullin-RING E3 ubiquitin ligases (CRLs) were the first wellknown NEDDylation target. 8 This protein family has a conserved NEDDylation site and requires NEDD8 covalent attachment to induce a conformational change in the structure that activates ubiquitin ligation to substrates. 9 Several studies have found that CRLs play an active role in the regulation of morphology, trafficking, functions, and mitochondrial degradation, with the ubiquitination protein system being primarily involved in the degradation of the mitochondrial compartment’s outer membrane. 6 Indeed, selective inhibition of CRL3 provides protection from liver damage induced by APAP in mice. 10 MLN4924 (also known as pevonedistat) is a small molecular inhibitor of the catalytic subunit of the NEDD8-activating enzyme (NAE-1), blocking the entire neddylation modification and inactivating all CRLs. The outcomes after blocking neddylation in mitochondrial function appear to vary depending on cell type. Some studies point out that MLN4924 induces oxidative stress and promotes autophagy in a variety of malignant cells, 11 whereas our group and others have demonstrated that this inhibitor blocks ROS in non-alcoholic fatty liver disease (NAFLD) 12 or suppresses basal but not maximal oxidative phosphorylation in pro-tumoral hepatocytes. 13 Considering that the role of neddylation in liver pathology has raised significant expectations, 12,14,15 we investigated the impact of neddylation in response to the damage caused by APAP overdose. Neddylation was found to be overrepresented in the liver biopsies from patients with AILI (APAP-induced liver injury) and in preclinical studies that resemble this pathology. Indeed, inhibiting NEDD8 conjugation with MLN4924 in animal models under APAP overdose resulted in a halt in liver injury and an increase in hepatic regeneration. The hepatic neddylome and ubiquitome were further characterized employing biotinylated ubiquitin ( bio UB) and biotinylated NEDD8 ( bio NEDD8) transgenic mice under APAP overdose model in the presence or absence of MLN4924. The findings revealed that the cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41 was overexpressed in the AILI model when neddylation was inhibited, resulting in high levels of cardiolipin (CL), which is required for optimal mitochondrial function. 16 Thus, neddylation inhibition halts AILI boosting anabolic cardiolipin pathway. RESULTS Alteration of NEDD8-protein homeostasis in patients with AILI and in preclinical models of hepatotoxicity by APAP overdose DILI is a complex liver pathology characterized by massive ROS production as well as inflammatory and necrotic processes in response to APAP overdose. 17 Disruptions in the NEDD8 proteome have been linked to oxidative stress-related liver Luis Alfonso Martı ´nez-Cruz, 1 Rube ´n Nogueiras, 9,10,11,12 Paula Iruzubieta, 13 Javier Crespo, 13 Steven Masson, 14,15 Misti Vanette McCain, 15 Helen L. Reeves, 14,15 Raul J. Andrade, 16,17 M. Isabel Lucena, 16,18 Ugo Mayor, 19,20 Naroa Goikoetxea-Usandizaga, 1,16 Irene Gonza ´lez-Recio, 1,22, *and Marı ´a L. Martı ´nez-Chantar 1,16,22,23, * 15 Newcastle University Translational and Clinical Research Institute, The Medical School, Newcastle University, NE2 4HH Newcastle upon Tyne, UK 16 Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Hepa ´ticas y Digestivas (CIBERehd), Carlos III National Health Institute, 28029 Madrid, Spain 17 Unidad de Gestio ´n Clı ´nica de Enfermedades Digestivas, Instituto de Investigacio ´n Biome ´dica de Ma ´laga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Ma ´laga, 29590 Ma ´laga, Spain 18 Servicio de Farmacologı ´a Clı ´nica, Instituto de Investigacio ´n Biome ´dica de Ma ´laga-IBIMA, Hospital Universitario Virgen de la Victoria, UICEC SCReN, Universidad de Ma ´laga, 29590 Ma ´laga, Spain 19 Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain 20 Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain 21 These authors contributed equally 22 Senior author 23 Lead contact *Correspondence: [email protected] (I.G.-R.), [email protected] (M.L.M.-C.) https://doi.org/10.1016/j.xcrm.2024.101653 2Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS 0 10 20 30 40 0 2 4 6 0.0 0.5 1.0 1.5 2.0 2.5 8DDEN 40x 0 1 2 3 20 40 60 80 A C DE B (legend on next page) Cell Reports Medicine 5, 101653, July 16, 2024 3 Article ll OPEN ACCESS pathologies. 12,15,18 Thus, the amount of free NEDD8 and NEDD8-conjugated proteins was assessed in liver samples from patients with AILI to ascertain whether abnormal neddylation appears dysregulated in APAP-associated liver damage. Comparing patients with AILI to healthy controls, a quantitative histological examination showed an increase in the liver neddylated proteome (Figure 1A; Table S1). The use of preclinical mouse models, mimicking APAP overdose in patients with AILI, has revealed key insights into the pathology. Gene expression assays highlighted significant alterations in the neddylation cycle in the livers of the animal model treated with the hepatotoxic compound 19 (Figure 1B). These findings were supported by western blot analyses, at 6, 24, and 48 h of APAP damage, which demonstrated an early-stage increase in neddylation profiles and in the E1 and E3 enzymes (Figure 1C). Moreover, serum samples from the animal models further validated the rise in neddylated proteins post-APAP overdose (Figure 1D). Primary hepatocytes exposed to a toxic APAP dose (10 mM) also exhibited a pronounced increase in neddylated cullins (Figure 1E), indicating a clear association between