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Intestinal macrophages with functional plasticity play essential roles in gut immune responses by increasing chemokines and cytokines, thereby contributing to the pathogenesis of inflammatory bowel disease (IBD). Poly(rC)-binding protein 1 (PCBP1), which is widely expressed in immune cells, binds to nucleic acids in mRNA processing, stabilization, translation and transcription. However, little is known about the influence of PCBP1 on macrophages and its specific mechanism in inflamed intestines. In this study, conditional depletion of Pcbp1 in macrophages protected mice from progression of dextran sulfate sodium induced colitis and resulted in significant alleviation of colitis. Pcbp1 deficiency markedly decreased C-C motif chemokine ligand 2 (CCL2) production by colonic CX3C motif chemokine receptor 1+ (CX3CR1+) macrophages and reduced accumulation of pro-inflammatory macrophages and production of pro-inflammatory cytokines, such as IL-6 and TNF-α, in the inflamed colon. RNA-immunoprecipitation analysis indicated that PCBP1 might interact with Ccl2 mRNA and regulate its expression in macrophages. PCBP1 expression in inflamed intestines also correlated significantly with IBD severity in patients, suggesting a critical involvement of PCBP1 in intestinal inflammation. We anticipate that our findings will facilitate the development of novel therapeutic approaches for IBD by targeting the specific function of immune cells in the local microenvironment, thereby helping to reduce adverse effects.

Post‐transcriptional regulation of cytokine production is crucial to ensure appropriate immune responses. We previously demonstrated that poly‐rC‐binding protein‐1 (PCBP1) can act as a trans‐acting factor to stabilize transcripts encoding sortilin, which mediates cytokine trafficking. Here, we report that PCBP2, which strongly resembles PCBP1, can stabilize sortilin transcripts in macrophages using the same mechanism employed by PCBP1. PCBP2 recognized the C‐rich element in the 3′ UTR of sortilin mRNA, and PCBP2 knockdown decreased sortilin transcripts, indicating that PCBP2 stabilizes sortilin mRNA by binding to its 3′ UTR. Zn2+ reversibly inhibited the nucleotide binding ability of PCBP2 in vitro. These findings suggest that both PCBP2 and PCBP1 may control the stability of sortilin transcripts by sensing intracellular Zn2+ levels in immune cells., We analyzed the function of poly‐rC‐binding protein‐2 (PCBP2), a paralog of PCBP1, in stabilizing the mRNA of sortilin, a cytokine trafficking mediator. PCBP2 binds to C‐rich elements in the 3′ UTR of sortilin mRNA; Zn2+ interferes with this interaction, suggesting that both PCBP1 and PCBP2 may regulate the stability of sortilin mRNA by sensing the intracellular Zn2+ levels.

Sortilin is a multifunctional sorting receptor involved in cytokine production in immune cells. To understand the mechanism of Sortilin-mediated cytokine trafficking, we determined the 2.45-Å structure of the dimerized Sortilin ectodomain (sSortilin or the Vps10-domain) crystallized at acidic pH. Substantial conformational changes upon dimerization lead to the intermolecular hydrophobic interaction between the conserved E455 and F137. Analysis of the electrostatic surface and size-exclusion chromatography revealed that sSortilin dimerization occurs due to an increase in hydrophobic interactions at the neutral dimer interface at acidic pH. The N682-attached N-glycan in the vicinity of the dimer interface implies its involvement in the dimerization. The disruption of Sortilin dimerization by mutations impairs efficient interferon-alpha secretion from cells. These results suggest the functional importance of Sortilin dimerization in cytokine trafficking.

Hypersensitivity pneumonitis (HP) is a pulmonary disease caused by repeated exposure to various aspiration antigens, including bacteria and fungi. Although TLRs are known to be required for the generation of HP triggered by bacteria, the significance of fungal receptors remains unclear. The present study aimed to investigate whether Dectin-1 and Dectin-2 contribute to the development of experimental HP triggered by the fungus Trichosporon asahii (T. asahii) that causes summer-type HP.We investigated the binding between Dectin-Fc protein and T. asahii by a dot blot assay. We performed the histological and flow cytometric analysis in the HP model using Dectin-1-deficient (Dectin-1(-/-)) and Dectin-2(-/-) mice. We also investigated Th17/Th1 responses in lung cells, and measured an IL-17-promoting cytokine IL-23 from bone marrow-derived dendritic cells (BMDCs) by ELISA.Dectin-1 bound more strongly to T. asahii than Dectin-2. Dectin-1(-/-) mice barely developed HP, whereas both wild-type mice and Dectin-2(-/-) mice developed similar lung diseases. Dectin-1 deficiency decreased the infiltration of neutrophils and monocyte-derived macrophages and repressed the expansion of lung CD4(+)IL-17A(+) cells. The production of IL-23 p19 was reduced in Dectin-1(-/-) BMDCs.These data suggested Dectin-1 plays a critical role in the development of fungus-induced HP.

Mast cells, basophils, and eosinophils are central effectors in allergic inflammatory disorders. These cells secrete abundant serine proteases as well as chemical mediators and cytokines; however, the expression profiles and functions of their endogenous inhibitors remain elusive. We found that murine secretory leukoprotease inhibitor (SLPI) is expressed in basophils and eosinophils but in not in mast cells. SLPI-deficient (Slpi-/-) basophils produce more cytokines than wild-type mice after IgE stimulation. Although the deletion of SLPI in basophils did not affect the release of chemical mediators upon IgE stimulation, the enzymatic activity of the serine protease tryptase was increased in Slpi-/- basophils. Mice transferred with Slpi-/- basophils were highly sensitive to IgE-mediated chronic allergic inflammation. Eosinophils lacking SLPI showed greater interleukin-6 secretion and invasive activity upon lipopolysaccharide stimulation, and the expression of matrix metalloproteinase-9 by these eosinophils was increased without stimulation. The absence of SLPI increases JNK1 phosphorylation at the steady state, and augments the serine phosphorylation of JNK1-downstream ETS transcriptional factor Elk-1 in eosinophils upon stimulation. Of note, SLPI interacts with a scaffold protein, JNK-interacting protein 3 (JIP3), that constitutively binds to the cytoplasmic domain of toll-like receptor (TLR) 4, suggesting that SLPI controls Elk-1 activation via binding to JIP3 in eosinophils. Mice transferred with Slpi-/- eosinophils showed the exacerbation of chitin-induced allergic inflammation. These findings showed that SLPI is a negative regulator in allergic effector cells and suggested a novel inhibitory role of SLPI in the TLR4 signaling pathways.

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.

Plasmacytoid dendritic cells (PDCs) produce type I interferons (IFNs) in response to viral nucleic acids to exert antiviral immunity. However, PDCs are related to the progress and severity of autoimmune diseases, such as systemic lupus erythematosus, because they respond to host DNA. Therefore, the regulation of PDC activation is critical for maintaining adequate immune responses. Here we show that an inhibitory major histocompatibility complex class I receptor, paired immunoglobulin-like receptor B (PIR-B), suppressed Fms-like tyrosine kinase 3 ligand-induced PDC differentiation in BM cells, as well as Toll-like receptor 9-mediated IFN-α production by PDCs, through the dephosphorylation of STAT1/STAT2. In particular, PIR-B inhibited IFN-α–mediated STAT phosphorylation, suggesting that PIR-B negatively regulates the positive feedback mechanism of IFN-α secretion triggered by Toll-like receptor 9. These results demonstrate a novel regulatory role for PIR-B in PDCs.

CnfU, a key iron-sulfur (Fe-S) cluster biosynthetic scaffold that is required for biogenesis of ferredoxin and photosystem I in chloroplasts, consists of two tandemly repeated domains in which only the N-terminal domain contains a conserved CXXC motif. We have determined the crystal structure of the metal-free dimer of AtCnfU-V from Arabidopsis thaliana at 1.35 A resolution. The N-terminal domains of the two monomers are linked together through two intermolecular disulfide bonds between the CXXC motifs. At the dimer interface, a total of four cysteine sulfur atoms provide a Fe-S cluster assembly site surrounded by uncharged but hydrophilic structurally mobile segments. The C-terminal domain of one monomer interacts with the N-terminal domain of the opposing monomer and thereby stabilizes dimer formation. Furthermore, Fe K-edge X-ray absorption spectroscopic analysis of the holo-CnfU dimer in solution suggests the presence of a typical [2Fe-2S]-type cluster coordinated by four thiolate ligands. Based on these data, a plausible model of the holo-AtCnfU-V dimer containing a surface-exposed [2Fe-2S] cluster assembled in the dimer interface was deduced. We propose that such a structural framework is important for CnfU to function as a Fe-S cluster biosynthetic scaffold.

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.

Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

Iron–sulfur proteins play physiologically important roles in a variety of metabolic processes in eukaryotes. In plants, iron–sulfur cluster biosynthesis is known to take place both in mitochondria and chloroplasts. However no components that mediate iron–sulfur cluster delivery in the plant cell cytosol have been identified so far. Here we report identification and characterization of a cytosolic Nbp35 homolog named AtNbp35 from Arabidopsis thaliana. AtNbp35-deficient Arabidopsis mutants were seedling lethal. Unlike the previously characterized yeast ScNbp35 which forms a heterotetramer with ScCfd1, AtNbp35 forms a homodimer in the cytosol and can harbor both [4Fe–4S] and [2Fe–2S] clusters on its amino- and carboxyl-terminal domains, respectively. Taken together, our data suggest that Nbp35 plays a pivotal role in iron–sulfur cluster assembly and delivery in the plant cell cytosol as a bifunctional molecular scaffold.

IscA has been proposed to be a scaffold protein of the iron-sulfur cluster biosynthetic machinery. We have identified the IscA homolog to be localized to plastids, termed AtIscA-I, in Arabidopsis thaliana. The AtIscA-I protein was apparently constitutively expressed in all tissues analyzed in Arabidopsis. The AtIscA-I protein exists in the stroma as a soluble protein which tends to form a homo-dimer and can host a [2Fe-2S]-like cluster. Complete loss of the protein from plastids did not cause any significant defect either in normal plant growth or in biogenesis of major iron-sulfur proteins, indicating this protein is not essential or redundant for these functions. In contrast, loss of one of the three plastid-localized CnfU scaffold proteins, AtCnfU-V, caused significant reduction in the level of AtIscA-I. These data suggest that efficient biogenesis of AtIscA-I scaffold requires function of another essential scaffold protein CnfU.