The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

SimR, a TetR-family transcriptional regulator (TFR), controls the export of simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. Simocyclinone is exported by a specific efflux pump, SimX and the transcription of simX is repressed by SimR, which binds to two operators in the simR-simX intergenic region. The DNA-binding domain of SimR has a classical helix-turn-helix motif, but it also carries an arginine-rich N-terminal extension. Previous structural studies showed that the N-terminal extension is disordered in the absence of DNA. Here, we show that the N-terminal extension is sensitive to protease cleavage, but becomes protease resistant upon binding DNA. We demonstrate by deletion analysis that the extension contributes to DNA binding, and describe the crystal structure of SimR bound to its operator sequence, revealing that the N-terminal extension binds in the minor groove. In addition, SimR makes a number of sequence-specific contacts to the major groove via its helix-turn-helix motif. Bioinformatic analysis shows that an N-terminal extension rich in positively charged residues is a feature of the majority of TFRs. Comparison of the SimR–DNA and SimR–simocyclinone complexes reveals that the conformational changes associated with ligand-mediated derepression result primarily from rigid-body rotation of the subunits about the dimer interface

Lilliput

Catalog for the exhibition Lilliput held at the Seton Hall University Walsh Gallery, June 8 - July 24, 2009. Curated by Jeanne Brasile and Asha Ganpat. Includes an essay by Jeanne Brasile and Asha Ganpat. Includes color illustrations

Fractalkine/CX3CL1: a potential new target for inflammatory diseases

A better understanding of the immunological processes governed by cytokines and chemokines has shaped our approach to the design of therapeutics for diseases such as rheumatoid arthritis (RA), atherosclerosis, and other inflammatory disorders. The discovery of chemokines and their receptors as integral components and regulators of inflammation has dramatically contributed to advances in treating these disease states. Among the different classes of chemokines, fractalkine/CX3CL1, with its unique functional and structural characteristics, has been found to participate in inflammation. This viewpoint summarizes the emerging role of fractalkine/CX3CL1 from the historical, functional, and clinical perspective and provides evidence to validate it as a potential therapeutic target in cardiovascular disease, rheumatoid arthritis, as well as other diseases related to vascular inflammation

Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity

Abstract Gut mucosa consists of stratified layers of microbes, semi-permeable mucus, epithelium and stroma abundant in immune cells. Although tightly regulated, interactions between gut commensals and immune cells play indispensable roles in homeostasis and cancer pathogenesis in the body. Thus, there is a critical need to develop a robust model for the gut mucosal microenvironment. Here, we report our novel co-culture utilizing 3D Flipwell system for establishing the stratified layers of discrete mucosal components. This method allows for analyzing synchronous effects of test stimuli on gut bacteria, mucus, epithelium and immune cells, as well as their crosstalks. In the present report, we tested the immuno-stimulatory effects of sepiapterin (SEP, the precursor of the cofactor of nitric oxide synthase (NOS)—BH4) on the gut mucosal community. We previously reported that SEP effectively reprogrammed tumor-associated macrophages and inhibited breast tumor cell growth. In our co-cultures, SEP largely promoted mucus integrity, bacterial binding, and M1-like polarization of macrophages. Conversely, these phenomena were absent in control-treated cultures. Our results demonstrate that this novel co-culture may serve as a robust in vitro system to recapitulate the effects of pharmacological agents on the gut mucosal microenvironment, and could potentially be expanded to test the effects outside the gut

RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

IL-36 cytokines have recently emerged as mediators of inflammation in autoimmune conditions including psoriasis vulgaris (PsV) and generalized pustular psoriasis (GPP). This study used RNA-seq to profile the transcriptome of primary epidermal keratinocytes (KCs) treated with IL-1B, IL-36A, IL-36B, or IL-36G. We identified some early IL-1B-specific responses (8 h posttreatment), but nearly all late IL-1B responses were replicated by IL-36 cytokines (24 h posttreatment). Type I and II interferon genes exhibited time-dependent response patterns, with early induction (8 h) followed by no response or repression (24 h). Altogether, we identified 225 differentially expressed genes (DEGs) with shared responses to all 4 cytokines at both time points (8 and 24 h). These involved upregulation of ligands (IL1A, IL1B, and IL36G) and activating proteases (CTSS) but also upregulation of inhibitors such as IL1RN and IL36RN. Shared IL-1B/IL-36 DEGs overlapped significantly with genes altered in PsV and GPP skin lesions, as well as genes near GWAS loci linked to autoimmune and autoinflammatory diseases (e.g., PsV, psoriatic arthritis, inflammatory bowel disease, and primary biliary cholangitis). Inactivation of MyD88 adapter protein using CRISPR/Cas9 completely abolished expression responses of such DEGs to IL-1B and IL-36G stimulation. These results provide a global view of IL-1B and IL-36 expression responses in epidermal KCs with fine-scale characterization of time-dependent and cytokine-specific response patterns. Our findings support an important role for IL-1B and IL-36 in autoimmune or autoinflammatory conditions and show that MyD88 adaptor protein mediates shared IL-1B/IL-36 responses

Serum matrix metalloproteinase-9 in children exposed to arsenic from playground dust at elementary schools in Hermosillo, Sonora, Mexico

Arsenic exposure in adults has been associated with increased serum matrix metalloproteinase-9 (MMP-9), a biomarker which is associated with chronic respiratory disease, lung inflammation, cardiovascular disease and cancer. The objective of this study was to evaluate the association between serum MMP-9 levels in children, urinary arsenic, arsenic chronic daily intake (CDI) and arsenic exposure from playground dust. This cross-sectional study examined 127 children from five elementary schools, in Hermosillo, Sonora, Mexico. Arsenic was analyzed in the dust using a portable X-ray fluorescence (XRF) analyzer. Total urinary arsenic was determined by inductively coupled plasma/optical emission spectrometry. Serum was analyzed for MMP-9 using ELISA. Arsenic levels in playground dust averaged 16.9 +/- 4.6 mg/kg. Urinary arsenic averaged 34.9 +/- 17.1 mu g/L. Arsenic concentration in playground dust was positively associated with serum MMP-9 levels in crude analyses and after adjustment (P < 0.01), MMP-9 and CDI were positively associated only after adjustment (P < 0.01), and no association was found between MMP-9 and urinary arsenic. In conclusion, our study showed an association in children between serum MMP-9 levels and playground dust arsenic concentrations. Therefore, exposure to arsenic in dust where children spend significant time may manifest toxic effects.12 month embargo; published online: 1 August 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Calcitonin receptor plays a physiological role to protect against hypercalcemia in mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]-induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress