TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation.

It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress.

Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress, and pathogen infection. In this study, we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility in worms. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.

Targeting EDEM protects against ER stress and improves development and survival in C. elegans.

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.

Sperm Redox System Equilibrium: Implications for Fertilization and Male Fertility.

Structural and regulatory requirements of mammalian spermatozoa in both development and function make them extremely unique cells. Looking at the complexity of spermatozoon structure and its requirements for both motility and quick breakdown within the post-fertilization environment, as well as its functional needs as an extremely streamlined cell with high energy requirements, demonstrate the high importance of oxidative-reductive processes. The oxidative state of the testis and epididymis during sperm development and maturation highly influences sperm structure, with a high dependence on disulfide bond formation, facilitated by thiol mediated processes. However, once functionally active, sperm transition to a new high-risk functional paradigm requiring low levels of reactive oxygen species (ROS) while also being highly susceptible to oxidative damage due to the high proportion of polyunsaturated fatty acids within the lipid bilayer of the plasmalemma and the lack of cytosolic antioxidant defenses. This chapter highlights how glutathione and thioredoxin systems mediate the oxidative environment of the male reproductive tract and facilitate the successful development, maturation and function of mammalian spermatozoa.

Reduction of mRNA export unmasks different tissue sensitivities to low mRNA levels during Caenorhabditis elegans development.

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes.

Biallelic Variants in UBA5 Reveal that Disruption of the UFM1 Cascade Can Result in Early-Onset Encephalopathy.

Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.

Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism.

In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.

The neuroprotective transcription factor ATF5 is decreased and sequestered into polyglutamine inclusions in Huntington's disease.

Activating transcription factor-5 (ATF5) is a stress-response transcription factor induced upon different cell stressors like fasting, amino-acid limitation, cadmium or arsenite. ATF5 is also induced, and promotes transcription of anti-apoptotic target genes like MCL1, during the unfolded protein response (UPR) triggered by endoplasmic reticulum stress. In the brain, high ATF5 levels are found in gliomas and also in neural progenitor cells, which need to decrease their ATF5 levels for differentiation into mature neurons or glia. This initially led to believe that ATF5 is not expressed in adult neurons. More recently, we reported basal neuronal ATF5 expression in adult mouse brain and its neuroprotective induction during UPR in a mouse model of status epilepticus. Here we aimed to explore whether ATF5 is also expressed by neurons in human brain both in basal conditions and in Huntington's disease (HD), where UPR has been described to be partially impaired due to defective ATF6 processing. Apart from confirming that ATF5 is present in human adult neurons, here we report accumulation of ATF5 within the characteristic polyglutamine-containing neuronal nuclear inclusions in brains of HD patients and mice. This correlates with decreased levels of soluble ATF5 and of its antiapoptotic target MCL1. We then confirmed the deleterious effect of ATF5 deficiency in a Caenorhabditis elegans model of polyglutamine-induced toxicity. Finally, ATF5 overexpression attenuated polyglutamine-induced apoptosis in a cell model of HD. These results reflect that decreased ATF5 in HD-probably secondary to sequestration into inclusions-renders neurons more vulnerable to mutant huntingtin-induced apoptosis and that ATF5-increasing interventions might have therapeutic potential for HD.

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage.

Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.

Selenoprotein TRXR-1 and GSR-1 are essential for removal of old cuticle during molting in Caenorhabditis elegans.

Selenoproteins, in particular thioredoxin reductase, have been implicated in countering oxidative damage occurring during aging but the molecular functions of these proteins have not been extensively investigated in different animal models. Here we demonstrate that TRXR-1 thioredoxin reductase, the sole selenoprotein in Caenorhabditis elegans, does not protect against acute oxidative stress but functions instead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting. We show that the oxidation state of disulfide groups in the cuticle is tightly regulated during the molting cycle, and that when trxr-1 and gsr-1 function is reduced, disulfide groups in the cuticle remain oxidized. A selenocysteine-to-cysteine TRXR-1 mutant fails to rescue molting defects. Furthermore, worms lacking SELB-1, the C. elegans homolog of Escherichia coli SelB or mammalian EFsec, a translation elongation factor known to be specific for selenocysteine in E. coli, fail to incorporate selenocysteine, and display the same phenotype as those lacking trxr-1. Thus, TRXR-1 function in the reduction of old cuticle is strictly selenocysteine dependent in the nematode. Exogenously supplied reduced glutathione reduces disulfide groups in the cuticle and induces apolysis, the separation of old and new cuticle, strongly suggesting that molting involves the regulated reduction of cuticle components driven by TRXR-1 and GSR-1. Using dauer larvae, we demonstrate that aged worms have a decreased capacity to molt, and decreased expression of GSR-1. Together, our results establish a function for the selenoprotein TRXR-1 and GSR-1 in the removal of old cuticle from the surface of epidermal cells.

Ferroptosis inhibition by oleic acid mitigates iron-overload-induced injury.

Iron overload, characterized by accumulation of iron in tissues, induces a multiorgan toxicity whose mechanisms are not fully understood. Using cultured cell lines, Caenorhabditis elegans, and mice, we found that ferroptosis occurs in the context of iron-overload-mediated damage. Exogenous oleic acid protected against iron-overload-toxicity in cell culture and Caenorhabditis elegans by suppressing ferroptosis. In mice, oleic acid protected against FAC-induced liver lipid peroxidation and damage. Oleic acid changed the cellular lipid composition, characterized by decreased levels of polyunsaturated fatty acyl phospholipids and decreased levels of ether-linked phospholipids. The protective effect of oleic acid in cells was attenuated by GW6471 (PPAR-α antagonist), as well as in Caenorhabditis elegans lacking the nuclear hormone receptor NHR-49 (a PPAR-α functional homologue). These results highlight ferroptosis as a driver of iron-overload-mediated damage, which is inhibited by oleic acid. This monounsaturated fatty acid represents a potential therapeutic approach to mitigating organ damage in iron overload individuals.

Peroxiredoxin 2 is required for the redox mediated adaptation to exercise.

Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle that promotes changes in gene transcription and mitochondrial biogenesis, required for the beneficial adaptive response. We demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive hormesis response to physiological levels of H2O2 in myoblasts and following exercise in C. elegans. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. Moreover, a swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of specific Cysteine residues revealed a more reduced redox state in the non-exercised prdx-2 mutant strain that become oxidized following exercise. In contrast, specific Cys residues from regulatory proteins become more reduced in the N2 strain following exercise, establishing the key regulatory role of PRDX-2 in a redox signalling cascade following endogenous ROS generation. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological redox stress.

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine.

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans.

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.

The human thioredoxin reductase-1 splice variant TXNRD1_v3 is an atypical inducer of cytoplasmic filaments and cell membrane filopodia.

Thioredoxin reductases are important selenoproteins maintaining cellular redox balance and regulating several redox dependent processes in apoptosis, cell proliferation and differentiation. Specific functions of dedicated splice variants may add further complexity to the functions of these proteins. We show here that a splice variant of human thioredoxin reductase 1, TXNRD1_v3, forms both dynamic cytoplasmic filaments and provokes instantaneous formation of dynamic cell membrane protrusions identified as filopodia. Using truncated versions of the protein we found that both the cytoplasmic filaments and the filopodia formation were exclusively dependent on the glutaredoxin domain of the protein. Interestingly, actin polymerization was required for filopodia formation triggered by TXNRD1_v3, but not for generation of cytoplasmic filaments. We conclude that the glutaredoxin domain of TXNRD1_v3 is an atypical regulator of the cell cytoskeleton that potently induces formation of highly ordered cytoplasmic filaments and cell membrane filopodia.

Cautionary note on the use of Caenorhabditis elegans to study muscle phenotypes caused by mutations in the human MYH7 gene.

Mutations in the human MYH7 gene, encoding a slow skeletal muscle/ß-cardiac myosin heavy chain, cause different types of myopathies. The nematode model Caenorhabditis elegans has frequently been employed to study the molecular and physiological consequences of MYH7 mutations in muscle function by introducing mutations into the unc-54 gene, the worm MYH7 ortholog. We report here that the C. elegans model is not appropriate for such studies if they involve expression of the UNC-54 protein (wild-type or fused to green fluorescent protein) above endogenous levels.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy.

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

The peroxisomal fatty acid transporter ABCD1/PMP-4 is required in the C. elegans hypodermis for axonal maintenance: A worm model for adrenoleukodystrophy.

Adrenoleukodystrophy is a neurometabolic disorder caused by a defective peroxisomal ABCD1 transporter of very long-chain fatty acids (VLCFAs). Its pathogenesis is incompletely understood. Here we characterize a nematode model of X-ALD with loss of the pmp-4 gene, the worm orthologue of ABCD1. These mutants recapitulate the hallmarks of X-ALD: i) VLCFAs accumulation and impaired mitochondrial redox homeostasis and ii) axonal damage coupled to locomotor dysfunction. Furthermore, we identify a novel role for PMP-4 in modulating lipid droplet dynamics. Importantly, we show that the mitochondria targeted antioxidant MitoQ normalizes lipid droplets size, and prevents axonal degeneration and locomotor disability, highlighting its therapeutic potential. Moreover, PMP-4 acting solely in the hypodermis rescues axonal and locomotion abnormalities, suggesting a myelin-like role for the hypodermis in providing essential peroxisomal functions for the nematode nervous system.

A Caenorhabditis elegans ortholog of human selenium-binding protein 1 is a pro-aging factor protecting against selenite toxicity.

Human selenium-binding protein 1 (SELENBP1) was originally identified as a protein binding selenium, most likely as selenite. SELENBP1 is associated with cellular redox and thiol homeostasis in several respects, including its established role as a methanethiol oxidase that is involved in degradation of methanethiol, a methionine catabolite, generating hydrogen sulfide (H2S) and hydrogen peroxide (H2O2). As both H2S and reactive oxygen species (such as H2O2) are major regulators of Caenorhabditis elegans lifespan and stress resistance, we hypothesized that a SELENBP1 ortholog in C. elegans would likely be involved in regulating these aspects. Here we characterize Y37A1B.5, a putative selenium-binding protein 1 ortholog in C. elegans with 52% primary structure identity to human SELENBP1. While conferring resistance to toxic concentrations of selenite, Y37A1B.5 also attenuates resistance to oxidative stress and lowers C. elegans lifespan: knockdown of Y37A1B.5 using RNA interference resulted in an approx. 10% increase of C. elegans lifespan and an enhanced resistance against the redox cycler paraquat, as well as enhanced motility. Analyses of transgenic reporter strains suggest hypodermal expression and cytoplasmic localization of Y37A1B.5, whose expression decreases with worm age. We identify the transcriptional coregulator MDT-15 and transcription factor EGL-27 as regulators of Y37A1B.5 levels and show that the lifespan extending effect elicited by downregulation of Y37A1B.5 is independent of known MDT-15 interacting factors, such as DAF-16 and NHR-49. In summary, Y37A1B.5 is an ortholog of SELENBP1 that shortens C. elegans lifespan and lowers resistance against oxidative stress, while allowing for a better survival under toxic selenite concentrations.

Downregulation of thioredoxin-1-dependent CD95 S-nitrosation by Sorafenib reduces liver cancer.

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.