Cell-binding IgM in CSF is distinctive of multiple sclerosis and targets the iron transporter SCARA5.

Intrathecal IgM production in multiple sclerosis is associated with a worse disease course. To investigate pathogenic relevance of autoreactive IgM in multiple sclerosis, CSF from two independent cohorts, including multiple sclerosis patients and controls, were screened for antibody binding to induced pluripotent stem cell-derived neurons and astrocytes, and a panel of CNS-related cell lines. IgM binding to a primitive neuro-ectodermal tumour cell line discriminated 10% of multiple sclerosis donors from controls. Transcriptomes of single IgM producing CSF B cells from patients with cell-binding IgM were sequenced and used to produce recombinant monoclonal antibodies for characterization and antigen identification. We produced five cell-binding recombinant IgM antibodies, of which one, cloned from an HLA-DR + plasma-like B cell, mediated antigen-dependent complement activation. Immunoprecipitation and mass spectrometry, and biochemical and transcriptome analysis of the target cells identified the iron transport scavenger protein SCARA5 as the antigen target of this antibody. Intrathecal injection of a SCARA5 antibody led to an increased T cell infiltration in an experimental autoimmune encephalomyelitis (EAE) model. CSF IgM might contribute to CNS inflammation in multiple sclerosis by binding to cell surface antigens like SCARA5 and activating complement, or by facilitating immune cell migration into the brain.

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (PflaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila.

Correction: Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011.

[This corrects the article DOI: 10.1371/journal.pone.0048494.].

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.

Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.

Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15)N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.