Computational Structural Biology of S-nitrosylation of Cancer Targets

Nitric oxide (NO) plays an essential role in redox signaling in normal and pathological cellular conditions. In particular, it is well known to react in vivo with cysteines by the so-called S-nitrosylation reaction. S-nitrosylation is a selective and reversible post-translational modification that exerts a myriad of different effects, such as the modulation of protein conformation, activity, stability, and biological interaction networks. We have appreciated, over the last years, the role of S-nitrosylation in normal and disease conditions. In this context, structural and computational studies can help to dissect the complex and multifaceted role of this redox post-translational modification. In this review article, we summarized the current state-of-the-art on the mechanism of S-nitrosylation, along with the structural and computational studies that have helped to unveil its effects and biological roles. We also discussed the need to move new steps forward especially in the direction of employing computational structural biology to address the molecular and atomistic details of S-nitrosylation. Indeed, this redox modification has been so far an underappreciated redox post-translational modification by the computational biochemistry community. In our review, we primarily focus on S-nitrosylated proteins that are attractive cancer targets due to the emerging relevance of this redox modification in a cancer setting

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-Methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a β-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite

Identification of dexamethasone-induced metabolic vulnerabilities in glioblastoma

Glioblastoma represents the most aggressive and common high-grade (IV) malignant brain tumour in adults. Despite many efforts have been done in order to ameliorate the clinical outcome of this disease, the standard of care has not substantially changed in the last 20 years, and is based on surgical resection followed by radiotherapy and chemotherapy with the alkylating agent temozolomide. Nonetheless, the aggressiveness and recurrence of this tumour make the prognosis still very poor and the median survival for glioblastoma patients is 12-18 months from the time of diagnosis. Since 1960’s, patients presenting with clinical symptoms associated with glioblastoma are invariably treated with dexamethasone, a potent anti-inflammatory drug that reduces the peritumoral oedema, and alleviates the neurological symptoms caused by the increased intracranial pressure. Given the clinical relevance of dexamethasone for the management of glioblastoma patients, several studies have been performed to understand the effects of dexamethasone on patients’ survival. A retrospective clinical analysis of three independent glioma patient cohorts found that steroids treatment associates with shorter survival. As indicated by the name, glucocorticoids regulate glucose metabolism. Dexamethasone increases glycaemia, and this has been linked to a shorter survival of glioblastoma patients. Nevertheless, because of its effectiveness, affordability and accessibility, dexamethasone will remain a mainstay of glioblastoma patients’ therapy. On these bases, we employed naïve patient-derived cell lines that have been demonstrated to retain the pathophysiological features of the tumour in the patients to identify the metabolic effects of dexamethasone on glioblastoma cells and characterize specific drug-induced vulnerabilities. In particular, we studied the metabolic reactions altered by dexamethasone treatment with the aim to exploit them as therapeutic targets. Therefore, we cultured the cells in serumfree physiological media (Plasmax) to retain the stem cells subpopulation and expose them to the same metabolites concentrations found in human plasma. We found that the glucocorticoid receptor, the dexamethasone main target, was expressed in all cell lines, and translocated to the nucleus upon dexamethasone treatment. While the effects of dexamethasone on proliferation were cell linedependent, we profiled a dexamethasone-dependent transcriptional signature common to all cell lines. Orthogonal transcriptomic and metabolomics analyses identified nicotinamide N-methyltransferase, NNMT, to be transcriptionally and functionally upregulated by dexamethasone. NNMT transfers methyl groups from S-adenosyl-methionine (SAM) to nicotinamide, producing N1-methylnicotinamide. Dexamethasone-mediated NNMT over-activation caused a shortage in SAM and an increase in N1-methylnicotinamide levels in all cell lines. Unexpectedly, dexamethasone treatment did not sensitize glioblastoma cells to sub-physiological and growth-limiting concentrations of methionine. We validated these findings in 10 naïve human glioblastoma cell lines and in glioblastoma tumours orthotopically implanted in immunocompromised mice. In vivo, dexamethasone decreased the methionine levels in tumour and contralateral brain tissue without altering its circulating levels. Dexamethasone administration significantly decreased tumour volume assessed by MRI and proliferation index. Notably, the levels of NNMT and N1-methylnicotinamide were markedly higher in tumour tissue compared to contralateral normal brain, and these differences were amplified by dexamethasone treatment. Moreover, we demonstrated that the activity of NNMT is increased in tumour tissues derived from glioblastoma patients, compared to adjacent oedematous brain tissue. These results suggest that NNMT activity could be targeted for the development of a novel PET tracer for the visualization of invasive tumours, aiding the diagnosis and the response to therapy of glioblastoma patients. Moreover, these tumour-specific dexamethasone-induced metabolic alterations may lead to a rationally designed therapeutic plan for glioblastomas with heterogeneous mutational status

TRAP1 and S-nitrosylation - molecular dynamics simulations

Collection of simulations used in the publication "S-nitrosylation affects TRAP1 structure and ATPase activity and contributes to mitochondrial homeostasis". The script and input files can be found in the associated Github repository of our group: https://github.com/ELELAB/TRAP1_activit

Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted IDH1-mutant astrocytomas.

peer reviewedBACKGROUND: Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance. METHODS: Proteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice. RESULTS: We find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice. CONCLUSIONS: We show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited

Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis

Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation