10. These authors contributed equally to the work.11. These authors contributed equally to the work. AbstractThe metazoan hypoxic response is regulated by the oxygen-dependent posttranslational hydroxylation of the -subunits of the hypoxia inducible factor (HIF)system. This identification has raised the question of whether other proteinhydroxylases are involved in the regulation of gene expression. Here we demonstrate that the splicing factor protein U2AF65 undergoes post-translational lysyl-5-hydroxylation as catalysed by the Fe(II) and 2-oxoglutarate dependent dioxygenaseJumonji domain-containing protein 6 (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a close human homologue of the hypoxia inducible factor asparaginyl-hydroxylase. Jmjd6 is shown to regulate alternative RNA splicing of endogenous and reporter genes. The ability of Jmjd6 to influence the splicing pattern of all endogenous genes investigated supports a specific role for Jmjd6 in regulating RNA splicing. Main textMetazoan cells respond to limiting oxygen by activation of the hypoxia inducible factor (HIF) system(1). The HIF subunits are regulated by Fe(II) and 2-oxoglutarate

A comparative mechanistic study of Cu-catalyzed oxidative coupling reactions of N-phenyltetrahydroisoquinoline with different nucleophiles was conducted. Two previously reported combinations of catalyst and oxidant were studied, CuCl(2)·2H(2)O/O(2) and CuBr/tert-butyl hydroperoxide (TBHP). On the basis of a synthetic study with different nucleophiles, the electrophilicity of the intermediate iminium ion was estimated and differences between the two methods were revealed. The key intermediate in the aerobic method is shown to be an iminium ion, formed through oxidation by copper(II), which can react with any nucleophile of sufficient reactivity. The role of oxygen is the reoxidation of the reduced catalyst. In the CuBr/TBHP system, an α-amino peroxide is proposed as a true intermediate within the catalytic cycle, formed from the amine and TBHP by a Cu-catalyzed radical reaction pathway and acting as a precursor to the iminium ion intermediate.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with chemokine-like functions that plays a pivotal role in the pathogenesis of inflammatory diseases by promoting leukocyte recruitment. We showed that MIF promotes the atherogenic recruitment of monocytes and T cells through its receptors CXCR2 and CXCR4. Effects of MIF on B cell recruitment have not been addressed. In this study, we tested the involvement of MIF in B cell chemotaxis and studied the underlying mechanism. We show that MIF promotes primary murine B cell chemotaxis in a dose-dependent manner, comparable to the B cell chemokines CXCL13 and CXCL12. Splenic B cells express CXCR4 and the receptor CD74 but not CXCR2. Inhibition of CXCR4 or CD74 or a genetic deficiency of Cd74 in primary B cells fully abrogated MIF-mediated B cell migration, implying cooperative involvement of both receptors. MIF stimulation of B cells resulted in a rapid increase in intracellular Ca2+ mobilization and F-actin polymerization. Intriguingly, the tyrosine kinase ZAP-70 was activated upon MIF and CXCL12 treatment in a CXCR4- and CD74-dependent manner. Pharmacological inhibition of ZAP-70 resulted in abrogation of primary B cell migration. Functional involvement of ZAP-70 was confirmed by small interfering RNA–mediated knockdown in Ramos B cell migration. Finally, primary B cells from ZAP-70 gene–deficient mice exhibited ablated transmigration in response to MIF or CXCL12. We conclude that MIF promotes the migration of B cells through a ZAP-70–dependent pathway mediated by cooperative engagement of CXCR4 and CD74. The data also suggest that MIF may contribute to B cell recruitment in vivo (e.g., in B cell–related immune disorders).

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4.