Noradrenergic ß-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na-K ATPase Subunit Expression in C6 Cells.

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na-K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na-K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on ß-adrenoceptor decreased Na-K ATPase activity and mRNA expression of the catalytic α2-Na-K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na-K ATPase expression. In contrast, noradrenaline acting on ß-adrenoceptor up-regulated the expression of regulatory ß2-Na-K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na-K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.

Sensitive detection of intracellular RNA of human telomerase by using graphene oxide as a carrier to deliver the assembly element of hybridization chain reaction.

Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.

GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons.

Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms--such as isoforms IV, IX, XII, XIV, and XV--also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H(+) at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H(+) shifts of granule cells following CO(2) removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H(+) dynamics in the cytosol, both in injected oocytes and in mouse neurons.

Intracellular ß-glucosidases CEL1a and CEL1b are essential for cellulase induction on lactose in Trichoderma reesei.

Lactose (1,4-O-ß-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular ß-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular ß-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis ß-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular ß-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.

Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects.

The ß-site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) were initially identified as transmembrane aspartyl proteases cleaving the amyloid precursor protein (APP). BACE1 is a major drug target for Alzheimer's disease because BACE1-mediated cleavage of APP is the first step in the generation of the pathogenic amyloid-ß peptides. BACE1, which is highly expressed in the nervous system, is also required for myelination by cleaving neuregulin 1. Several recent proteomic and in vivo studies using BACE1- and BACE2-deficient mice demonstrate a much wider range of physiological substrates and functions for both proteases within and outside of the nervous system. For BACE1 this includes axon guidance, neurogenesis, muscle spindle formation, and neuronal network functions, whereas BACE2 was shown to be involved in pigmentation and pancreatic ß-cell function. This review highlights the recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer's disease. The protease BACE1 is a major drug target in Alzheimer disease. Together with its homolog BACE2, both proteases have an increasing number of functions within and outside of the nervous system. This review highlights recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer disease.

Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells.

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.

Cutting edge: NADPH oxidase modulates MHC class II antigen presentation by B cells.

Phagocyte NADPH oxidase plays a key role in pathogen clearance via reactive oxygen species (ROS) production. Defects in oxidase function result in chronic granulomatous disease with hallmark recurrent microbial infections and inflammation. The oxidase's role in the adaptive immune response is not well understood. Class II presentation of cytoplasmic and exogenous Ag to CD4(+) T cells was impaired in human B cells with reduced oxidase p40(phox) subunit expression. Naturally arising mutations, which compromise p40(phox) function in a chronic granulomatous disease patient, also perturbed class II Ag presentation and intracellular ROS production. Reconstitution of patient B cells with a wild-type, but not a mutant, p40(phox) allele restored exogenous Ag presentation and intracellular ROS generation. Remarkably, class II presentation of epitopes from membrane Ag was robust in p40(phox)-deficient B cells. These studies reveal a role for NADPH oxidase and p40(phox) in skewing epitope selection and T cell recognition of self Ag.

Adaptor protein APPL1 couples synaptic NMDA receptor with neuronal prosurvival phosphatidylinositol 3-kinase/Akt pathway.

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

Participation of 47C>T SNP (Ala-9Val polymorphism) of the SOD2 gene in the intracellular environment of human peripheral blood mononuclear cells with and without lipopolysaccharides.

The outcome of sepsis occurs due to influence of environmental and genetic factors besides genes variants whose expression support its outcome or not. Oxidative stress is associated to the pathogenicity of sepsis, occurring when there is a reactive species overproduction associated with inflammation. The aim of this study was to characterize the cellular redox status of human peripheral blood mononuclear cells (PBMCs) with either -9Ala (AA) or -9Val (VV) SOD2 genotypes and evaluate their response to oxidative stress induced by lipopolysaccharide (LPS). The PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers for each allele) and the following assays were performed: antioxidant enzyme activities (superoxide dismutase; catalase; glutathione peroxidase), total radical-trapping antioxidant parameter, non-enzymatic antioxidant capacity (total antioxidant reactivity), and quantification of conjugated dienes (lipid peroxidation). At basal conditions (i.e., not stimulated by LPS), cells from 47C allele carriers showed higher activities of CAT and SOD, as well as higher TAR compared to 47T allele. However, when 47CC cells were challenged with LPS, we observed a higher shift toward a pro-oxidant state compared to 47TT cells. The CAT activity and lipid peroxidation were increased in cells with both alleles, but SOD activity increased significantly only in 47TT cells. These results demonstrate that SOD2 polymorphisms are associated with different cellular redox environments at both basal and LPS-stimulated states, and identification of this polymorphism may be important for a better understanding of pro-inflammatory conditions.

Analysis of intracellular enzyme activity by surface enhanced Raman scattering.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal), by wild type ß-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

Stromal cell-derived factor-1 signaling via the CXCR4-TCR heterodimer requires phospholipase C-ß3 and phospholipase C-γ1 for distinct cellular responses.

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-ß3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-ß3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-ß3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.

Leishmania inhibitor of serine peptidase 2 prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages.

Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.

The role of glycogen synthase kinase 3 in regulating IFN-ß-mediated IL-10 production.

The ability of IFN-ß to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-ß stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-ß to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-ß activity. IFN-ß-mediated suppression of GSK3-ß promoted IL-10, because IL-10 production by IFN-ß-stimulated dendritic cells (DC) expressing an active GSK3-ß knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-ß augmented IL-10 production. IFN-ß increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-ß to induce IL-10 from DC. IL-10 production by IFN-ß-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-ß induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-ß.

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice.

Diabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.

Zinc is a novel intracellular second messenger.

Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcepsilon receptor I [FcepsilonRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave. The zinc wave was dependent on calcium influx and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation. The results suggest that the zinc wave is involved in intracellular signaling events, at least in part by modulating the duration and strength of FcepsilonRI-mediated signaling. Collectively, our findings indicate that zinc is a novel intracellular second messenger.

In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish.

The zebrafish genome contains a large number of genes encoding potential cytokine receptor genes as judged by homology to mammalian receptors. The sequences are too divergent to allow unambiguous assignments of all receptors to specific cytokines, and only a few have been assigned functions by functional studies. Among receptors for class II helical cytokines-i.e., IFNs that include virus-induced Ifns (Ifn-) and type II Ifns (Ifn-γ), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)-only the Ifn--specific complexes have been functionally identified, whereas the receptors for the two Ifn-γ (Ifn-γ1 and Ifn-γ2) are unknown. In this work, we identify conditions in which Ifn-γ1 and Ifn-γ2 (also called IFNG or IFN-γ and IFN-gammarel) are induced in fish larvae and adults. We use morpholino-mediated loss-of-function analysis to screen candidate receptors and identify the components of their receptor complexes. We find that Ifn-γ1 and Ifn-γ2 bind to different receptor complexes. The receptor complex for Ifn-γ2 includes cytokine receptor family B (Crfb)6 together with Crfb13 and Crfb17, whereas the receptor complex for Ifn-γ1 does not include Crfb6 or Crfb13 but includes Crfb17. We also show that of the two Jak2 paralogues present in the zebrafish Jak2a but not Jak2b is involved in the intracellular transmission of the Ifn-γ signal. These results shed new light on the evolution of the Ifn-γ signaling in fish and tetrapods and contribute toward an integrated view of the innate immune regulation in vertebrates.

Francisella acid phosphatases inactivate the NADPH oxidase in human phagocytes.

Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.

Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.