Dynamique de l'Onzin au sein de la voie endocytaire : une étude biochimique et morphologique.

A proteomic analysis of the membrane fraction of rat liver highly enriched in lysosomes lead to the discovery of Onzin among the most abundant proteins. We showed that this small highly conserved cystein rich protein is mainly present in the immune organs and in the digestive tract. The intracellular localization of Onzin in mouse liver has been studied using centrifugation techniques, especially inducing in vivo, with different substances (yeast invertase, Triton WR-1339 and latex beads), a shift in the density of lysosomes and phagosomes. We conclude that Onzin is present in the endocytic pathway, associated with a compartment in dynamic relationship with the lysosomes of Kupffer cells. We confirmed those results by biochemical and morphological approaches using mouse peritoneal macrophages.Une analyse protéomique de la fraction membranaire d'un échantillon foie de rat fortement enrichi en lysosomes a mené à la découverte de l'Onzin parmi les protéines les plus abondantes. Nous avons montré que cette petite protéine riche en cystéines et fortement conservée entre différentes espèces est majoritairement présente dans les organes lymphoïdes et dans le tube digestif. La distribution intracellulaire de l'Onzin dans le foie de souris a été étudiée par des techniques de centrifugation, notamment en induisant in vivo par différentes substances (invertase de levure, Triton WR-1339 et billes de latex) le changement de la densité des lysosomes et des phagosomes. Nous déduisons de ces expériences que l'Onzin est associée à un compartiment de la voie endocytaire en relation dynamique avec le lysosome des cellules de Kupffer. Ces résultats ont également été confirmés et précisés par des approches biochimiques et morphologiques en utilisant des macrophages péritonéaux de souris

Dynamique de l'Onzin au sein de la voie endocytaire (une étude biochimique et morphologique)

Une analyse protéomique de la fraction membranaire d'un échantillon foie de rat fortement enrichi en lysosomes a mené à la découverte de l'Onzin parmi les protéines les plus abondantes. Nous avons montré que cette petite protéine riche en cystéines et fortement conservée entre différentes espèces est majoritairement présente dans les organes lymphoïdes et dans le tube digestif. La distribution intracellulaire de l'Onzin dans le foie de souris a été étudiée par des techniques de centrifugation, notamment en induisant in vivo par différentes substances (invertase de levure, Triton WR-1339 et billes de latex) le changement de la densité des lysosomes et des phagosomes. Nous déduisons de ces expériences que l'Onzin est associée à un compartiment de la voie endocytaire en relation dynamique avec le lysosome des cellules de Kupffer. Ces résultats ont également été confirmés et précisés par des approches biochimiques et morphologiques en utilisant des macrophages péritonéaux de souris.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)

Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026–1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein ‘p76’ and it appeared later in the NCBI protein database as the ‘hypothetical protein LOC196463’. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ~20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes