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SDH-generated superoxide is the upstream mediator of α-TOS-induced cell death in siGSNOR HepG2 cells.

PDF file - 77K, Glutamine-deprivation up-regulates MCT-1 levels

Supplementary figures legends

WB densitometry and statistical analyses

PDF file - 98K, Complex II inhibition is responsible for 3-BrPA-induced metabolic-oxidative stress upon glutamine deprivation

PDF file - 180K, MCT-1 up-regulation induced by glutamine deprivation is responsible for 3-BrPA chemo-potentiation

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. Cancer Res; 72(17); 4526–36. ©2012 AACR.

PDF file - 211K

PDF file - 147K, Autophagy contributes to the execution of 3-BrPA-induced cell death upon glutamine withdrawal

PDF file - 154K, Glutamine deprivation-induced 3-BrPA chemo-potentiation does not depend on synergistic impairment of glutaminolysis and glycolysis

Supplementary materials and methods

PDF file - 127K, Chemo-potentiation of 3-BrPA cytotoxicity is not associated with the activation of apoptosis

PDF file - 55K, Increased oxidative stress underlies 3-BrPA chemo-potentiation induced by glutamine-deprivation

Angiogenesis is the process of blood vessel growth. The angiogenic switch consists of new blood vessel formation that, in carcinogenesis, can lead to the transition from a harmless cluster of dormant cells to a large tumorigenic mass with metastatic potential. Hypoxia, i.e. the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating hypoxia-inducible factor-1 (HIF-1), a transcription factor triggering de novo angiogenesis. HIF-1 and the angiogenic molecules that are expressed upon its activation are modulated by redox status. Modulations of the redox environment can influence the angiogenesis signaling at different levels, thereby impinging on the angiogenic switch. This review provides a molecular overview of the redox-sensitive steps in angiogenic signaling, the main molecular players involved and their crosstalk with the unfolded protein response (UPR). New classes of inhibitors of these modulators which might act as antiangiogenic drugs in cancer are also discussed.

S-nitrosylation of proteins is a nitric oxide (NO)-based post-translational modification of cysteine residues. By removing the NO moiety from S-nitrosothiol adducts, denitrosylases restore sulfhydryl protein pool and act as downstream tuners of S-nitrosylation signaling. Alterations in the S-nitrosylation/denitrosylation dynamics are implicated in many pathological states, including cancer ontogenesis and progression, skeletal muscle myogenesis and function. Here, we aim to provide and link different lines of evidence, and elaborate on the possible role of S-nitrosylation/denitrosylation signaling in rhabdomyosarcoma, one of the most common pediatric mesenchymal malignancy.

S-nitrosylation, the post-translational modification of cysteines by nitric oxide, has been implicated in several cellular processes and tissue homeostasis. As a result, alterations in the mechanisms controlling the levels of S-nitrosylated proteins have been found in pathological states. In the last few years, a role in cancer has been proposed, supported by the evidence that various oncoproteins undergo gain- or loss-of-function modifications upon S-nitrosylation. Here, we aim at providing insight into the current knowledge about the role of S-nitrosylation in different aspects of cancer biology and report the main anticancer strategies based on: (i) reducing S-nitrosylation-mediated oncogenic effects, (ii) boosting S-nitrosylation to stimulate cell death, (iii) exploiting S-nitrosylation through synthetic lethality.

