Gamma-secretase is a functional component of phagosomes

Gamma-secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are gamma-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of gamma-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The gamma-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, gamma-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-gamma stimulates gamma-secretase-dependent cleavages on phagosomes and that gamma-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines.     

Structure-based model for light-harvesting properties of nucleic acid nanostructures

Programmed self-assembly of DNA enables the rational design of megadalton-scale macromolecular assemblies with sub-nanometer scale precision. These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient in silico evaluation of nucleic acid nanostructures for diverse light-harvesting and photonic applications.     

Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas

We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.     

Modulation of the phagosome proteome by interferon-gamma

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.     

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains

CXCR7 is an atypical chemokine receptor that signals through β-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of β-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 μM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.     

Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.Phagocytosis, the mechanism by which large particles are internalized, leads to the formation of phagosomes, a specialized organelle in which the engulfed material is degraded (1, 2). In mammals, various cells including macrophages, neutrophils and dendritic cells display remarkable phagocytic activities, rapidly eliminating microorganisms, foreign inert particles, and apoptotic cells. The killing of microorganisms by professional phagocytes precludes the emergence of infectious diseases. This innate immune process is followed by the degradation of microbes in a highly concentrated mixture of hydrolases, activated by the acidic pH generated in the phagosome lumen, generating antigenic peptides that are displayed at the cell surface, enabling their recognition by T lymphocytes (3). The peptides not loaded on MHC molecules are fully degraded in phagolysosomes and the end products are likely recycled from phagosomes by a variety of transporters (1). The establishment of these functional properties involves a complex remodeling of phagosomes, referred to as phagolysosome biogenesis (4, 5). This highly regulated process requires the fusion of nascent phagosomes with trans Golgi-derived vesicles, early endosomes, late endosomes and ultimately lysosomes (1, 2). These fusion events are believed to alter significantly the proteome of phagosomes during phagolysosome biogenesis and regulate their functional properties (6).The capacity to kill and degrade microbes is one of the many functions that phagosomes acquire during phagolysosome biogenesis. In a previous study, we identified more than 140 proteins associated with phagosomes (7), leading to the proposal of novel mechanisms to explain phagosomal functions such as antigen cross-presentation (8). This proteomics study also shown the presence on phagosomes of proteins known to segregate into lipid rafts at the cell surface, such as flotillin-1 and prohibitin, leading to the proposal that membrane microdomains might also assemble on phagosomes. At the plasma membrane, these structures constitute foci of specialized functions, notably for signal transduction (9). Further biochemical and morphological analyses confirmed the presence of membrane microdomains on phagosomes (10). The role of membrane microdomains and the molecular nature of these structures in phagosomes is still poorly understood. Recent data indicated that two phagosomal protein complexes, V-ATPase and NADPH oxidase may use membrane microdomains as assembly platforms (11). Furthermore, the potential involvement of phagosome microdomains in innate immunity was highlighted by the finding that at least two unrelated pathogens, the Gram-negative bacteria Brucella and the intracellular parasite Leishmania donovani, target phagosome lipid rafts as a strategy to evade host-defense mechanisms (12–14). Hence, the molecular characterization of the detergent-soluble and -insoluble fractions isolated from phagosomes should provide unique insights into the mechanisms used by pathogens to alter the functional properties of this organelle. Different approaches have been used to study membrane microdomains, including imaging techniques such as fluorescence resonance energy transfer, fluorescence photoactivation localization microscopy, as well as cell fractionation procedures using non-ionic detergents to enrich detergent-resistant membrane domains (15). Imaging approaches highlighted the fact that cholesterol-enriched membrane microdomains are dynamic microscopic structures of less than 20 nm in range. On the other hand, detergent-based fractionation approaches have been extensively used to identify key components of membrane microdomains, including series of signaling factors (16–18). Although the exact nature and the level of correspondence of the membrane microdomains studied by the morphological and biochemical approaches is still actively debated, similar sets of proteins have been identified in these structures (15).In the present study we used quantitative proteomics approach to characterize, for the first time, the modifications of lipid rafts proteins occurring during the biogenesis of an intracellular organelle. Our data indicate that segregation of sets of proteins in sub-regions of the phagosome membrane occurs throughout the biogenesis and maturation of phagolysosome, introducing the concept that spatiotemporal reorganization of the phagosome proteome plays a key role in the establishment of the functional properties of this organelle.     

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca²⁺-independent, as inhibition of Ca²⁺ channels or culture in the absence of Ca²⁺ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.