aberrant neddylation and hepatic damage. Collectively, these findings underscore the critical role of atypical neddylation in the context of liver injury in both human AILI cases and animal models. Inhibition of neddylation resolves APAP toxicity in mice To gain insight into the functional consequences that neddylation has in the liver of patients with AILI and in mice under a toxic dose of APAP, we inhibited the NEDD8-conjugated activity with the NAE-1 inhibitor MLN4924 in the preclinical mice model under APAP-induced toxicity. Twenty-four hours after APAP administration, these mice received MLN4924 in a single dose (Figure S1A). Mice were sacrificed 24 h after MLN4924 treatment, and the effect was confirmed by immunohistochemistry (IHC) and western blot analyses of the NEDD8 proteome in these mice (Figures 2A and S1B). Animals treated with MLN4924 under APAP overdose presented a significant reduction of neddylated proteins in comparison to the liver of non-treated mice and exposure to the hepatotoxic compound (Figure 2A). The image quantification at a higher magnification is shown in Figure S1C. In line with these results, H&E staining and TUNEL assay revealed the presence of necrotic areas in the APAP-treated mice, which were significantly reduced with ML4924 treatment (Figures 2B and 2C). Inflammatory response is a well-known process during DILI. 19 Analysis of macrophage F4/80 staining reveals a lower number of Kupffer cells under NAE-1 inhibition (Figure 2D). The image quantification at a higher magnification is shown in Figure S1C. Consistently, the levels of tumor necrosis factor and interleukin-6 were measured in the serum of these animal models (Figure 2E). Inhibiting neddylation significantly reduced the levels of both cytokines as well as the serum transaminases (Figures 2E and 2F). To assess the influence of neddylation inhibition on inflammatory responses, a new preclinical study was performed. Mice were administered MLN4924 shortly after the induction of hepatic damage, precisely 6 h following an APAP overdose, as depicted in Figure S2A. Subsequent evaluation at 18 h post-treatment revealed a marked decrease in necrotic areas and TUNEL-positive staining in the treated animals (Figure S2A). Concurrently, there was a notable attenuation of the inflammatory process, as evidenced by a significant reduction in F4/80 staining (Figure S2A). This reduction was also correlated with decreased transaminase levels, indicating a mitigation of liver injury (Figure S2B). The outcomes revealed that MLN4924 treatment leads to a significant decrease of NEDD8 levels, which is closely linked with a reversal of the principal characteristics of DILI, including necrosis and inflammatory processes. Proteomic insights into the APAP mouse model treated with neddylation inhibition drug The CRLs family are well-known NEDD8 substrates. 12 Cullin neddylation causes a conformational change in the E3 ubiquitin ligases, increasing their activity over their substrates, which is typically ubiquitination for protein degradation. 14 Other noncullin NEDD8 substrates, including p53, HuR, LKB1, and Akt, have been found to be regulated in stability, localization, and function for this PTM. 13,20,21 As a result, neddylation regulates multiple molecular pathways at the same time. 22 The landscape of hepatic neddylated proteins under the pathological condition of AILI was then delineated to elucidate how APAP toxicity affects this PTM. For that purpose, we employed whole-body genetically modified mice that express a bio NEDD8 construct (Figure S3A), which do not show any alteration in necrotic areas or the serum markers compared to wild-type (WT) mice (Figures S3B and S3C). Furthermore, when genes associated with APAP metabolism and oxidative stress were examined, no significant changes were observed in either animal model (Figure S3D). Similarly, analysis of the neddylome profile revealed no discernible alterations (Figure S3E). Biotin pull-down experiments were then performed in bio NEDD8 mice to see how the hepatic biotin-neddylated proteome is modulated following 24 h of APAP with/without MLN4924. The volcano plot analysis of the hepatic neddylome Figure 1. Global NEDD8 is characterized in patients with AILI and in preclinical mice models with an APAP overdose (A) Liver immunohistochemical staining and respective quantification of NEDD8 in a cohort of patients with AILI (N= 12) compared to a healthy group (n= 4). Scale bar corresponds to 50 mm. (B) mRNA expression levels of NEDD8 pathway (Nedd8,Uba3,Nae-1,Ube2m,Ube2f,Rbx1,Rnf7,Cbl,Dcund1d1,Dcund1d2,Dcun1d3,Mdm2,Atxn3,Senp8, Cops5,Usp21,Uchl3, and Uchl1) in mice treated with 360 mg/kg of APAP (n= 4) for 48 h and compared with a control group (n= 4). (C) Protein expression levels of NEDD8 and the enzymes involved in the NEDDylation pathway NAE-1, UBA3, CBL, DCUN1D3, and MDM2 in liver from mice treated with a single dose of 360 mg/kg of APAP (n= 3) for 6, 24, and 48 h and compared with a control group (n= 3). b-Actin was used as a loading control. (D) NEDD8 serum levels was determined in APAP overdose mice models (n= 5) for 48 h and compared to a control group (n= 5). (E) Protein expression levels of NEDD8 in primary hepatocytes treated with 10 mM of APAP overdose for 1, 3, and 6 h. GAPDH was used as a loading control. Triplicates were used for experimental condition. Data are shown as mean ±SEM. *p< 0.05, **p< 0.01, and ***p< 0.001 are shown (Student’s test). 4Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS A B C D EF (legend on next page) Cell Reports Medicine 5, 101653, July 16, 2024 5 Article ll OPEN ACCESS because of the APAP response in comparison to the control group revealed the proteins that were significantly modulated, as shown in Figure S3F. Furthermore, ‘‘the Database for Annotation, Visualization, and Integrated Discovery’’ (DAVID) (https:// david.ncifcrf.gov/) analysis showed biological processes such as peroxide catabolism and natural killer cell-mediated toxicity, which could be linked to reactive oxygen production and the inflammatory response seen in AILI (Figure S3G). Further investigation revealed that peroxidase activity was also significantly represented in molecular function (Figure S3G). In these Bio NEDD8 mice, MLN4924 effects were represented in a volcano plot (Figure 3A), and the analysis performed by the DAVID analysis identified the overrepresented biological processes, which included processes related to response to oxidative stress; for instance, TAM41, a CDP-DAG synthase required for cardiolipin biosynthesis in mitochondria. 16 Functional enrichment analysis of differentially neddylated proteins showed molecular function associated with cellular iron homeostasis (Figure 3B). These findings support previous research indicating the importance of cardiolipin in maintaining mitochondrial and cellular iron homeostasis. 23 Western blot from the liver of APAP or APAP plus MLN4924 versus WT mice showed a TAM41 upregulation, while in the mice treated with APAP, the levels of this protein were significantly diminished (Figure 3C). To better understand how neddylation affects TAM41 levels, we mapped the ubiquitinated proteome after APAP damage. For this, we used transgenic mice that expressed whole-body bio UB (Figure S3A). The phenotype of these animals was comparable to that of the WT. 24 To isolate the hepatic biotin-ubiquitin proteome, bio UB mice were treated with APAP in the presence or absence of MLN4924 and sacrificed 48 h after APAP overdose (Figure 3D). In these experimental circumstances, the western blot against TAM41 demonstrated that ubiquitinated forms of this protein occur in control mice and in the group that received APAP and MLN4924 treatments, which corresponds to inhibition in the neddylation of cullins (Figure 3D). To further corroborate these data, immunoprecipitation assays targeting NEDD8 were conducted, and the concentrations of TAM41 were quantified within these extracts. It was observed that TAM41 levels tended to diminish following APAP administration. However, this reduction was significantly abrogated upon treatment with MLN4924, indicating its potential in reversing the effects of APAP (Figures 3EandS3H). To enhance our understanding of TAM41’s regulatory mechanisms in a cellular context, we treated primary hepatocytes with the translational inhibitor cycloheximide, either alone or in combination with the proteasome inhibitor MG132 and the neddylation inhibitor MLN4924. This approach builds on previous observations that indicated TAM41 undergoes both neddylation and ubiquitination. It was found that a 12-h inhibition of protein synthesis by cycloheximide increases TAM41 stability, suggesting a protective effect against degradation pathways. Interestingly, when MG132 was applied, it not only confirmed this protective effect but also accelerated the stabilization process, observable at just 8 h. In a parallel finding, the introduction of MLN4924 during the early phase of treatment raised TAM41 protein levels (Figure S3I). Finally, in line with these results, we found that the levels of cardiolipin in the livers from WT mice treated with APAP plus MLN4924 were significantly upregulated as compared to the experimental group without MLN4924 therapy (Figure 3F). Cytochrome coxidase is the terminal complex of eukaryotic oxidative phosphorylation in mitochondria. The electrochemical gradient formed during the process is used to generate chemical energy in the form of ATP to power vital cellular processes. 25 It has already been reported that the identification of cardiolipinbinding sites on cytochrome coxidase at the entrance of proton channels modulates its activity. 26 Regarding the role of cardiolipin in cytochrome coxidase, our findings show that in isolated membranes, blocking neddylation in primary hepatocytes treated with APAP restores its activity in comparison to untreated cells (Figure 3G). These findings highlighted the significance of increasing TAM41 levels in the presence of NAE-1 inhibitor MLN4924, inducing cardiolipin levels, and modulating cytochrome coxidase activity, which could maintain oxidative phosphorylation in mitochondria. Targeting neddylation reduces mitochondrial dysfunction in the liver under APAP overdose Mitochondrial dysfunction is one of the main hallmarks of liver injury triggered by APAP intoxication, 17 and its recovered function has been described as a potential therapy to improve liver regeneration after damage induction. 27,28 In line with the proteomic analysis conducted on preclinical APAP models revealing significant impairment of mitochondrial function due to neddylation, the TUNEL assay demonstrated a marked reduction in apoptotic response in hepatocytes treated with MLN4924 compared to those treated solely with APAP (Figure 4A). Moreover, MLN4924 treatment substantially elevated active mitochondrial levels, as demonstrated by MitoTracker measurements, while concomitantly resulting in a marked reduction in ROS production, as determined by MitoSOX analysis (Figure 4A). These results collectively imply a promising therapeutic potential for MLN4924 in ameliorating Figure 2. Pharmacological neddylation inhibition reduces liver damage Comparison of control mice (n= 5) versus mice treated with 360 mg/kg of APAP overdose (n= 5) versus with 360 mg/kg of APAP overdose and 24 h later with 60 mg/kg MLN4924 (n= 5). (A) Liver immunohistochemical staining and respective quantification of NEDD8 (n= 5). Scale bar corresponds to 100 mm. (B) Liver necrosis areas over the total area in percentage was assessed by H&E staining and a total of 5 pictures per animal were evaluated in a total of 5 mice per group (n= 5). Scale bar corresponds to 200 mm. (C) Cell death was determined by TUNEL assay liver tissue (n= 5). Scale bar corresponds to 100 mm. (D) Inflammation was assessed by F4/80 staining (n= 5). Scale bar corresponds to 200 mm. (E) Tumor necrosis factor (TNF) and interleukin-6 (IL-6) levels were determined by ELISA assay in mice serum after 48 h of APAP overdose (n= 5 per group). (F) Transaminase ALT and AST levels were measured in mice serum after 48 h of APAP overdose (n= 5 per group). Data are shown as mean ±SEM. *p< 0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001 are shown (Student’s test). 6Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS -2 -1 0 1 2 0 1 2 3 4 Rpl13a Arl8b Cyp4a12a Tamm41 Otc Pkm Actr1a Lyz1 Aco2 Gstm1 F13b Ube2d3 0246 012345 0.0 0.5 1.0 1.5 2.0 0.00 0.01 0.02 0.03 0.04 0 5 10 15 0.4 0.6 0.8 1.0 1.2 A C D EFG B (legend on next page) Cell Reports Medicine 5, 101653, July 16, 2024 7 Article ll OPEN ACCESS APAP-induced hepatocyte damage through enhanced mitochondrial function and diminished ROS generation. In line with these findings, genes related with mitochondrial fusion and fission biogenesis as well as mitophagy 29 were significantly downregulated under APAP treatment in WT primary hepatocytes, while blocking neddylation seemed to avoid this tendency (Figure 4B). NAPQI emerges asa particularly deleterious byproduct resulting from the catabolism of APAP. 30 Its formation instigates GSH depletion, leading to mitochondrial dysfunction and necrosis, consequently intensifying the inflammatory response within the hepatic tissue. 17 Importantly, in the context of mice under MLN4924 treatment during APAP overdose, there was an increase in the ratio of reduced GSH to oxidized GSH (GSH/GSSG), as shown in Figure 4C. Accordingly, heme oxygenase 1 and nitric oxide synthase 2 expression was also altered because of redox cellular regulation under neddylation inhibition (Figure 4D). Overall, these results underline the significance of neddylation disruptions in mitochondrial activity in DILI and how blocking this PTM restores hepatocyte survival by reducing ROS and reestablishing energetic metabolism. Tam41 modulates the beneficial effects of MLN4924 treatment in primary hepatocytes exposed to APAP overdose To gain insight into the functional consequences of increased TAM41 levels due to PTM, we incubated primary hepatocytes in a medium with and without APAP for 6 h in the presence and absence of MLN4924 treatment and Tam41 silencing (Figure S4A). Blocking neddylation abrogates APAP cell death; in contrast, Tam41 inhibition reversed the beneficial effect, with concomitant increase in the TUNEL assay (Figures 5A and S4B). These data are further sustained by lower mitochondrial activity and greater ROS production, assessed by MitoTracker and MitoSOX respectively, in the lack of Tam41 (Figures 5A and S4B). Under these circumstances, the absence of Tam41 abolished the induction of mitochondrial membrane potential resulting from MLN4924 treatment (Figure 5B), according to a reduction in cardiolipin levels (Figure 5C). These findings align with the observed increases in extracellular ATP levels in APAP-treated hepatocytes and in the absence of Tam41 but in the presence of MLN4924, serving as an indicator of the apoptotic response. Conversely, intracellular ATP levels were significantly reduced in APAP-treated cells, and this effect was reversed upon blocking neddylation. Silencing of Tam41 abolished the restoration of ATP levels. (Figure 5D). Taken together, these results suggest that the absence of neddylation confers protection against liver damage induced by APAP overdose, mediated in part by Tam41. Overdoses of APAP have been demonstrated to disrupt the activities of complex I and complex II in rat hepatocytes both in vitro and in vivo, leading to heightened oxidative stress and reduced ATP production. 30,31 In this context, we investigated the activity of complex I in the presence or absence of MLN4924 and modulation of Tam41 expression. It was observed that blocking neddylation restored the activity of complex I, which was impaired during APAP overdose (Figure 5E), whereas silencing of Tam41 reversed this effect. Based on these findings and recognizing the critical dependence of complex I activity on the NAD/NADH ratio, both molecules were evaluated under the same experimental conditions described earlier. 32 The results revealed an increase in NAD levels and a reduction in NADH levels, leading to a higher NAD/NADH ratio when neddylation was blocked, thereby mitigating the effects of APAP. Conversely, the absence of Tam41 once again hindered these regulatory effects (Figure 5F). To gain deeper insights into the mechanism behind APAP injury, we utilized Tamm41 (GenBank: NM_026894) Mouse Tagged ORF Clone (OriGene) to induce Tam41 overexpression in primary hepatocytes (Figure S4C). The results prominently demonstrate the protective effect conferred by increased levels of Tam41 during APAP overdose (Figures 5G and S4D). These observations underscore the significance of neddylation in mitochondrial function, with Tam41 playing a key mediating role. TAM41 shows a critical role in the protective mechanisms of neddylation inhibition against AILI The role of TAM41 was assessed in preclinical animal models exposed to APAP-induced toxicity. Following an APAP overdose, mice received a single dose of MLN4924 24 h later. Twelve hours post-MLN4924 treatment, Tam41 was silenced in the mice. The Figure 3. Proteomic characterization of neddylated proteins in transgenic Bio NEDD8 mice treated with 360 mg/kg of APAP overdose and 24 h later administered MLN4924 treatment (A) Volcano plot representation of specific proteins regulated in Bio NEDD8 mice treated with APAP overdose and 24 h later with 60 mg/kg MLN4924 (n=4)in comparison with APAP overdose mice group (n= 3) after 24 h of APAP overdose. (B) Gene Ontology (GO) biological processes and GO molecular functions upregulated and downregulated in Bio NEDD8 mice treated with APAP overdose and 24 h later with MLN4924 (n= 4) in comparison with APAP overdose mice group (n= 3). (C) Protein expression levels of TAM41 in total liver homogenate of WT mice (n= 3), treated with APAP overdose and 24 h later with MLN4924 (n= 3) compared with a control group (n= 3). b-Actin was used as a loading control. (D) Protein expression levels of TAM41 after pull-down enrichment of biotinylated proteins in Bio NEDD8 mice treated with APAP overdose (n= 3) and 24 h later with MLN4924 (n= 3) compared with a control group (n= 3). b-Actin was used as a loading control from the input protein extract. Protein expression levels of TAM41 after pull-down enrichment of biotinylated proteins in Bio UB mice treated with APAP overdose (n= 3) and 24 h later with MLN4924 (n= 3) compared with a control group (n= 3). b-Actin was used as a loading control from the input protein extract. (E) Quantification of the western blot obtained from the immunoprecipitation of NEDD8 and western blot against TAM41 in in WT mice treated with APAP overdose (n= 3) and 24 h later with MLN4924 (n= 3) compared with a control group (n= 3). (F) Hepatic cardiolipin levels in WT mice treated with APAP overdose (n= 5) and 24 h later with MLN4924 (n= 5) compared with a control group (n= 5). (G) Cytochrome coxidase activity was determined in WT mice hepatocytes treated with APAP overdose (n= 4) and 24 h later with MLN4924 (n= 4) compared with a control group (n= 4). Data are shown as mean ±SEM. *p< 0.05, **p< 0.01 and ***p< 0.001 are shown (Student’s test). 8Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS A B D E C (legend on next page) Cell Reports Medicine 5, 101653, July 16, 2024 15 Article ll OPEN ACCESS Fund (ERDF); and Grupos consolidados Gobierno Vasco IT1476-22 to P.A. We would like to acknowledge Begon ˜a Rodrı ´guez Iruretagoyena for her technical support. AUTHOR CONTRIBUTIONS Conceptualization, C.G.-P., M.S.-M., I.G.-R., and M.L.M.-C.; funding acquisition, L.A.M.-C. and M.L.M.-C.; experiments, C.G.-P., M.S.-M., J.S., L.M., C.C., C.M.R.-G., L.E.Z.-P., P.P.-S., M.E., R.R.-A., S.L.-O., M.M.-G., T.C.D., M.P., I.A., D.X., P.A., F.E., L.A.M.-C., R.N., P.I., J.C., S.M., M.V.M., H.L.R., R.J.A., M.I.L., N.G.-U., U.M., and I.G.-R.; supervision, I.G.-R. and M.L.M.-C.; writing – original draft and review and editing, C.G.-P., M.S.-M., I.G.-R., and M.L.M.-C. All authors have revised and approved the final version of the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: September 27, 2023 Revised: February 28, 2024 Accepted: June 19, 2024 Published: July 16, 2024 REFERENCES 1. Suk, K.T., and Kim, D.J. (2012). Drug-induced liver injury: present and future. Clin. Mol. Hepatol. 18, 249–257. https://doi.org/10.3350/cmh. 2012.18.3.249. 2. Lee, W.M. (2020). 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Pharmacological neddylation inhibition improves mitochondrial function and regenerative response in preclinical mice model treated with an APAP overdose and 24 h later with MLN4924 treatment In the preclinical mice model under APAP-induced toxicity of 360 mg/kg (n= 5), 24 h after APAP administration, mice received MLN4924 in a single dose (n= 5). Mice were sacrificed 48 h after APAP overdose. (A) PCNA expression by immunohistochemistry (n= 5). Scale bar corresponds to 100 mm. (B) mRNA expression levels of Areg,Btc,Ereg,Hb-egf,Egf,Hgf,Tgfa, and Tgfb(n= 4). (C) Protein expression levels of P-cMET, cMET, p-EGFR, EGFR, cyclin D1, and PCNA. b-Actin was used as a loading control (n= 5). In the preclinical mice model under APAP-induced toxicity of 600 mg/kg (n= 5), 6 h after APAP administration, mice received MLN4924 in a single dose (n= 5). Mice were sacrificed 24 h after APAP overdose. (D) Liver necrosis was assessed by H&E staining. Cell death was evaluated by TUNEL. 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Antioxidants 11, 897. 