Marjan Gucek

National Institutes of Health, 베서스다, Maryland, United States

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Publications (3)9.9 Total impact

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    ABSTRACT: CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163(-/-) mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163(-/-) mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163(-/-) mice had the same phenotype as HDM-challenged Cd163(-/-) mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163(-/-) mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163(-/-) mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163(-/-) mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.Mucosal Immunology advance online publication, 16 September 2015; doi:10.1038/mi.2015.94.
    Mucosal Immunology 09/2015; DOI:10.1038/mi.2015.94 · 7.37 Impact Factor
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    ABSTRACT: Background: Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. Results: Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. Conclusion: The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. Significance: Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.
    Journal of Biological Chemistry 04/2013; 288(19). DOI:10.1074/jbc.M113.466193 · 4.57 Impact Factor
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    ABSTRACT: Claudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localization and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used MS analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localized to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites which were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localization is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in TER. Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.
    Journal of Cell Science 07/2012; 125(20). DOI:10.1242/jcs.111237 · 5.43 Impact Factor