Salvatore Rizza

Glutamine Deprivation Enhances Antitumor Activity of 3-Bromopyruvate through the Stabilization of Monocarboxylate Transporter-1

Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherapy.

S-Nitrosoglutathione Reductase Deficiency-Induced S-Nitrosylation Results in Neuromuscular Dysfunction

AIMS Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. RESULTS We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. INNOVATION Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. CONCLUSION These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

Established Principles and Emerging Concepts on the Interplay between Mitochondrial Physiology and S-(De)nitrosylation: Implications in Cancer and Neurodegeneration

S-nitrosylation is a posttranslational modification of cysteine residues that has been frequently indicated as potential molecular mechanism governing cell response upon redox unbalance downstream of nitric oxide (over)production. In the last years, increased levels of S-nitrosothiols (SNOs) have been tightly associated with the onset of nitroxidative stress-based pathologies (e.g., cancer and neurodegeneration), conditions in which alterations of mitochondrial homeostasis and activation of cellular processes dependent on it have been reported as well. In this paper we aim at summarizing the current knowledge of mitochondria-related proteins undergoing S-nitrosylation and how this redox modification might impact on mitochondrial functions, whose impairment has been correlated to tumorigenesis and neuronal cell death. In particular, emphasis will be given to the possible, but still neglected implication of denitrosylation reactions in the modulation of mitochondrial SNOs and how they can affect mitochondrion-related cellular process, such as oxidative phosphorylation, mitochondrial dynamics, and mitophagy.

S-nitrosylation of the mitochondrial chaperone TRAP1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.

S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson’s Disease and Amyotrophic Lateral Sclerosis

Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based in vitro models of neurodegeneration, namely, Parkinsons disease (PD) and familial amyotrophic lateral sclerosis (fALS). In particular, we provide evidence that GSNOR-knocking down protects SH-SY5Y against PD toxins, while, by contrast, its upregulation is required for G93A-SOD1 expressing cells resistance to NO-releasing drugs. Although completely opposite, both conditions are characterized by Nrf2 localization in the nuclear compartment: in the first case induced by GSNOR silencing, while in the second one underlying the antinitrosative response. Overall, our results demonstrate that GSNOR expression has different effect on neuronal viability in dependence on the stimulus applied and suggest that GSNOR could be a responsive gene downstream of Nrf2 activation.

To eat, or NOt to eat: S-nitrosylation signaling in autophagy.

Autophagy is the main catabolic cellular process through which cells adapt their needs (e.g., growth and proliferation) to environmental availability of nutrients (e.g., amino acid and glucose) and growth factors. The rapid activation of the autophagy response essentially depends on protein post‐translational modifications (PTMs), which act as molecular switches triggering signaling cascades. Deregulation of autophagy contributes to pathological conditions, such as cancer and neurodegeneration. Therefore, understanding how PTMs affect the occurrence of autophagy is of the highest importance for clinical applications. Besides phosphorylation and ubiquitylation, which represent the best known examples of PTMs, redox‐based modifications are also emerging as contributing to the regulation of intracellular signaling. Of note, S‐nitrosylation of cysteine residues is a redox PTM and is the principal mechanism of nitric oxide‐based signaling. Results emerging in recent years suggest that NO has a role in modulating autophagy. However, the function of S‐nitrosylation in autophagy regulation remains still unveiled. By this review, we describe the upstream events regulating autophagy activation focusing on recently published evidence implying a S‐nitrosylation‐dependent regulation.

S-Nitrosation and Ubiquitin-Proteasome System Interplay in Neuromuscular Disorders

Protein S-nitrosation is deemed as a prototype of posttranslational modifications governing cell signaling. It takes place on specific cysteine residues that covalently incorporate a nitric oxide (NO) moiety to form S-nitrosothiol derivatives and depends on the ratio between NO produced by NO synthases and nitrosothiol removal catalyzed by denitrosating enzymes. A large number of cysteine-containing proteins are found to undergo S-nitrosation and, among them, the enzymes catalyzing ubiquitination, mainly the class of ubiquitin E3 ligases and the 20S component of the proteasome, have been reported to be redox modulated in their activity. In this review we will outline the processes regulating S-nitrosation and try to debate whether and how it affects protein ubiquitination and degradation via the proteasome. In particular, since muscle and neuronal health largely depends on the balance between protein synthesis and breakdown, here we will discuss the impact of S-nitrosation in the efficiency of protein quality control system, providing lines of evidence and speculating about its involvement in the onset and maintenance of neuromuscular dysfunctions.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy

Significance The free radical theory of aging remains controversial. Accumulation of mitochondrial damage is commonly accepted as an age-related phenomenon associated with the inescapable side effects of oxidative metabolism. However, to date, molecular determinants of this phenomenon have not been identified. Previous evidence indicates that engineered mice deficient in the denitrosylase S-nitrosoglutathione reductase (GSNOR) show features of aging. Here, we show that due to epigenetic events, GSNOR expression declines with age, ultimately resulting in the accumulation of damaged mitochondria. By contrast, centenarians maintain high GSNOR expression. Collectively, these data suggest that GSNOR may act as a longevity protein countering defects in mitochondrial physiology that arise from age-related epigenetic deregulation. S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

Chronicles of a reductase: Biochemistry, genetics and physio-pathological role of GSNOR

S-nitrosylation is a major redox posttranslational modification involved in cell signaling. The steady state concentration of S-nitrosylated proteins depends on the balance between the relative ability to generate nitric oxide (NO) via NO synthase and to reduce nitrosothiols by denitrosylases. Numerous works have been published in last decades regarding the role of NO and S-nitrosylation in the regulation of protein structure and function, and in driving cellular activities in vertebrates. Notwithstanding an increasing number of observations indicates that impairment of denitrosylation equally affects cellular homeostasis, there is still no report providing comprehensive knowledge on the impact that denitrosylation has on maintaining correct physiological processes and organ activities. Among denitrosylases, S-nitrosoglutathione reductase (GSNOR) represents the prototype enzyme to disclose how denitrosylation plays a crucial role in tuning NO-bioactivity and how much it deeply impacts on cell homeostasis and human patho-physiology. In this review we attempt to illustrate the history of GSNOR discovery and provide the evidence so far reported in support of GSNOR implications in development and human disease.

Tumor Suppressor Roles of the Denitrosylase GSNOR

Nitric oxide (NO) is a gaseous pleiotropic molecule that can both induce irreversible oxidative damages and modulate physiological signal transductions by transient protein modifications, the most important of which is the S-nitrosylation of cysteine residues. Being noxious and healthy, the role of NO in cancer is seemingly contradictory, as at low concentrations it mediates tumor growth and proliferation whereas at high concentrations it promotes apoptosis and cancer growth inhibition. However, it is becoming evident that when endogenously produced, such as upon inducible nitric oxide synthase (NOS) activation, NO acts to sustain tumorigenesis. Similarly, although less explored, defects and deficiency in the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) have been associated with the development and malignancy of liver and breast cancers, suggesting a primary role for NO signaling-that is, S-nitrosylation, being deeply involved in neoplastic transformation and progression. In this review, we summarize past and recent evidence on the role of S-nitrosylation and GSNOR in different processes that contribute to cell transformation when deregulated, such as DNA damage repair, energetic metabolism, and cell death. We also outline possible S-nitrosylation-targeted proteins that might contribute to tumorigenesis, and, finally, we speculate on the role of GSNOR in regulating the oncogenic effects induced downstream.