Phosphorylation at the disordered N-end makes HuR accumulate and dimerize in the cytoplasm.

Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.

The lipopolysaccharide-TLR4 axis regulates hepatic glutaminase 1 expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis.

INTRODUCTION: Ammonia is a pathogenic factor implicated in the progression of metabolic-associated steatotic liver disease (MASLD). The contribution of the glutaminase 1 (GLS) isoform, an enzyme converting glutamine to glutamate and ammonia, to hepatic ammonia build-up and the mechanisms underlying its upregulation in metabolic-associated steatohepatitis (MASH) remain elusive. METHODS: Multiplex transcriptomics and targeted metabolomics analysis of liver biopsies in dietary mouse models representing the whole spectra of MASLD were carried out to characterize the relevance of hepatic GLS during disease pathological progression. In addition, the acute effect of liver-specific GLS inhibition in hepatic ammonia content was evaluated in cultured hepatocytes and in in vivo mouse models of diet-induced MASLD. Finally, the regulatory mechanisms of hepatic GLS overexpression related to the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) axis were explored in the context of MASH. RESULTS: In mouse models of diet-induced MASLD, we found that augmented liver GLS expression is closely associated with the build-up of hepatic ammonia as the disease progresses from steatosis to steatohepatitis. Importantly, the acute silencing/pharmacological inhibition of GLS diminishes the ammonia burden in cultured primary mouse hepatocytes undergoing dedifferentiation, in steatotic hepatocytes, and in a mouse model of diet-induced steatohepatitis, irrespective of changes in ureagenesis and gut permeability. Under these conditions, GLS upregulation in the liver correlates positively with the hepatic expression of TLR4 that recognizes LPS. In agreement, the pharmacological inhibition of TLR4 reduces GLS and hepatic ammonia content in LPS-stimulated mouse hepatocytes and hyperammonemia animal models of endotoxemia. CONCLUSIONS: Overall, our results suggest that the LPS/TLR4 axis regulates hepatic GLS expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis.

Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination.

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

Effect of Gracilaria vermiculophylla Macroalga on Non-Alcoholic Fatty Liver Disease in Obese Rats.

Marine algae are valuable sources of bioactive compounds that have the potential to be used in the management of various pathologies. Despite the increasing prevalence of NAFLD, the absence of an approved effective pharmacological treatment with demonstrable effectiveness persists. In this context, the aim of the present study is to assess the effect of Gracilaria vermiculophylla red seaweed dietary supplementation on hepatic lipid accumulation, as well as on oxidative stress, inflammation and fibrosis- related markers on obese fa/fa Zucker rats fed with a standard diet, supplemented or not with 2.5% or 5% dehydrated Gracilaria vermiculophylla. After a six-week supplementation with the macroalga, no significant reduction in hepatic total lipid content or hepatic triglyceride content was observed. However, both doses were able to diminish hepatic NEFA concentration by reducing de novo lipogenesis and increasing mitochondrial biogenesis. Moreover, supplementation with the dose of 2.5% improved some oxidative stress and inflammation-related markers. Supplementation with the dose of 5% did not exert these clear beneficial effects. Thus, this study demonstrates that while Gracilaria vermiculophylla may not mitigate hepatic steatosis, it could exert protective effects on the liver by reducing NEFA content and enhancing oxidative stress and inflammation parameters.

Spatial metabolomics and its application in the liver.

Hepatocytes work in highly structured, repetitive hepatic lobules. Blood flow across the radial axis of the lobule generates oxygen, nutrient, and hormone gradients, which result in zoned spatial variability and functional diversity. This large heterogeneity suggests that hepatocytes in different lobule zones may have distinct gene expression profiles, metabolic features, regenerative capacity, and susceptibility to damage. Here, we describe the principles of liver zonation, introduce metabolomic approaches to study the spatial heterogeneity of the liver, and highlight the possibility of exploring the spatial metabolic profile, leading to a deeper understanding of the tissue metabolic organization. Spatial metabolomics can also reveal intercellular heterogeneity and its contribution to liver disease. These approaches facilitate the global characterization of liver metabolic function with high spatial resolution along physiological and pathological time scales. This review summarizes the state of the art for spatially resolved metabolomic analysis and the challenges that hinder the achievement of metabolome coverage at the single-cell level. We also discuss several major contributions to the understanding of liver spatial metabolism and conclude with our opinion on the future developments and applications of these exciting new technologies.

Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma.

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-ß) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-ß-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-ß. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-ß, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-ß-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-ß in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-ß1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-ß that may contribute to tumor progression, we found that NOX4 is also required for TGF-ß-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-ß-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-ß-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-ß signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-ß pathway.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Magnesium and Liver Metabolism Through the Lifespan.