Significance: Cysteines have an essential role in redox signaling, transforming an oxidant signal into a biological response. Among reversible cysteine post-translational modifications, S-nitrosylation acts as a redox-switch in several pathophysiological states, such as ischemia/reperfusion, synaptic transmission, cancer, and muscular dysfunctions. Recent Advances: Growing pieces of in vitro and in vivo evidence argue for S-nitrosylation being deeply involved in development and aging, and playing a role in the onset of different pathological states. New findings suggest it being an enzymatically regulated cellular process, with deep impact on mitochondrial structure and function, and in cellular metabolism. In light of this, the recent discovery of the denitrosylase S-nitrosoCoA (coenzyme A) reductase takes on even greater importance and opens new perspectives on S-nitrosylation as a general mechanism of cellular homeostasis. Critical Issues: Based on these recent findings, we aim at summarizing and elaborating on the established and emerging crucial roles of S-nitrosylation in mitochondrial metabolism and mitophagy, and provide an overview of the pathophysiological effects induced by its deregulation. Future Directions: The identification of new S-nitrosylation targets, and the comprehension of the mechanisms through which S-nitrosylation modulates specific classes of proteins, that is, those impinging on diverse mitochondrial functions, may help to better understand the pathophysiology of aging, and propose lines of intervention to slow down or extend the onset of aging-related diseases.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT. This work was partially supported by the European Foundation for the Study of Diabetes (EFSD/Lilly, 2017 and EFSD/Boehringer Ingelheim European Research Programme on ‘‘Multi-System Challenges in Diabetes’’) and the Italian Ministry of Health (GR-2018-12367588) to D.L.-B.; Associazione Italiana per la Ricerca sul Cancro (AIRC) under IG 2019 - ID. 23562 project to K.A.; MIUR ‘‘Progetto Eccellenza’’ to Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, and NUTRAGE (CNR FOE 2019, DSB.AD004.271) to A.S; Italian Foundation of Multiple Sclerosis (grant 2017/R/8), the Italian Ministry of Health (grant GR-2016-02362380) and the MAI Award grant to V. Chiurchiu; National Institutes of Health (NIH) common fund (DP5 OD028125) and the Burroughs Wellcome Fund (1019648) to J.R.B.; NIH K01DK125608 to F.S.; R01DK102898 and R01DK122808 to Y.- H.T.; and NIH RO1 DK121805 and AHA 19TPA34910079 to B.Z. A.H. was supported by RTI2018-095497-B-I00 from MICINN, HR17_00527 from La Caixa Foundation, and TNE-18CVD04 from the Leducq Foundation. M.S-D was supported by a fellowship PRE2019-08746 from the Ministerio de Ciencia e Innovacio´ n. M.R. was partially supported by a fellowship from AIRC (IG 2019 - ID. 23562) and by the Italian Ministry of Health (SG-2019-12368589). V.C. is part of the PhD Program in Evolutionary Biology and Ecology, Department of Biology, University of Rome Tor Vergata. Sí

Nitric oxide (NO) is an essential signaling molecule in the regulation of multiple cellular processes. It is endogenously synthesized by NO synthase (NOS) as the product of L-arginine oxidation to L-citrulline, requiring NADPH, molecular oxygen, and a pterin cofactor. Two NOS isoforms are constitutively present in cells, nNOS and eNOS, and a third is inducible (iNOS). Despite their biological relevance, the details of their complex structural features and reactivity mechanisms are still unclear. In this review, we summarized the contribution of computational biochemistry to research on NOS molecular mechanisms. We described in detail its use in studying aspects of structure, dynamics and reactivity. We also focus on the numerous outstanding questions in the field that could benefit from more extensive computational investigations., Graphical Abstract Unlabelled Image

Background: Hyperbaric exposure (HE) is proven to be a stressor to several mechanisms in living cells. Even if after homeostasis restoration, harmful effects are expected, in particular a presence of free radicals. These latter are the stimulus to negative phenomenon as inflammation or cancer. In Italy, with 7500 km of sea shores, a large quantity of workers is exposed to HE during occupational activities. A deep knowledge of HE and bodily effects is not well defined; hence a multidisciplinary assessment of risk is needed. To detect one or more indicators of HE a research group is organised, under the INAIL sponsorship. The research project focused on the oxidative stress (OxS) and this paper details on the possible protocol to estimate, with a large amount of techniques on several human liquids, the relationship between OxS and HE. Specific attention will be paid to identify confounding factors and their influence. Methods: Blood and urine will be sampled. Several lab techniques will be performed on samples, both targeted, to measure the level of well-known biomarkers, and untargeted. Regard the formers: products of oxidation of DNA and RNA in urine; inflammation and temperature cytokines and protein carbonyles in blood. Untargeted evaluation will be performed for a metabolomics analysis in urine. Confounding factors: temperature, body fat, fitness, allergies and dietary habits. These factors will be assessed, directly or indirectly, prior and after HE. The final scope of the project is to determine one or more indicators that relates to HE in hits twofold nature: depth and duration. Conclusion: The relationship between OxS and HE is not deeply investigated and literature proposes diverging results. The project aims to define the time dependence of biomarkers related to OxS, to rise knowledge in risk assessment in workers exposed to HE.