18 Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG, HRP-linked Antibody Cell Signaling Cat#t7076S; RRID_ AB:3105896 Anti-rabbit IgG, HRP-linked Antibody Cell Signaling Cat#7074S; RRID: AB_3105897 Cyclin D1 Cell Signaling Cat#2978; RRID: AB_2259616 Dcund1d3 Santa Cruz Biotechnology Cat#sc-514506; RRID: AB_3105891 Egfr Merk Cat#06-847; RRID: AB_2096607 F4/80 Bio-Rad Cat#MCA497BB; RRID: AB_323893 Gapdh [6C5] Abcam Cat#ab8245; RRID: AB_2107448 Mdm2 Proteintech Cat#66511-1-Ig; RRID: AB_2881874 Met (c-Met) [EP1454Y] - N-terminal Abcam Cat#ab51067; RRID: AB_880695 Nae1 Cell Signaling Cat#14321; RRID: AB_2798448 NEDD8 antibody for IHC Cell Signaling Cat#2745; RRID: AB_10695300 NEDD8 antibody IP and WB Abcam Cat#ab81264; RRID: AB_1640720 Pcna Santa Cruz Biotechnology Cat#sc-25280; RRID: AB_628109 Purified Mouse IgG1, kIsotype Control BD Pharmingen Cat#557273; RRID: AB_396613 pY1068-Egfr Cell Signaling Cat#3777; RRID: AB_2096270 pY1230/1234/1235-cMet MerckMillipore Cat#07-810; RRID: AB_568851 Tam41 Abcam Cat#ab230359; RRID: AB_3105893 Total OXPHOS Cocktail (V-ATP5A, IIIUQCRC2, II-SDHB, IV-COXII, I-NDUFB8) Abcam Cat#ab110411; RRID: AB_2756818 Uba3 Santa Cruz Biotechnology Cat#sc-377272; RRID: AB_3105894 b-actin Sigma-Aldrich Cat#A2228; RRID: AB_476697 Biological samples Paraffin-embedded liver AILI patients Supplied by MD PhD Helen L Reeves Newcastle Hospital NHS Foundation Trust (Newcastle, England) Paraffin-embedded liver healthy transplant donors Supplied by MD PhD Javier Crespo Marque ´s de Valdecilla University Hospital (Santander, Spain) Chemicals, peptides, and recombinant proteins 2-hydroxypropyl-b-cyclodextrin Sigma-Aldrich Cat# H107-100G CAS. 128446-35-5 2-Propanol Sigma-Aldrich Car#I9516 CAS 67-63-0 Acetaminophen (APAP) Sigma-Aldrich Cat# A7085-100G CAS. 103-90-2 Acetic Acid Sigma-Aldrich Car# W200603 CAS 64-19-7 Acetonitrile Sigma-Aldrich Cat# 34851 CAS 75-05-8 Calcium chloride Sigma-Aldrich Cat# C5670 CAS 10043-52-4 Chloroform Sigma-Aldrich Cat#C2431 CAS 67-66-1 Cycloheximide Sigma-Aldrich C1988-1g Collagenase type IV, CLS-4 Worthington Cat#LS004188 Corning Collagen I, Rat Tail, Corning Cat#354236 Dako EnVIsion system, Peroxidase Dako Cat#K5007 (Continued on next page) Cell Reports Medicine 5, 101653, July 16, 2024 e1 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER D-Glucose Sigma-Aldrich Cat#G7021 CAS 50-99-7 DharmaFECT 1 Transfection Reagent Dharmacon Cat#T-2001-01 Dihydroxiethidium (DHE) Sigma Cat#D-7008-10mg Dimethyl pimelimidate dihydrochloride (DMP) Sigma Cat#80490 DL-Dithiothreitol (DTT) Sigma-Aldrich Cat# DTT-RO CAS 3483-12-3 DPX Mountant for histology Sigma-Aldrich Cat#06522 Dulbecco’s Phosphate-Buffered Saline (PBS) Gibco Cat# 14190144 EnVision+ System HRP Dako Cat#K4001 Eosin B Sigma-Aldrich Cat#2853 Ethanol Sigma-Aldrich Cat#E7023 CAS 64-17-5 Fetal Bovine Serum (FBS) GIBCO Cat#A38401 Fluoromount g with DAPI Southern Biotech Cat# 0100-01 Formic Acid PanReac AppliChem Cat#1002641000 CAS 64-18-6 Glycol ether diamine tetraacetic acid (EGTA) Sigma-Aldrich Cat# E3889 CAS 67-42-5 Guanidine hydrochloride Sigma-Aldrich Cat#G3272 CAS 50-01-1 Harris Hematoxylin Bio-Optica Cat#05–06005/L Histo-Clear I Solution Electron Microscopy Sciences Cat#64110-004 Hydrogen Peroxide 30% w/v PanReac AppliChem #Cat121076 CAS 7722-84-1 Igepal Sigma-Aldrich Cat#I8896 CAS 9002-93-1 InvivofectamineTM 3.0 Reagent Invitrogen Cat#IVFs3001 iodoacetamide (IAA) Sigma-Aldrich Cat#I6125 CAS 144-48-9 Isoflurane Baxter SL Cat#NR60378 jetPRIME Polyplus Cat#114-15 L-glutamine GIBCO Cat#25030-024 Mayer’s Haematoxylin Sigma-Aldrich Cat# MHS16 Methanol PanReac AppliChem #Cat1610911714 CAS 67-56-1 MG132 Sigma-Aldrich 1211877-36-9 Minimum Essential Media GIBCO Cat#31095029 N-Ethylmaleimide Sigma Cat#23030 OCT Pioneer Cat#PRC/OCT Oxygen Air Liquide SLU Cat#ESCG101710 Paraformaldehyde 4% solution in PBS Santa Cruz Cat#Sc-281692 CAS 30525-89-4 Penicillin-Streptomycin-Glutamine (100x) GIBCO Cat#10378016 Pevonedistat (MLN4924) MeDChemExpress, MCE Cat#HY-70062 CAS. 905579-51-3 Phenylmethylsulfonyl fluoride (PMSF) Sigma-Aldrich Cat#52332 CAS 329-98-6 Phosphatase inhibitor cocktail Sigma-Aldrich Cat#P2850 Ponceau S Solution Sigma-Aldrich Cat#P7170-1L CAS 6226-79-5 Potassium chloride Sigma-Aldrich Cat#G7021 CAS 7447-40-7 (Continued on next page) e2 Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Potassium ethylenediaminetetraacetate dibasic (EDTA) Sigma-Aldrich Cat#E9884 CAS 25102-12-9 Protease Inhibitor cocktail Sigma-Aldrich Cat#P8340, Protease Inhibitor Cocktail Tablets Roche Diagnostics Cat#11697498001 Sodium azide Sigma-Aldrich Cat# S8032 CAS 26628-22-8 Sodium bicarbonate Sigma-Aldrich Cat#S5761 CAS 144-55-8 Sodium chloride PanReac AppliChem Cat#1064041000 CAS 7647-14-5 Sodium deoxycholate Sigma-Aldrich Cat# 30970 CAS 302-95-4 Sodium dodecyl sulfate Sigma-Aldrich Cat#L5750 CAS 151-21-3 Sodium fluoride Sigma-Aldrich Cat#S7920-100G CAS 7681-49-4 Sodium orthovanadate Sigma-Aldrich Cat#S6508-50G CAS 13721-39-6 Sodium phosphate dibasic Sigma-Aldrich Cat# 71640 CAS 7558-79-4 Sodium phosphate monobasic Sigma-Aldrich Cat# S0751 CAS 7558-80-7 Tris Base Fisher Cat#BP152-1 Triton X-100 Sigma-Aldrich Cat# 100-500mL Trizol Invitrogen Cat#15596026 Tween 20 Sigma-Aldrich Cat# P9416 Urea Sigma-Aldrich Cat#U5378 CAS 57-13-6 b-mercapthoethanol Sigma-Aldrich Cat#M3148 CAS 60-24-2 Critical commercial assays 3,30-Diaminobenzidine (DAB) Sigma-Aldrich Cat#D6815 A special microcentrifuge filter VivaClean Mini 0.8 mL m PES Sartorius Cat# VK01P042 ATPliteTM luminescence ATP detection kit Perkin Elmer Cat#6016943 Bead Ruptor Homogenizer OMNI International N/A Cardiolipin Assay kit Sigma-AldrichCat#MAK362 Clarity Western ECL substrate Bio-Rad Cat#170–5061 Complex I Enzyme Activity Assay Kit Abcam Cat#ab109721 DNase I, amplification Grade Invitrogen (ThermoFischer) Cat#18068015 GOT/AST IFCC. Enzymatic - UV Spinreact Cat#41270 GPT/ALT IFCC. Enzymatic - UV Spinreact Cat#41280 IL-6 Mouse ELISA kit Invitrogen TM Cat#KMC0061 In situ cell death detection kit Roche Cat#11684795910 Micro BCA Protein Assay Kit Thermo Fisher Scientific Cat#23235 MitoSOXTM Red MItochondrial Superoxide Indicator Invitrogen (ThermoFischer) Cat#M36008 MitoTrackerTM