Immuno-purification of a dimeric subcomplex of the respiratory NAHD-CoQ reductase of Rhodobacter capsulatus equivalent to the FP fraction of the mitochondrial complex I

AbstractThe Rhodobacter capsulatus genes encoding the NUOE and NUOF subunits, equivalent to the 24 kDa and 51 kDa subunits of the mammalian mitochondrial complex I, have been sequenced. According to the nucleotide sequence, the NUOE subunit is 389 amino acids long and has a molecular mass of 41.3 kDa. In comparison to the mitochondrial equivalent subunit, NUOE is extended at the C terminus by more than 150 amino acids. The NUOF subunit is 431 amino acids long and has a molecular mass of 47.1 kDa. A subcomplex containing both the NUOE and NUOF subunits was extracted by detergent treatment of R. capsulatus membranes and immuno-purified. This subcomplex is homologous to the mitochondrial FP fragment. Mass spectrometry after trypsin treatment of the NUOE subunit validates the atypical primary structure deduced from the sequence of the gene

Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

Détection et quantification par PCR en temps réel et balises moléculaires du virus de l'hépatite B et de ses variants résistants à la Lamivudine

PARIS-BIUP (751062107) / SudocSudocFranceF

Affinity Purification of Soluble Lysosomal Proteins for Mass Spectrometric Identification

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Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells

International audienc

Towards a human repertoire of monocytic lysosomal proteins

International audienceThe lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one‐dimensional (1‐D) and two‐dimensional (2‐D) gel electrophoresis, protein identification by N‐terminal sequencing, matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2‐D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH$_4$Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose‐6‐phosphate, they were purified on an affinity support bearing mannose‐6‐phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well‐known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A‐stimulated genes) are described here for the first time as mannose‐6‐phosphate‐containing proteins

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

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry

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Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins

International audienceAs a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid-interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure