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Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which inToxoplasma gondiiincludes MIC8 and the microneme-associated CRMP complex. Here, we examine the function of the microneme protein CLAMP, uncovering its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP-deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. Nevertheless, SPATR, which appears to act as an accessory factor inToxoplasma, is essential duringPlasmodium falciparumblood stages. Our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human “mammalian target of rapamycin” (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kβ) and cyclic guanosine monophosphate–dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kβ in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kβ. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kβ and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

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.

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-1beta, IL-12, and TNF-alpha) 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.

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.

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.

Since B-1 cells were first described, their origin and function remain controversial. Given the ability to produce natural antibodies and large amounts of IL-10, there is a consensus about their role in innate immunity. More recently, however, B-1 cells have been associated to adaptive immunity as well, due to the demonstration of immunological memory and antigen presentation capability. Here we demonstrate that adoptive transfer of pre-sensitized B-1b cells (obtained from OVA-sensitized mice) to naïve B-1 deficient animals, drastically affects the ability of transplanted animals to mount an adaptive response upon immunization with OVA. In contrast to naïve B-1 populated mice, mice transplanted with sensitized B-1 exhibit lower anti-OVA antibody levels, milder footpad swelling in response to OVA subcutaneous injection and reduced granulomatous reaction to OVA-coated beads. Moreover, we show that these pre-sensitized B-1 cells, when acting as APCs, induce poor T cell proliferation in vitro when compared with macrophages or B-1 cells obtained from naïve mice. This property may be due in part to insufficient expression of the co-stimulatory molecule CD86, necessary for optimal antigen presentation. In conclusion, our data suggest a novel role for B-1 cells as part of suppressor mechanisms in the immune system.

The anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua. It is hypothesized to attenuate allergic asthma via inhibition of multiple signaling pathways. We used a comprehensive approach to elucidate the mechanism of action of artesunate by designing a novel biotinylated dihydroartemisinin (BDHA) to identify cellular protein targets of this anti-inflammatory drug. By adopting an untargeted proteomics approach, we demonstrated that artesunate may exert its protective anti-inflammatory effects via direct interaction with multiple proteins, most importantly with a number of mitochondrial enzymes related to glucose and energy metabolism, along with mRNA and gene expression, ribosomal regulation, stress responses and structural proteins. In addition, the modulatory effects of artesunate on various cellular transcription factors were investigated using a transcription factor array, which revealed that artesunate can simultaneously modulate multiple nuclear transcription factors related to several major pro- and anti-inflammatory signalling cascades in human bronchial epithelial cells. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2), a key promoter of antioxidant mechanisms, which is inhibited by the Kelch-like ECH-associated protein 1 (Keap1). Our results demonstrate that, like other electrophilic Nrf2 regulators, artesunate activates this system via direct molecular interaction/modification of Keap1, freeing Nrf2 for transcriptional activity. Altogether, the molecular interactions and modulation of nuclear transcription factors provide invaluable insights into the broad pharmacological actions of artesunate in inflammatory lung diseases and related inflammatory disorders.

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. © 2014 American Chemical Society., U.S. Department of Energy: DE-FG02-97ER25308 National Science Foundation

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.

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.

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPARγ) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPARγ could exert opposite effects in the immune response and the possible implications for SLE. Increased PPARγ mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPARγ/GAPDH mRNA = 2.21 ± 0.49 vs. 0.57 ± 0.14, respectively ( p

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.

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.

SUMMARY 1 The role of haemodynamic changes in left ventricular remodelling has been poorly investigated, especially in the context of volume overload cardiac hypertrophy. Low diastolic blood pressure and high left ventricular filling pressure are expected to affect coronary driving pressure negatively and thereby put in jeopardy subendocardial perfusion in particular. The consequences to global left ventricular remodelling remain undetermined. The aim of the present study was to investigate the role of coronary driving pressure in the development of subendocardial remodelling and the conceivable effects on cardiac function, using a rat model of aortocaval fistula. 2 Wistar rats, weighing 330–350 g, were submitted to aortocaval fistula (ACF group) or sham (control group) operations. Two haemodynamic measurements were determined following surgery, the initial measurement at week 1 and the final measurement at week 8. Cytokine expression, myeloperoxidase (MPO) activity, metalloproteinase expression and activity and fibrosis were assessed in two distinct left ventricular myocardial layers: the subendocardium (SE) and the non-subendocardium (non-SE). 3 The ACF group showed lower initial and final coronary driving pressure and lower final +dP/dt and –dP/dt compared with the control group. Multivariate analyses disclosed initial coronary driving pressure as the only haemodynamic parameter independently associated with SE fibrosis (R2 = 0.76; P

Melanoma patients experience inferior survival after bio-chemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS), and elevated levels of nitrotyrosine, a product derived from nitric oxide (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 anti-cancer 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 decreased its cytotoxic potency. Activity of the pro-apoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/S-nitrisothiols, whereas lower activity resulted from cisplatin combined with stabilization of S-nitrisothiols. 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 (PHD2), the enzyme responsible for targeting the pro-survival transcription factor HIF-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. Citation Format: Luiz C. Godoy, Chase C.T. Anderson, Rajdeep Chowdhury, Laura J. Trudel, Gerald N. Wogan. Melanoma fights cisplatin with NO: S-nitrosation as a mechanism supporting drug-resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2013-894

Exposure to nitric oxide (NO•) during inflammation has been implicated in the development of various forms of cancer, including melanoma. Clinical and epidemiologic studies indicate that expression of inducible nitric oxide synthase (NOS2), as well as NO• production by tumor cells, correlate with patient mortality. NO• favors resistance to drug-induced apoptosis in melanoma cells and may do so by caspases inactivation through S-nitrosation, which could represent a mechanism responsible for NO• inhibition of apoptosis. Here we further investigate how S-nitrosation impacts apoptotic and survival pathways in human melanoma. We demonstrate that A375, SK-Mel 100 and SK-Mel 28 cells constitutively express NOS2 and that an inhibitor of NO• synthesis decreases cell proliferation. Moreover, this same treatment, or the pre-incubation with the NO• scavenger carboxy-PTIO, leads to enhanced toxicity of cisplatin, suggesting that endogenous NO• regulates growth and enhances anti-apoptotic mechanisms in melanoma cells. This anti-apoptotic effect may be due to protein S-nitrosation promoted by endogenous NO•, because disruption of nitrosothiols by dithiothreitol promotes a remarkable increase in cisplatin-induced cell death in comparison with cells treated with cisplatin alone (80% vs. 20% cell death, respectively). On the other hand, when accumulation of cellular nitrosothiols is favored via inhibition of thioredoxin reductase by 1-chloro-2,4-dinitrobenzene, increased resistance to cisplatin is seen in A375 cells. Using the biotin switch technique (BST), a low, yet detectable level of endogenous protein S-nitrosation is seen in A375 cells. The properties conferred by exogenous NO• to melanoma cells were studied in a controlled-delivery system to simulate NO• concentrations known to occur in inflamed tissues. Using this system, the toxicity of NO• was shown to be lower in A375, SK-Mel 100 and SK-Mel 28 cells when compared to other non-melanoma cell lines. The possible protective effect of a chronic exposure to a non-toxic dose of exogenous NO• was investigated. A375 cells were treated with 0.64 µM NO• or argon (control) for 24 hours and then cisplatin was added to the system. Cell viability was assessed 24 hours later with trypan blue staining. While nearly 50% cell death was seen in argon+cisplatin-treated cells, no significant cell death occurred under NO•+cisplatin treatment, which points to an anti-apoptotic effect of low, sustained doses of NO• in this system. Finally, using the BST, the level of global protein S-nitrosation in the cells exposed to 0.64 µM NO• was shown to be considerably increased. Collectively, these results show that NO• favors survival and proliferation of melanoma cells in vitro, possibly by S-nitrosation of components of the signaling cascades. Identification of the proteins susceptible to this NO•-mediated modification will help to devise rational strategies for disease intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 204.