Green FM Invitrogen (ThermoFischer) Cat#M7514 M-MLV Reverse Transcriptase (200 U/mL) Invitrogen (ThermoFischer) Cat#28025013 NAD/NADH Assay Kit Abcam Cat#ab65348 NEDD8 mouse ELISA kit MyBiosource Cat#MBS109530 NeutrAvidin-Agarose beads ThermoScientific Cat#29200 (Continued on next page) Cell Reports Medicine 5, 101653, July 16, 2024 e3 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Pre-equilibrated PD10 columns GE Healthcare Cat#28-9232-45 Protein A/G PLUS-Agarose Santa Cruz Biotechnology Cat#2003 Protein Assay Dye Reagent Concentrate (Bradford) Bio-Rad Cat#500-0006 Protein G Sepharose 4 Fast Flow Cytiva Cat#GE17-0618-01 Purified Mouse IgG1, kIsotype Control BD Pharmingen Cat#557273 SDH Activity Assay Kit Merck Cat#MAK197 SYBRSelect Master Mix Applied Biosystems (ThermoFischer) Cat#t44720903 Tetramethyldhodamine Ethyl Ester Perchlorate (TMRE) ThermoFisher Scientific Cat#T669 Tumor Necrosis Factor (TNF) ELISA Assay DuoSet II kit R&D Systems Cat#DY410 TUNEL Assay Kit – HRP – DAB Abcam Cat#ab206386 Vector Vip DAB substrate Vectorlabs Cat#SK-4105 Vector Vip purple substrate Vectorlabs Cat#SK-4600 Experimental models: Cell lines Mice primary hepatocytes Charles River Laboratories C57BL/6NCtrl Experimental models: Organisms/strains C57BL/6 mouse Charles River Laboratories C57BL/6NCtrl Bio NEDD8 transgenic mice CIC bioGUNE animal facility C57Bl6xCBA F1 Bio UB transgenic mice CIC bioGUNE animal facility C57Bl6xCBA F1 Global 14% Protein Rodent Maintenance diet Envirgo Cat#2014C Oligonucleotides Primers for qPCR, See Table S2 This paper N/A SilencerTM Negative Control No. 1 siRNA Ambion (ThermoFischer) Cat# 4404021 siTam41 (Fw 50-GUCUGUGAUUUAU AGUUCAtt-30,Rv3 0-UGAACUAUAA AUCACAGACtt-50) Ambion (ThermoFischer) In vitro Cat#4390771 siTam41 (Fw 50-GGAGAUAGAUAA AAGCCCAtt-30,Rv3 0-UGGGCUUUUA UCUAUCUCCag-50) Ambion (ThermoFischer) In vivo Cat #4457308 Recombinant DNA Plasmid: Overexpression of mice Tam41 (NM_026894) OriGene Cat#MR212613 Plasmid: Empty vector (pcDNA3.1- (empty)-TAG) AddGene Cat#138209 Software and algorithms BioRender Science Suite Inc https://www.biorender.com RRID:SCR_018361 Image Lab 6.0.1 software Bio-Rad Laboratories https://www.bio-rad.com/es-es/product/ image-lab-software?ID=KRE6P5E8Z RRID: N/A ImageJ NIH https://imagej.nih.gov/ij/index.html RRID SCR_003070 PEAKS X software Bioinformatics solutions https://www.bioinfor.com/peaks-studio/ RRID:SCR_022841 Perseus Max Planck Institute of Biochemistrry https://www.maxquant.org/perseus/ RRID: N/A Prism 9 GraphPad software GraphPad software https://www.graphpad.com/ RRID: SCR_00278 (Continued on next page) e4 Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Marı ´a Luz Martı ´nez-Chantar ([email protected]). Materials availability Mouse lines, Bio NEDD8 and Bio UBQ, previously generated and used in this study are available upon request to Marı ´a Luz Martı ´nezChantar ([email protected]). This study did not generate new unique reagents. Data and code availability dThis paper does not report original code. dThe mass spectrometry data have been deposited to the ProteomeXchange Consortium (https://proteomecentral. proteomexchange.org) via the iPRoX partner repository: PXD053221. dAny additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Human samples and clinical information This research project was performed in accordance with the ethical code of the World Medical Association, the Declaration of Helsinki, and with local and national laws. The Newcastle and North Tyneside Regional Ethics Committee, the Newcastle Academic Health Partners Bioresource (NAHPB), the Newcastle upon Tyne NHS Foundation Trust Research and Development (R&D) department (Reference numbers: 10/H0906/41; NAHPB Project 48; REC12/NE/0395; R&D 6579; Human Tissue Act licence 12534) and Research Ethics Committee of IDIVAL Cantabria (Code 2017.052) approved the study procedures, and a written informed consent was obtained before inclusion in the study of each patient. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Other AXIO Imager A1 Manual Carl Zeiss N/A AXIO Imager D1 Upright Fluorescence Microscope Carl Zeiz N/A BEH C18 Column Waters Corp Cat#186002350 ChemiDoc Imaging System Bio-Rad Laboratories N/A Cryostate Leica Biossystems Cat# CM 1850 UV Nitrocellulose transfer membrane ThermoFischer Cat# LC2009 Precellys Tissue Homogenizer Bertin Instruments N/A Selectra Junio Spinlab 100 analyser Vital Scientific Spinreact N/A Spectra M2 BioNova N/A SpeedVacTM Thermofischer SPD131DDA SYNAPT G2 HDMS TOF Waters Corp N/A Thin-layer chromatography (TLC) silica sheets 20 320cm Merck Millipore Cat#1055530001 TimsTOF Pro with PASEF coupled online to an Evosep ONE Bruker Daltonics N/A Tissue Homogenizer FasPrep N/A Type 50.4 Ti Fixed-Angle Titanium Rotor Beckamn Coulter Cat#377299 UPLC-MS N/A N/A ViiA 7 Real-Time PCR System Applied Biosystems (ThermoFischer) Cat#4453545 Whatman paper 3MM Chr GE Healthcare Cat#3030-931 ImageQuant LAS 4000 imaging system GE Healthcare N/A Cell Reports Medicine 5, 101653, July 16, 2024 e5 Article ll OPEN ACCESS A total of 12 liver samples, with and average age of 36 ±13 years, from 7 male and 5 female patients with severe AILI (acetaminophen-induced liver injury) which undergoing urgent liver transplantation were included in this study (Table S1). The diagnosis of AILI, established in the Newcastle Hospitals NHS Foundation Trust (Newcastle, England), was based on clinical data, features of liver histology and exclusion of other possible causes of liver injury (viral hepatitis, biliary diseases, alcohol abuse, non-alcoholic fatty liver disease, autoimmune liver diseases, and hereditary diseases). All stated intentional paracetamol ingestion and had detectable paracetamol blood levels after ingestion. Additionally, 4 liver biopsies from organ transplant donors without liver lesions were used as controls for immunostaining analyses. The healthy biopsies samples were obtained from Marque ´s de Valdecilla University Hospital, Santander. Finally, the immunohistochemical analysis of global Neddylation levels was performed in paraffin-embedded liver tissue 8mm sections from 12 AILI patients and 4 healthy controls. Preclinical studies and animal maintenance All procedures were carried out in accordance with the CIC bioGUNE Animal Care and Use Committee and the local authority (Diputacio ´n de Bizkaia), under the codes P-CBG-CBBA-0218 and P-CBG-CBBA-1421, respectively, according to the criteria established by the European Union. The animals used for the experimentation, male three-month-old C57BL/6J wild-type mice were acquired from Charles River Laboratories and accommodated into the AALAC-accredited CIC bioGUNE animal facilities and maintained at 21 ±1C, 45 ±10% humidity and 12/12h light/dark cycles and fed a standard diet (Harlan Tekland, Envigo #2014C) with water and ad libitum. Mice were starved for 12h and then acetaminophen or APAP (Sigma-Aldrich, Cat# A7085) was given by intraperitoneal injection as a single dose of 360 mg/kg or 600 mg/kg depending on the experiment’s objectives which is specify in the figure legend along the manuscript and in the following section. Bio NEDD8 and Bio UBIQUITIN ( Bio UB) transgenic mice model In the present work, Bio NEDD8 and Bio UB transgenic mice (C57Bl6xCBA F1) were used to characterize the proteins undergoing these two post-translational modifications during APAP overdose and their modulation after neddylation inhibition. The generation of these Bio UB mice has been previously described in. 50 The Bio UB and Bio NEDD8 mice have a random genetic insertion of ubiquitin or NEDD8 sequences, respectively, that are conjugated with biotin sequence and BirA enzyme. Preclinical model of acetaminophen overdose and treatments Mice fasted for 12 h received a single dose of APAP 360 mg/kg by intraperitoneal injection. After 24 h of APAP overdose mice were randomly divided into two groups: one was administered subcutaneously with 60 mg/kg of MLN4924 (MeDChemExpress, Cat#HY70062) dissolved in vehicle solution of 2-hydroxypropyl-b-cyclodextrin (Sigma-Aldrich, Cat# H107), and the other group with vehicle solution as it was describe in our previous publications. 12,18 Finally, mice were sacrificed at 48 h after APAP administration. Moreover, the same experiment was performed but MLN4924 was given after 6 h of APAP 360 mg/kg or 600 mg/kg overdose administration and mice were sacrificed at 24h of APAP overdose. The set of experiments to silence Tam41 in vivo was performed in mice fasted for 12h and treated with 360 mg/kg APAP. After 12h of overdose animals were divided into 3 groups (n= 4) one administered with the SiTam41 (50-GGAGAUAGAUAAAAGCCCAtt -30, 30-UGGGCUUUUAUCUAUCUCCag -50) (Thermo Fisher Scientific, Custom, Catalog #4457308), other two with siCtrl (ThermoFischer, Cat# 4404021). 250nm of specific in vivo siRNA were resuspended in 625mL of Nuclease-Free Water (Merck, Cat#W4502). Mice received 1.7 mg/kg of specific in vivo siRNA complexed with Invivofectamine 3.0 Reagent (Invitrogen, USA, Cat#IVFs3001) following the manufacturer’s instructions and resuspended in sterile PBS through tail vein injection, which allows a specific silencing in the liver. 28 Then, two groups (siTam41 and SiCtrl) were treated with 60 mg/kg of MLN4924 after 24h of APAP overdose. Finally, mice were sacrificed at 48 h after APAP administration. Serum and liver samples were cryopreserved, and part of the liver tissue was maintained in Paraformaldehyde 4% solution in PBS (Santa Cruz, Cat#Sc-281692) or embedded in the OCT Embedding matrix of the frozen section (Pioneer, Cat#PRC/OCT) to further histological studies. Isolation and culture of primary hepatocytes Protocol to obtained primary hepatocyte was previously approved by CIC bioGUNE Animal Care and Use Committee and the local authority (Diputacio ´n de Bizkaia) according to the criteria established by the European Union. Primary hepatocytes from male threemonth-old C57BL/6J wild-type mice acquired from Charles River (St Germain sur l’Arbresle, France) and maintained at the CIC bioGUNE Animal Facilities were isolated by perfusion with Collagenase type IV (Worthington, Cat#LS004188). Briefly, mice were anesthetized with isoflurane inhalator (1.5% isoflurane in O 2 ; Baxter SL, Cat#NR60378). Following, the abdominal cavity was opened and the catheter was introduced into the vena cava. Liver was perfused with buffer I (13stock solution (Sigma-Aldrich: D-Glucose Cat#G7021, KCl Cat#G7021, NaHCO 3 Cat#S5761 and NaCl Cat#1064041000 from PanReac AppliChem), 5 mM EGTA (SigmaAldrich Cat# E3889) (37 C, oxygenated), and portal vein was cut. Then, liver was washed with buffer II (13stock solution) and subsequently, liver was perfused with buffer III (13stock solution, 2 mM CaCl 2 (Sigma-Aldrich, Cat# C5670), collagenase type I (Worthington) (37 C, oxygenated). After the perfusion, liver was placed in a Petri dish containing Minimum Essential Medium (MEM; Gibco, #Cat31095029) with 1% penicillin (100 U/ml), streptomycin (100U/ml), Amphotericin (100U/ml) (Anti-Anti; Gibco, e6 Cell Reports Medicine 5, 101653, July 16, 2024 Article ll OPEN ACCESS