Sojin Shikano

Molecular Cloning of a Dendritic Cell-associated Transmembrane Protein, DC-HIL, That Promotes RGD-dependent Adhesion of Endothelial Cells through Recognition of Heparan Sulfate Proteoglycans

We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells, but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore, DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial migration of DC.

Expression of NF-κB in epidermis and the relationship between NF-κB activation and inhibition of keratinocyte growth

Summary Background Nuclear factor‐κB (NF‐κB) is a transcription factor involved in a number of signalling pathways in many cell types. NF‐κB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation.

Chronic inhibition of cardiac Kir2.1 and hERG potassium channels by celastrol with dual effects on both ion conductivity and protein trafficking

A high percentage of drugs and drug candidates has been found to cause cardiotoxicity by reducing potassium conductance, more commonly known as QT prolongation. However, some compounds do not show direct block of ionic flow, suggesting that other mechanisms may also lead to reduction of potassium currents. Using the functional recovery after chemobleaching (FRAC) assay, we have examined a collection of drugs and drug-like compounds for potential perturbation of cardiac potassium channel trafficking. Here we report that a significant number of inhibitory compounds displayed effects on channel expression on the cell surface. Further investigation of celastrol (3-hydroxy-24-nor-2-oxo-1 (10),3,5,7-friedelatetraen-29-oic acid), a cell-permeable dienonephenolic triterpene compound, revealed its potent inhibitory activity on both Kir2.1 and hERG potassium channels, causal to QT prolongation. In addition to acute block of ion conduction, celastrol also alters the rate of ion channel transport and causes a reduction of channel density on the cell surface. In contrast, celastrol has no effects on trafficking of either CD4 or CD8 membrane proteins. Furthermore, the potency for reducing surface expression is ∼5-10-fold more effective than that for either direct acute inhibition or reported cytoprotectivity via activation of the heat shock transcription factor 1. Because the reduction of potassium channel activity is a common form of druginduced cardiotoxicity, the potent inhibition of cell surface expression by celastrol underscores a need to evaluate drug candidates for their chronic effects on biogenesis of potassium channels. Our results suggest that chronic exposure to certain drugs may be an important aspect of acquired QT prolongation.

Genetic isolation of transport signals directing cell surface expression

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.

Epidermal Langerhans cell-targeted gene expression by a dectin-2 promoter

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5′-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV Co-receptor GPR15

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15S359A mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins

To maintain its functional integrity, a cell senses and reacts to the acute or chronic changes in the environment under physiological and pathological conditions. This typically involves cell surface membrane proteins such as receptors, ion channels, and structural proteins, whose surface expression level is regulated at multiple different steps of their biosynthesis and trafficking. Protein trafficking is mediated by a series of dynamic interactions between the sorting motifs of cargo proteins and the cellular machineries that recognize these motifs. While the constitutive trafficking of many cargo proteins relies on intrinsic sorting signals, post-translational modification of cargo proteins often serves as a key switch that enables the spatio-temporal regulation of their trafficking. Protein phosphorylation is one of the most intensively studied post-translational modifications that control the membrane trafficking. However, molecular mechanisms by which phosphorylation signal regulates the protein localization are diverse and remain not fully understood. The 14-3-3 proteins had been identified to specifically recognize phosphorylated serine or threonine residues, and thus represents one of the most distinct effector molecules that function downstream of the phosphorylation signal by kinases. This chapter will focus on the emerging role of 14-3-3 proteins in the phosphorylation-dependent control of cell surface membrane protein trafficking.