Ultrasmall Manganese Ferrites for In Vivo Catalase Mimicking Activity and Multimodal Bioimaging.

Manganese ferrite nanoparticles display interesting features in bioimaging and catalytic therapies. They have been recently used in theranostics as contrast agents in magnetic resonance imaging (MRI), and as catalase-mimicking nanozymes for hypoxia alleviation. These promising applications encourage the development of novel synthetic procedures to enhance the bioimaging and catalytic properties of these nanomaterials simultaneously. Herein, a cost-efficient synthetic microwave method is developed to manufacture ultrasmall manganese ferrite nanoparticles as advanced multimodal contrast agents in MRI and positron emission tomography (PET), and improved nanozymes. Such a synthetic method allows doping ferrites with Mn in a wide stoichiometric range (Mnx Fe3-x O4 , 0.1 ≤ x ≤ 2.4), affording a library of nanoparticles with different magnetic relaxivities and catalytic properties. These tuned magnetic properties give rise to either positive or dual-mode MRI contrast agents. On the other hand, higher levels of Mn doping enhance the catalytic efficiency of the resulting nanozymes. Finally, through their intracellular catalase-mimicking activity, these ultrasmall manganese ferrite nanoparticles induce an unprecedented tumor growth inhibition in a breast cancer murine model. All of these results show the robust characteristics of these nanoparticles for nanobiotechnological applications.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies.

The APPswe/PS1A246E mutations in an astrocytic cell line leads to increased vulnerability to oxygen and glucose deprivation, Ca2+ dysregulation, and mitochondrial abnormalities.

Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APPSWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APPSWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca2+ concentration ([Ca2+ ]c ) were potentiated in the APPSWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APPSWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders.

Reactive oxygen species, nutrition, hypoxia and diseases: Problems solved?

Within the last twenty years the view on reactive oxygen species (ROS) has changed; they are no longer only considered to be harmful but also necessary for cellular communication and homeostasis in different organisms ranging from bacteria to mammals. In the latter, ROS were shown to modulate diverse physiological processes including the regulation of growth factor signaling, the hypoxic response, inflammation and the immune response. During the last 60-100 years the life style, at least in the Western world, has changed enormously. This became obvious with an increase in caloric intake, decreased energy expenditure as well as the appearance of alcoholism and smoking; These changes were shown to contribute to generation of ROS which are, at least in part, associated with the occurrence of several chronic diseases like adiposity, atherosclerosis, type II diabetes, and cancer. In this review we discuss aspects and problems on the role of intracellular ROS formation and nutrition with the link to diseases and their problematic therapeutical issues.

Nitrosothiols in the immune system: signaling and protection.

SIGNIFICANCE: In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling. RECENT ADVANCES: Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function. CRITICAL ISSUES: Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward. FUTURE DIRECTIONS: Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapies.

Specificity in S-nitrosylation: a short-range mechanism for NO signaling?

SIGNIFICANCE: Nitric oxide (NO) classical and less classical signaling mechanisms (through interaction with soluble guanylate cyclase and cytochrome c oxidase, respectively) operate through direct binding of NO to protein metal centers, and rely on diffusibility of the NO molecule. S-Nitrosylation, a covalent post-translational modification of protein cysteines, has emerged as a paradigm of nonclassical NO signaling. RECENT ADVANCES: Several nonenzymatic mechanisms for S-nitrosylation formation and destruction have been described. Enzymatic mechanisms for transnitrosylation and denitrosylation have been also studied as regulators of the modification of specific subsets of proteins. The advancement of modification-specific proteomic methodologies has allowed progress in the study of diverse S-nitrosoproteomes, raising clues and questions about the parameters for determining the protein specificity of the modification. CRITICAL ISSUES: We propose that S-nitrosylation is mainly a short-range mechanism of NO signaling, exerted in a relatively limited range of action around the NO sources, and tightly related to the very controlled regulation of subcellular localization of nitric oxide synthases. We review the nonenzymatic and enzymatic mechanisms that support this concept, as well as physiological examples of mammalian systems that illustrate well the precise compartmentalization of S-nitrosylation. FUTURE DIRECTIONS: Individual and proteomic studies of protein S-nitrosylation-based signaling should take into account the subcellular localization in order to gain further insight into the functional role of this modification in (patho)physiological settings.

Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.

Adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by hypoxia-inducible factors (HIFs). There is abundant evidence that production of reactive oxygen species (ROS) increases during hypoxia, which contributes to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS can transduce signals by reversibly modifying the redox state of cysteine residues in certain proteins. Using the "redox fluorescence switch" (RFS), a thiol redox proteomic technique that fluorescently labels reversibly oxidized cysteines, we analyzed endothelial cells subjected to acute hypoxia and subsequent reoxygenation. We observed a general increase in cysteine oxidation during hypoxia, which was reversed by reoxygenation, and two-dimensional electrophoresis revealed the differential oxidation of specific proteins. Using complementary derivatization techniques, we confirmed the modification of individual target proteins and identified specific cysteine residues that were oxidized in hypoxic conditions, thereby overcoming several limitations associated with fluorescence derivatization. These findings provide an important basis for future studies of the role of these modifications in HIF activation and in other acute adaptive responses to hypoxia.