Within the organism, the liver is the main organ responsible for metabolic homeostasis and xenobiotic transformation. To maintain an adequate liver weight-to-bodyweight ratio, this organ has an extraordinary regenerative capacity and is able to respond to an acute insult or partial hepatectomy. Maintenance of hepatic homeostasis is crucial for the proper functioning of the liver, and in this context, adequate nutrition with macro- and micronutrient intake is mandatory. Among all known macro-minerals, magnesium has a key role in energy metabolism and in metabolic and signaling pathways that maintain liver function and physiology throughout its life span. In the present review, the cation is reported as a potential key molecule during embryogenesis, liver regeneration, and aging. The exact role of the cation during liver formation and regeneration is not fully understood due to its unclear role in the activation and inhibition of those processes, and further research in a developmental context is needed. As individuals age, they may develop hypomagnesemia, a condition that aggravates the characteristic alterations. Additionally, risk of developing liver pathologies increases with age, and hypomagnesemia may be a contributing factor. Therefore, magnesium loss must be prevented by adequate intake of magnesium-rich foods such as seeds, nuts, spinach, or rice to prevent age-related hepatic alterations and contribute to the maintenance of hepatic homeostasis. Since magnesium-rich sources include a variety of foods, a varied and balanced diet can meet both macronutrient and micronutrient needs.

Nicotinamide N-methyltransferase (NNMT) regulates the glucocorticoid signaling pathway during the early phase of adipogenesis.

Obesity is associated with adipose tissue dysfunction through the differentiation and expansion of pre-adipocytes to adipocytes (hyperplasia) and/or increases in size of pre-existing adipocytes (hypertrophy). A cascade of transcriptional events coordinates the differentiation of pre-adipocytes into fully differentiated adipocytes; the process of adipogenesis. Although nicotinamide N-methyltransferase (NNMT) has been associated with obesity, how NNMT is regulated during adipogenesis, and the underlying regulatory mechanisms, remain undefined. In present study we used genetic and pharmacological approaches to elucidate the molecular signals driving NNMT activation and its role during adipogenesis. Firstly, we demonstrated that during the early phase of adipocyte differentiation NNMT is transactivated by CCAAT/Enhancer Binding Protein beta (CEBPB) in response to glucocorticoid (GC) induction. We found that Nnmt knockout, using CRISPR/Cas9 approach, impaired terminal adipogenesis by influencing the timing of cellular commitment and cell cycle exit during mitotic clonal expansion, as demonstrated by cell cycle analysis and RNA sequencing experiments. Biochemical and computational methods showed that a novel small molecule, called CC-410, stably binds to and highly specifically inhibits NNMT. CC-410 was, therefore, used to modulate protein activity during pre-adipocyte differentiation stages, demonstrating that, in line with the genetic approach, chemical inhibition of NNMT at the early stages of adipogenesis impairs terminal differentiation by deregulating the GC network. These congruent results conclusively demonstrate that NNMT is a key component of the GC-CEBP axis during the early stages of adipogenesis and could be a potential therapeutic target for both early-onset obesity and glucocorticoid-induced obesity.

Research Priorities for Precision Medicine in NAFLD.

NAFLD is a multisystem condition and the leading cause of chronic liver disease globally. There are no approved NAFLD-specific dugs. To advance in the prevention and treatment of NAFLD, there is a clear need to better understand the pathophysiology and genetic and environmental risk factors, identify subphenotypes, and develop personalized and precision medicine. In this review, we discuss the main NAFLD research priorities, with a particular focus on socioeconomic factors, interindividual variations, limitations of current NAFLD clinical trials, multidisciplinary models of care, and novel approaches in the management of patients with NAFLD.

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver.

The deficiency of CITRIN, the liver mitochondrial aspartate-glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(-/-) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (-/-) mice expressing liver specific transgenic aralar had a small (~ 4-6 nmoles x mg prot-1 x min-1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(-/-) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(-/-) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Structural basis of the inhibition of cystathionine γ-lyase from Toxoplasma gondii by propargylglycine and cysteine.

Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2 S). Importantly, inhibition of the enzyme and consequently of its H2 S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CßS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme-substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL-N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor d,l-propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed.

Impaired Function of Solute Carrier Family 19 Leads to Low Folate Levels and Lipid Droplet Accumulation in Hepatocytes.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Isolation of the Hepatic Ubiquitome/NEDDylome by Streptavidin Pull-Down Assay in the Biotinylated Ubiquitin (bioUb)/Biotinylated NEDD8 (bioNEDD8) Transgenic Mice.

In the last years, our group and others have uncovered the role of ubiquitin (Ub) and ubiquitin-like proteins such as the neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-mediated modifications in several types of liver disease, including nonalcoholic fatty liver disease, liver fibrosis, and hepatocellular carcinoma. For this purpose, we have taken advantage of biotinylated ubiquitin (bioUb) and biotinylated NEDD8 (bioNEDD8) mice, transgenic mouse models in which ubiquitin and NEDD8, respectively, are biotinylated in vivo. Using these genetic tools and pull-down assays that exploit the strong biotin-streptavidin interaction, denaturing lysis conditions, and stringent washing procedures, only proteins modified by Ub or NEDD8 are isolated from mammalian tissues in vivo. Here, we report a protocol of streptavidin pull-down of ubiquitinated and NEDDylated liver proteins using the bioUb and bioNEDD8 mice that can potentially be used to characterize both the hepatic ubiquitome and NEDDylome in different models of liver injury.