Apoptotic cells were shown to induce dendritic cell immune tolerance. We applied a proteomic approach to identify molecules that are secreted from apoptotic monocytes, and thus may mediate engulfment and immune suppression. Supernatants of monocytes undergoing apoptosis were collected and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and differentially expressed proteins were identified using tandem mass spectrometry. Thrombospondin-1 (TSP-1) and its cleaved 26-kDa heparin-binding domain (HBD) were identified. We show that TSP-1 is expressed upon induction of monocyte apoptosis in a caspase-dependent pattern and the HBD is cleaved by chymotrypsinlike serine protease. We further show that CD29, CD36, CD47, CD51, and CD91 simultaneously participate in engulfment induction and generation of an immature dendritic cell (iDC) tolerogenic and phagocytic state. We conclude that apoptotic cell TSP-1, and notably its HBD, creates a signalosome in iDCs to improve engulfment and to tolerate engulfed material prior to the interaction with apoptotic cells. IntroductionIn recent years, it has become apparent that upon induction of apoptosis, apoptotic cells play an active role in their own engulfment by signaling professional phagocytes and/or antigenpresenting cells, without triggering an inflammatory or autoimmune response. [1][2][3][4][5] This process seems to play an important role in homeostasis, resolution of inflammation, and peripheral tolerance induction. 4,[6][7][8] Apoptotic cells have been shown to signal the innate immune system in a variety of ways. "Eat me" signals on apoptotic cells serve as markers for phagocytes to specifically recognize these cells and subsequently ingest them. Such signals can appear on apoptotic cell membranes. Direct signals include alteration in cell surface phospholipid composition, 9 changes in cell surface glycoprotein expression, distinct alterations in cell surface charge, 10,11 or expression of specific molecules. 12 Alternatively, certain serum or phagocyte-derived proteins can opsonize an apoptotic cell surface and signal phagocytes to engulf the opsonized cells. 4,[13][14][15][16][17] Viable cells actively express "do not eat me" signals by restriction of phosphatydilserine to the inner leaflet of their membrane, or "stay away" signals using CD31 expression. 18 Recently, attention has been given not only to apoptotic cell membrane changes and phagocyte receptors, but also to the release of a membrane-derived phospholipid, lysophosphatidylcholine, which acts as a "find me" signal that is important for phagocytic cell recruitment. 19 Most of these mechanisms suggest efficient identification and clearance of cells undergoing apoptosis, with noninflammatory and nonautoimmune consequences.We decided to further explore whether apoptotic cells can actively express and secrete molecules that have a physiological significance for their own engulfment and for the environmental immune suppression. We examined whether apoptosis-induced immune suppress...

Presynaptic voltage-gated K ϩ (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kv␣1.1 and Kv␤ subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knockdown of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kv␤1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.

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Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.

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