Luiz C. Godoy

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Nitric oxide activation of Keap1/Nrf2 signaling in human colon carcinoma cells

The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (•NO)-induced Nrf2–Kelch-like ECH-associated protein 1 (Keap1) signaling and its role in counteracting •NO-induced apoptosis of human colon cancer HCT116 cells. Nrf2 was localized in the cytoplasm in control cells; •NO triggered its rapid nuclear accumulation, transcriptional activation, and up-regulation of HO-1, NQO1, and GCL, but not GST A4 and P1 subunits. Nrf2 accumulation in the nucleus was also associated with enhanced transcription and posttranscriptional modifications. (S)-nitrosation of Keap1 may contribute to nuclear accumulation of Nrf2 by facilitating its dissociation from Keap1, thus initiating •NO-mediated Nrf2–Keap1 signaling. •NO-mediated induction of ARE-dependent genes occurred well before apoptosis, as judged by caspase 3 activation. Collectively, these results show that the Nrf2–Keap1 signaling pathway mediates protective cellular responses to mitigate •NO-induced damage and may contribute to the relative resistance of HCT116 to •NO-induced cytotoxicity.

B-1 cells facilitate Paracoccidioides brasiliensis infection in mice via IL-10 secretion.

Protective immunity in paracoccidioidomycosis is mainly mediated by cellular immunity. The role of B cells in this disease, in particular B-1 cells, is poorly understood. The aim of this study was to characterize the participation of B-1 cells in resistance or susceptibility of BALB/c and BALB/Xid mice to P. brasiliensis (Pb) pulmonary infection. BALB/Xid, which lacks B-1 cells, exhibited higher resistance to infection when compared with BALB/c mice. However, adoptive transfer of B-1 cells to BALB/Xid mice drastically increased the susceptibility of these animals to Pb infection. The fungal burden in BALB/c and B-1-reconstituted BALB/Xid was significantly higher as compared to BALB/Xid strain. Compact, well-organized granulomas were observed in the lungs of BALB/Xid mice, whereas large lesions with necrotic center with a plethora of fungi developed in BALB/c mice. It was also shown that B-1 cells impair phagocytosis of Pb by macrophages in vitro via secretion of IL-10, which was increased upon stimulation with a purified Pb antigen, gp43. Finally, in vivo blockade of IL-10 led to a better control of infection by the highly susceptible B10.A mouse. These findings suggest that B-1 cells play a major role in resistance/susceptibility to Pb infection in murine models, most likely via production of IL-10.

Mechanism-Based Triarylphosphine-Ester Probes for Capture of Endogenous RSNOs

Nitrosothiols (RSNOs) have been proposed as important intermediates in nitric oxide (NO•) metabolism, storage, and transport as well as mediators in numerous NO-signaling pathways. RSNO levels are finely regulated, and dysregulation is associated with the etiology of several pathologies. Current methods for RSNO quantification depend on indirect assays that limit their overall specificity and reliability. Recent developments of phosphine-based chemical probes constitute a promising approach for the direct detection of RSNOs. We report here results from a detailed mechanistic and kinetic study for trapping RSNOs by three distinct phosphine probes, including structural identification of novel intermediates and stability studies under physiological conditions. We further show that a triarylphosphine-thiophenyl ester can be used in the absolute quantification of endogenous GSNO in several cancer cell lines, while retaining the elements of the SNO functional group, using an LC–MS-based assay. Finally, we demonstrate that a common product ion (m/z = 309.0), derived from phosphine–RSNO adducts, can be used for the detection of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological samples. Collectively, these findings establish a platform for the phosphine ligation-based, specific and direct detection of RSNOs in biological samples, a powerful tool for expanding the knowledge of the biology and chemistry of NO•-mediated phenomena.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein

The clearance of apoptotic cells by phagocytes is a fundamental process during tissue remodeling and resolution of inflammation. In turn, the phagocytosis of apoptotic cells generates signals that suppress pro-inflammatory activation of macrophages. These events occur during the resolution phase of inflammation and therefore the malfunctioning of this process may lead to inflammation-related tissue damage. Here, we demonstrate that the calcium-binding protein S100A9, normally abundant in the cytoplasm of neutrophils and also released by apoptotic neutrophils, is involved in the suppression of macrophages after the uptake of apoptotic neutrophils. Both, spontaneous and induced production of inflammatory species (nitric oxide, hydrogen peroxide and TNF-alpha) as well as the phagocytic activity were inhibited when macrophages were in presence of apoptotic neutrophils, conditioned medium from neutrophil cultures or a peptide corresponding to the C-terminal region of S100A9 protein. On the other hand, macrophages kept in the conditioned medium of neutrophils that was previously depleted of S100A9 were shown to resume the activated status. Finally, we demonstrate that the calcium-binding property of S100A9 might play a role in the suppression process, since the stimulation of intracellular calcium release with ionomycin significantly reversed the effects of the uptake of apoptotic neutrophils in macrophages. In conclusion, we propose that S100A9 is a novel component of the regulatory mechanisms of inflammation, acting side-by-side with other suppressor factors generated upon ingestion of apoptotic cells.

Spatiotemporal Intracellular Nitric Oxide Signaling Captured Using Internalized, Near-Infrared Fluorescent Carbon Nanotube Nanosensors

Fluorescent nanosensor probes have suffered from limited molecular recognition and a dearth of strategies for spatial-temporal operation in cell culture. In this work, we spatially imaged the dynamics of nitric oxide (NO) signaling, important in numerous pathologies and physiological functions, using intracellular near-infrared fluorescent single-walled carbon nanotubes. The observed spatial-temporal NO signaling gradients clarify and refine the existing paradigm of NO signaling based on averaged local concentrations. This work enables the study of transient intracellular phenomena associated with signaling and therapeutics.

Nitric Oxide Produced Endogenously Is Responsible for Hypoxia- Induced HIF-1α Stabilization in Colon Carcinoma Cells

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1&bgr;, IL-12, and TNF-&agr;) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Role of distinct immune components in the radiation-induced abrogation of systemic lupus erythematosus development in mice

The New Zealand Black × New Zealand White F1 [(NZB/NZW) F1] mouse develops an autoimmune condition resembling aspects of human systemic lupus erythematosus (SLE). We investigated the effects of a novel prophylactic thoraco-abdominal gamma irradiation protocol on the onset and evolution of lupus in these animals. Survival of irradiated mice was higher when compared with nonirradiated mice. Kidney lesions were milder and autoantibody levels were lower in irradiated mice. To identify possible mechanisms involved in the radiation-induced improvement of disease, distinct components of humoral and cellular immune responses were evaluated. Because B-1 cells are known to be involved in various autoimmune diseases, we investigated the participation of these cells in SLE progression. Unexpectedly, B-1 cells were not depleted in (NZB/NZW) F1, even after several rounds of irradiation. No alterations were found in viability and physiology of B-1 cells in SLE animals with the exception of constitutive overexpression of the anti-apoptotic molecule Bcl-2, which may account for the observed radioresistance. Thus, a role for B-1 cells in murine SLE cannot be excluded, since the irradiation protocol did not effectively eliminate these cells. Additionally, we demonstrate a marked delay in the ability of splenocytes to repopulate the spleen after irradiation in (NZB/NZW) F1, in contrast to leucocytes in other cellular compartments. The implications of these findings for the fate of SLE in this model are discussed. Lupus (2007) 16, 947—954.