Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

Hydropersulfides (RSSH) attenuate doxorubicin-induced cardiotoxicity while boosting its anticancer action.

Cardiotoxicity is a frequent and often lethal complication of doxorubicin (DOX)-based chemotherapy. Here, we report that hydropersulfides (RSSH) are the most effective reactive sulfur species in conferring protection against DOX-induced toxicity in H9c2 cardiac cells. Mechanistically, RSSH supplementation alleviates the DOX-evoked surge in reactive oxygen species (ROS), activating nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways, thus boosting endogenous antioxidant defenses. Simultaneously, RSSH turns on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of mitochondrial function, while decreasing caspase-3 activity to inhibit apoptosis. Of note, we find that RSSH potentiate anticancer DOX effects in three different cancer cell lines, with evidence that suggests this occurs via induction of reductive stress. Indeed, cancer cells already exhibit much higher basal hydrogen sulfide (H2S), sulfane sulfur, and reducing equivalents compared to cardiac cells. Thus, RSSH may represent a new promising avenue to fend off DOX-induced cardiotoxicity while boosting its anticancer effects.

The reaction of hydropersulfides (RSSH) with S-nitrosothiols (RS-NO) and the biological/physiological implications.

S-Nitrosothiol (RS-NO) generation/levels have been implicated as being important to numerous physiological and pathophysiological processes. As such, the mechanism(s) of their generation and degradation are important factors in determining their biological activity. Along with the effects on the activity of thiol proteins, RS-NOs have also been reported to be reservoirs or storage forms of nitric oxide (NO). That is, it is hypothesized that NO can be released from RS-NO at opportune times to, for example, regulate vascular tone. However, to date there are few established mechanisms that can account for facile NO release from RS-NO. Recent discovery of the biological formation and prevalence of hydropersulfides (RSSH) and their subsequent reaction with RS-NO species provides a possible route for NO release from RS-NO. Herein, it is found that RSSH is capable of reacting with RS-NO to liberate NO and that the analogous reaction using RSH is not nearly as proficient in generating NO. Moreover, computational results support the prevalence of this reaction over other possible competing processes. Finally, results of biological studies of NO-mediated vasorelaxation are consistent with the idea that RS-NO species can be degraded by RSSH to release NO.

Nitric Oxide and Cancer: When to Give and When to Take Away?

The mechanistic roles of nitric oxide (NO) during cancer progression have been important considerations since its discovery as an endogenously generated free radical. Nonetheless, the impacts of this signaling molecule can be seemingly contradictory, being both pro-and antitumorigenic, which complicates the development of cancer treatments based on the modulation of NO fluxes in tumors. At a fundamental level, low levels of NO drive oncogenic pathways, immunosuppression, metastasis, and angiogenesis, while higher levels lead to apoptosis and reduced hypoxia and also sensitize tumors to conventional therapies. However, clinical outcome depends on the type and stage of the tumor as well as the tumor microenvironment. In this Viewpoint, the current understanding of the concentration, spatial, and temporal dependence of responses to NO is correlated with potential treatment and prevention technologies and strategies.

Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

Nitric Oxide in Macrophage Immunometabolism: Hiding in Plain Sight.

Nitric Oxide (NO) is a soluble endogenous gas with various biological functions like signaling, and working as an effector molecule or metabolic regulator. In response to inflammatory signals, immune myeloid cells, like macrophages, increase production of cytokines and NO, which is important for pathogen killing. Under these proinflammatory circumstances, called "M1", macrophages undergo a series of metabolic changes including rewiring of their tricarboxylic acid (TCA) cycle. Here, we review findings indicating that NO, through its interaction with heme and non-heme metal containing proteins, together with components of the electron transport chain, functions not only as a regulator of cell respiration, but also a modulator of intracellular cell metabolism. Moreover, diverse effects of NO and NO-derived reactive nitrogen species (RNS) involve precise interactions with different targets depending on concentration, temporal, and spatial restrictions. Although the role of NO in macrophage reprogramming has been in evidence for some time, current models have largely minimized its importance. It has, therefore, been hiding in plain sight. A review of the chemical properties of NO, past biochemical studies, and recent publications, necessitates that mechanisms of macrophage TCA reprogramming during stimulation must be re-imagined and re-interpreted as mechanistic results of NO exposure. The revised model of metabolic rewiring we describe here incorporates many early findings regarding NO biochemistry and brings NO out of hiding and to the forefront of macrophages immunometabolism.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.