Dendritic cells constitutively secrete a population of small (50–90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1α) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

No abstract

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSDlocalized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission. Neurons are highly polarized cells, specializing in the reception of numerous, independent signal inputs and rapid integration of these inputs into an electrochemical response. The major sites of signal input are synapses, which are highly ordered cell junctions formed between two neurons and are typically unidirectional in fast excitatory chemical neurotransmission in the mammalian CNS. The response to neurotransmitter (NT) 1 release at the synapse is provided by a protein matrix of NT receptors and supporting proteins collectively known as the postsynaptic density (PSD) (for review, see Refs. 1-3). The PSD has several proposed functions including: signal amplification, cytoskeletal anchorage, biochemical signaling regulation, and NT receptor clustering (1, 4 -6).Changes in size and composition of the PSD correlate with changes in synaptic strength (7,8), including alterations that are stably maintained such as long-term potentiation (LTP), a physiologically relevant increase in synaptic efficacy and a model for learning and memory (9, 10). Therefore, an understanding of the protein composition of the PSD is a prerequisite for modeling the molecular interactions regulating synaptic strength.The structure of PSDs purified from rodent brains using gradient centrifugation and Triton X-100 extraction has been shown by electron microscopy (EM) to be virtually identical to the "in vivo" PSD structure (4, 11). Gel electrophoresis, enzymatic activity assays, and EM experiments have demonstrated that this procedure yields a highly pure, membranefree PSD fraction (11,12). Recent proteomic studies have investigated the composition of the PSD by SDS-PAGE or two-dimensional gel electrophoresis (2DE) coupled with MS (13-16). Li et al. (16) also performed shotgun proteomics using cysteine-containing peptides selected using ICAT techniques. However, each of these investigations identified less than one-third of previously described and biochemically confirmed PSD components, pointing to limitations in the techniques used. A recent paper by Yoshimura et al. (17) reports the identification by mass spectrometry of 492 proteins in the PSD, which suggests that the PSD is more ...

Exosomes are nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types. They represent an "in vitro" discovery, but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition is unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. The potential of dendritic cell-derived exosomes (Dex) as cell-free cancer vaccines is addressed in this review. Lessons learned from the pioneering clinical trials allowed reassessment of the priming capacities of Dex in preclinical models, optimizing clinical protocols, and delineating novel, biological features of Dex in cancer patients.

No abstract

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.