Sphingosine 1-phosphate modulates antigen capture by murine langerhans cells via the S1P2 receptor subtype

Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions

The mechanism of nucleosome traversal by RNA polymerase II: Roles for template uncoiling and transcript elongation factors

RNA polymerase II traverses nucleosomes rapidly and efficiently in the cell but it has not been possible to duplicate this process in the test tube. A single nucleosome has generally been found to provide a strong barrier to transcript elongation in vitro. Recent studies have shown that effective transcript elongation can occur on nucleosomal templates in vitro, but this depends on both facilitated uncoiling of DNA from the octamer surface and the presence of transcription factors that maintain polymerase in the transcriptionally competent state. These findings indicate that the efficiency and rate of transcription through chromatin could be regulated through controlled DNA uncoiling. These studies also demonstrate that nucleosome traversal need not result in nucleosome displacement

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

<div><p>Pathogenic bacteria may modify their surface to evade the host innate immune response. <em>Yersinia enterocolitica</em> modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, <em>Y. enterocolitica</em> expresses a tetra-acylated lipid A consistent with the 3′-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that <em>Y. enterocolitica</em> encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo₂-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of <em>lpxR</em> is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of <em>Y. enterocolitica</em> virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the <em>lpxR</em> mutant grown at 21°C. Mechanistically, our data revealed that the expressions of <em>flhDC</em> and <em>rovA</em>, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the <em>lpxR</em> mutant. Finally, we establish that the low inflammatory response associated to <em>Y. enterocolitica</em> infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.</p> </div

Transition Step during Assembly of HIV Tat:P-TEFb Transcription Complexes and Transfer to TAR RNA

Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription