Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology.

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane-based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s-1) and for sugar translocation (2 s-1 - 30 s-1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.

A cytochrome P450-derived epoxygenated metabolite of anandamide is a potent cannabinoid receptor 2-selective agonist.

Oxidation of the endocannabinoid anandamide by cytochrome P450 (P450) enzymes has the potential to affect signaling pathways within the endocannabinoid system and pharmacological responses to novel drug candidates targeting this system. We previously reported that the human cytochromes P450 2D6, 3A4, and 4F2 are high-affinity, high-turnover anandamide oxygenases in vitro, forming the novel metabolites hydroxyeicosatetraenoic acid ethanolamides and epoxyeicosatrienoic acid ethanolamides. The objective of this study was to investigate the possible biological significance of these metabolic pathways. We report that the 5,6-epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective cannabinoid receptor 2 (CB2) agonist. The K(i) values for the binding of 5,6-EET-EA to membranes from Chinese hamster ovary (CHO) cells expressing either recombinant human CB1 or CB2 receptor were 11.4 microM and 8.9 nM, respectively. In addition, 5,6-EET-EA inhibited the forskolin-stimulated accumulation of cAMP in CHO cells stably expressing the CB2 receptor (IC(50) = 9.8 +/- 1.3 nM). Within the central nervous system, the CB2 receptor is expressed on activated microglia and is a potential therapeutic target for neuroinflammation. BV-2 microglial cells stimulated with low doses of interferon-gamma exhibited an increased capacity for converting anandamide to 5,6-EET-EA, which correlated with increased protein expression of microglial P450 4F and 3A isoforms. Finally, we demonstrate that 5,6-EET-EA is more stable than anandamide in mouse brain homogenates and is primarily metabolized by epoxide hydrolase. Combined, our results suggest that epoxidation of anandamide by P450s to form 5,6-EET-EA represents an endocannabinoid bioactivation pathway in the context of immune cell function.

Th17 cells in human disease.

SUMMARY: Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4(+) T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma. Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection. Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans. As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms. Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection.

Demethylation of CD40LG on the inactive X in T cells from women with lupus.

Why systemic lupus erythematosus primarily affects women is unknown. Recent evidence indicates that human lupus is an epigenetic disease characterized by impaired T cell DNA methylation. Women have two X chromosomes; one is inactivated by mechanisms including DNA methylation. We hypothesized that demethylation of sequences on the inactive X may cause gene overexpression uniquely in women, predisposing them to lupus. We therefore compared expression and methylation of CD40LG, a B cell costimulatory molecule encoded on the X chromosome, in experimentally demethylated T cells from men and women and in men and women with lupus. Controls included TNFSF7, a methylation-sensitive autosomal B cell costimulatory molecule known to be demethylated and overexpressed in lupus. Bisulfite sequencing revealed that CD40LG is unmethylated in men, while women have one methylated and one unmethylated gene. 5-Azacytidine, a DNA methyltransferase inhibitor, demethylated CD40LG and doubled its expression on CD4(+) T cells from women but not men, while increasing TNFSF7 expression equally between sexes. Similar studies demonstrated that CD40LG demethylates in CD4(+) T cells from women with lupus, and that women but not men with lupus overexpress CD40LG on CD4(+) T cells, while both overexpress TNFSF7. These studies demonstrate that regulatory sequences on the inactive X chromosome demethylate in T cells from women with lupus, contributing to CD40LG overexpression uniquely in women. Demethylation of CD40LG and perhaps other genes on the inactive X may contribute to the striking female predilection of this disease.

Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.

OBJECTIVE: To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues. METHODS: Human class II major histocompatibility complex (MHC)-typed FLS were used as APCs for murine class II MHC-restricted CD4 T cell hybridomas. Interferon-gamma (IFNgamma)-treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2. RESULTS: Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFNgamma, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFNgamma-dependent and MHC-restricted manner. CONCLUSION: RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

Cells of the synovium in rheumatoid arthritis. T lymphocytes.

Recent findings have substantiated the importance of T lymphocytes to the pathogenesis of rheumatoid arthritis (RA). Here, we review emerging data regarding genetic predisposition, spontaneous animal models of arthritis, and cell-cell interactions that implicate T cells as driving synovial inflammation and joint destruction. Information regarding the proinflammatory role of interleukin-17-producing T cells and the functional state of regulatory T cells both in animal models and in patients with RA is also discussed. In light of the overwhelming evidence that disrupted T-cell homeostasis greatly contributes to joint pathology in RA, the therapeutic potential of targeting activators of pro-inflammatory T cells or their products is compelling.

Interleukin-17 as a molecular target in immune-mediated arthritis: immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin-4.

OBJECTIVE: Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin-4 (IL-4) reduce the incidence and severity of murine collagen-induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL-4 DCs on cytokine production by subsets of T helper cells. METHODS: Male DBA mice ages 6-8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL-4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon-gamma (IFNgamma) and IL-17 responses were measured. Antibodies to IL-4, IL-12, and IL-23, and recombinant IL-4, IL-12, and IL-23 were used to further study the regulation of T cell cytokine production by IL-4 DCs. RESULTS: Splenic T cells obtained during the initiation phase of arthritis produced less IL-17 when cultured in the presence of IL-4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNgamma and tumor necrosis factor). T cell IL-17 production after collagen rechallenge was not inhibited by a lack of IL-23, since IL-4-mediated suppression of IL-17 was not reconstituted by IL-23, an otherwise potent inducer of IL-17 production by T cells. Although IL-4 DCs can produce increased quantities of IL-12 and IFNgamma, suppression of IL-17 production by IL-4 DCs was independent of both. While IL-17 production by T cells obtained during the initiation phase of arthritis was regulated by IL-4 DCs, IL-17 production by T cells obtained during end-stage arthritis was not altered. CONCLUSION: Our data suggest that IL-4 DCs exert a therapeutic effect on collagen-induced arthritis by targeting IL-17. IL-17 suppression by IL-4 DCs is robust and is not reversed by IL-23. Timing might be important in IL-17-targeted therapy, since IL-17 production by T cells obtained during end-stage arthritis did not respond to suppression by IL-4 DCs.