First large-scale comparative proteomics/phosphoproteomics study characterizing some of the key steps that contributed to the remodeling of phagosomes that occurred during evolution. Comparison of profiling analyses of isolated phagosomes from three distant organisms (Dictyostelium, Drosophila, and mouse) revealed a protein core that defines a potential ‘ancient' phagosome and a set of 50 proteins that emerged while adaptive immunity was already well established.Gene duplication events of mouse phagosome paralogs occurred mostly in Bilateria and Euteleostomi, coinciding with the emergence of innate and adaptive immunity, and thus, provided the functional innovations needed for the establishment of these two crucial evolutionary steps of the immune system.Phosphoproteomics of isolated phagosomes from the same three distant species indicate that the phagosome phosphoproteome has been extensively modified during evolution. Still, some phosphosites have been maintained for >1.2 billion years, and thus, highlight their particular significance in the regulation of key phagosomal functions.

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actindependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actinbinding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.

Actin dynamics and myosin contractile forces are necessary to form and close the phagocytic cup. A myosin I, MyoK, a myosin-Arp2/3 linker, Abp1, and a Rac-dependent kinase, PakB form a circuit that regulates phagocytosis. MyoK is phosphorylated by PakB and positively regulates uptake, whereas binding of Abp1 negatively regulates PakB and MyoK.

Proteomics analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present study describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3 to 5 exoribonuclease involved in ribosome biogenesis at the level of 5.8 S rRNA maturation, more specifically in the processing of the 12 S precursor rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Nuclei of eukaryotic cells are highly organized organelles in which more than 30 different kinds of transient structures, called nuclear domains or nuclear bodies, support the nuclear functions (1). Since the 1960s, it has been known that nucleoli, the most prominent of the nuclear domains, are the sites of ribosome biogenesis. This function gives rise to their characteristic ultrastructural organization in three main compartments, the fibrillar center and the dense fibrillar and granular components (2).Ribosome biogenesis is a very complex process that involves the synthesis of ribosomal RNAs (rRNAs) 1 coupled with their extensive processing, the assembly of these rRNAs with ribosomal proteins, and the export of the resulting preribosomal subunits from nuclei to cytoplasm where the final maturation occurs (3). rRNA processing mainly consists of base modifications and cleavages (4). Precursor rRNAs (pre-rRNAs) contain external and internal sequences that are not present within mature rRNAs. Removal of these sequences is achieved through sequential endonucleolytic and exonucleolytic cleavages, producing mature 18 S, 5.8 S, and 28 S rRNAs. Studies in yeast have identified several factors responsible for such cleavages: the ribonucleoprotein complex RNase MRP and Ngl2p catalyzing endoribonucleolytic processing, Xrn1p and Rat1p that are 5Ј to 3Ј exonucleases, Rex1p and Rex2p that are 3Ј to 5Ј exonucleases, and a protein complex named exosome composed of 3Ј to 5Ј exoribonucleases and putative RNA-binding proteins (5-9). However, the list of the catalytic factors required for the complete processing of pre-rRNAs into mature species is still incomplete.Correct rRNA maturation that provides finely folded and packaged mature rRNAs is crucial because it is highly probable that eukaryotic ribosomes are, as it has clearly been demonstrated for prokaryote ribosomes, ribozymes (10, 11). In yeast, incorrectly maturated rRNAs lead to translational infidelity of ribosomes (12). In mammals, defects in pre-rRNA processing are therefore likely to entail the development of severe pathologies. Indeed results on DKC1, the gene altered in dyskeratosis congenita, indicated that altered rRNA processing plays a direct role in tumorigenesis (13). Recently Gleizes and co-workers (14) showed that ribosome biogenesis and notably pre-rRNA processing...