Although all ferredoxin-NADP+ reductases (FNRs) catalyze the same reaction. i. e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant type and the glutathione reductase type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties.Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP+ to its 4-oxo derivative, and the properties of plant-type FNRs from non- Subject: revision of ms ABBI-07-950Dear Editor, we would like to thank the Reviewers for their criticisms and suggestions that hopefully allowed us to improve our article. We carefully revised the manuscript according to the Reviewer's suggestions. The overall length of the paper has been significantly reduced by shortening longer chapters and by deleting three figures.The major changes in the revised version of the manuscript are as follows: Fig. 1, GR-type FNRs represent a broader group that include both the AdR-like and the ONFR-like enzymes. Thus, we prefer to maintain the distinction between the two terms (GR-type and AdR-like).2) While most of the chapters have similar lengths, the last two chapters are significantly longer than the previous six ones, creating an unpleasant imbalance in the presentation. The authors should try to either condense or split in multiple chapters the two long ones, in order to have a more armonious and balanced presentation of the topics. Large chapters ("Specific features of Plasmodium falciparum FNR" and "Ferredoxin binding and electron-transfer") were significantly shortened (by 35% and 22%, respectively). Previous very long paragraphs were broken into smaller ones.3) The work would significantly gain in strength upon adding a small summary paragraph at the end of the review article. Alternatively, the authors may consider a paragraph in which they present open questions that can now be raised and answered based upon current knowledge. A Conclusion chapter has been added. However, in order to limit the ms length, this chapter is not a summary or a list of open questions, but includes just some concluding remarks. 5) A list of abbreviations should be included. For example, what is NMN?A list of abbreviations has been added. 6) On page 4 line 15, both n and S should be defined.The definition of the parameters n and S has been given, and a reference added. 7) The authors should specify clearly which enzymes have been wrongly identified as adrenodoxin reductase-like enzymes (page 6).What we found is that some AdR-like proteins have been wrongly identified as other enzymes, and not the opposite. We think that, for the sake of brevity, it is not possible to give a comprehensive list of incorrectly identified entries within this review article. We have provide...

Quantitative analysis of Ca 2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca 2+ -dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca 2+ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca 2+ release kinetics with an off-rate estimated at around 700 s −1 and appropriate Ca 2+ binding affinity, likely attributable to local Ca 2+ -induced conformational changes around the designed Ca 2+ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca 2+ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca 2+ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca 2+ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca 2+ homeostasis.

BackgroundKv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined.MethodsWe performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events.ResultsLPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation.ConclusionsWe have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0906-6) contains supplementary material, which is available to authorized users.

The modification of human myoglobin (HMb) by reaction with nitrite and hydrogen peroxide has been investigated. This reaction is important because NO(2) (-) and H(2)O(2) are formed in vivo under conditions of oxidative and nitrative stress, where protein derivatization has been often observed. The abundance of HMb in tissues and in the heart makes it a potential source and target of reactive species generated in the body. The oxidant and nitrating species produced by HMb/H(2)O(2)/NO(2) (-) are nitrogen dioxide and peroxynitrite, which can react with exogenous substrates and endogenous protein residues. Tandem mass analysis of HMb modified by stoichiometric amounts of H(2)O(2) and NO(2) (-) indicated the presence of two endogenous derivatizations: oxidation of C110 to sulfinic acid (76 %) and nitration of Y103 to 3-nitrotyrosine (44 %). When higher concentrations of NO(2) (-) and H(2)O(2) were used, nitration of Y146 and of the heme were also observed. The two-dimensional gel-electrophoretic analysis of the modified HMbs showed spots more acidic than that of wild-type HMb, a result in agreement with the formation of sulfinic acid and nitrotyrosine residues. By contrast, the reaction showed no evidence for the formation of protein homodimers, as observed in the reaction of HMb with H(2)O(2) alone. Both HMb and the modified HMb are active in the H(2)O(2)/NO(2) (-)-dependent nitration of exogenous phenols. Their catalytic activity is quite similar and the endogenous modifications of HMb therefore have little effect on the reactivity of the protein intermediates.

In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP + reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors. These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis. We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmodium falciparum in recombinant form, and recently we solved the crystal structure of the P. falciparum reductase. Here we report on the functional properties of the latter enzyme, which differ markedly from those of homologous FNRs. In the physiological reaction, P. falciparum FNR displays a k cat five-fold lower than those usually determined for plastidic-type FNRs. By rapid kinetics, we found that hydride transfer between NADPH and proteinbound FAD is slower in the P. falciparum enzyme. The redox properties of the enzyme were determined, and showed that the FAD semiquinone species is highly destabilized. We propose that these two features, i.e. slow hydride transfer and unstable FAD semiquinone, are responsible for the poor catalytic efficiency of the P. falciparum enzyme. Another unprecedented feature of the malarial parasite FNR is its ability to yield, under oxidizing conditions, an inactive dimeric form stabilized by an intermolecular disulfide bond. Here we show that the monomer-dimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H 2 O 2 , glutathione, and lipoate. This finding suggests that modulation of the quaternary structure of P. falciparum FNR might represent a regulatory mechanism, although this needs to be verified in vivo.

Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL-4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL-4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Further, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B cell-deficient mice, confirming a B cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy.

Atomic co-ordinates and structure factors for the T67R/S92D metMbCN mutant have been deposited with the Protein Data Bank, under accession codes 1h1x and r1h1xsf, respectively. Protein engineering and cofactor replacement have been employed as tools to introduce/modulate peroxidase activity in sperm whale Mb (myoglobin). Based on the rationale that haem peroxidase active sites are characterized by specific charged residues, the Mb haem crevice has been modified to host a haem-distalpropionate Arg residue and a proximal Asp, yielding the T67R/S92D Mb mutant. To code extra conformational mobility around the haem, and to increase the peroxidase catalytic efficiency, the T67R/S92D Mb mutant has been subsequently reconstituted with protohaem-L-histidine methyl ester, yielding a stable derivative, T67R/S92D Mb-H. The crystal structure of T67R/S92D cyano-metMb (1.4 A resolution; R factor, 0.12) highlights a regular haem-cyanide binding mode, and the role for the mutated residues in affecting the haem propionates as well as the neighbouring water structure. The conformational disorder of the haem propionate-7 is evidenced by the NMR spectrum of the mutant. Ligand-binding studies show that the iron(III) centres of T67R/S92D Mb, and especially of T67R/S92D Mb-H, exhibit higher affinity for azide and imidazole than wild-type Mb. In addition, both protein derivatives react faster than wild-type Mb with hydrogen peroxide, showing higher peroxidase-like activity towards phenolic substrates. The catalytic efficiency of T67R/S92D Mb-H in these reactions is the highest so far reported for Mb derivatives. A model for the protein-substrate interaction is deduced based on the crystal structure and on the NMR spectra of protein-phenol complexes.

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