As mitochondria are vulnerable to oxidative damage and represent the main source of reactive oxygen species (ROS), they are considered key tuners of ROS metabolism and buffering, whose dysfunction can progressively impact neuronal networks and disease. Defects in DNA repair and DNA damage response (DDR) may also affect neuronal health and lead to neuropathology. A number of congenital DNA repair and DDR defective syndromes, indeed, show neurological phenotypes, and a growing body of evidence indicate that defects in the mechanisms that control genome stability in neurons acts as aging-related modifiers of common neurodegenerative diseases such as Alzheimer, Parkinson's, Huntington diseases and Amyotrophic Lateral Sclerosis. In this review we elaborate on the established principles and recent concepts supporting the hypothesis that deficiencies in either DNA repair or DDR might contribute to neurodegeneration via mechanisms involving mitochondrial dysfunction/deranged metabolism.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel—the MYC pathway and the cyclin D–cyclin-dependent kinase (CDK)–retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D–CDK–RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1–cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis. AMBRA1-mediated degradation of cyclin D through CRL4–DDB1 regulates cell proliferation and prevents replication stress in neurodevelopment and cancer.

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., Hydrogen peroxide and pro‐oxidant conditions activate ATM via oxidation of Cys2991. The resulting phospho‐signal activates CHK2 and p53 and culminates in enhanced translation of the denitrosylase GSNOR to sustain mitophagy and protect the cells against oxidative stress.

Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma. Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma.

Mitophagy is a selective process aimed at removing damaged or burned-out mitochondria; it is activated upon different stimuli and plays a fundamental role in preventing overproduction of reactive oxygen species (ROS) that might be generated by dysfunctional mitochondria. From this angle, mitophagy can be considered a fully-fledged antioxidant process. Such a surrogate antioxidant function is recently emerging, being shared among many molecular pathways and players that are usually not included among - and, formally, do not directly act as - antioxidants. ATM (ataxia telangiectasia mutated) is a prototype of this class of "neglected" antioxidants. In spite of its well-known role in DNA damage response, many phenotypes of ataxia telangiectasia (A-T) patients are, indeed, related to chronic oxidative stress, arguing for an additional antioxidant role of ATM. In a recent study, we discovered the mechanism through which ATM exerts antioxidant activity. In particular, we provided evidence that this involves ADH5/GSNOR (alcohol dehydrogenase 5 (class III), chi polypeptide), which, in turn, sustains mitophagy via PARK2 denitrosylation, and protects the cell from detrimental effects due to ROS.

Mitophagy is a selective process aimed at removing damaged or burned-out mitochondria; it is activated upon different stimuli and plays a fundamental role in preventing overproduction of reactive oxygen species (ROS) that might be generated by dysfunctional mitochondria. From this angle, mitophagy can be considered a fully-fledged antioxidant process. Such a surrogate antioxidant function is recently emerging, being shared among many molecular pathways and players that are usually not included among – and, formally, do not directly act as – antioxidants. ATM (ataxia telangiectasia mutated) is a prototype of this class of “neglected” antioxidants. In spite of its well-known role in DNA damage response, many phenotypes of ataxia telangiectasia (A-T) patients are, indeed, related to chronic oxidative stress, arguing for an additional antioxidant role of ATM. In a recent study, we discovered the mechanism through which ATM exerts antioxidant activity. In particular, we provided evidence that this involves ADH5/GSNOR (alcohol dehydrogenase 5 (class III), chi polypeptide), which, in turn, sustains mitophagy via PARK2 denitrosylation, and protects the cell from detrimental effects due to ROS.