Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection

BACKGROUND Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, −10.1 percentage points; 95% confidence interval [CI], −15.9 to −4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, −9.9 percentage points; 95% CI, −15.5 to −4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, −11.6 percentage points; 95% CI, −17.4 to −5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, −10.7 percentage points; 95% CI, −16.4 to −5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239.

Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor.

International audienceFur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain

Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.

International audienceSelective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

International audienceThe Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment

IL-33 acts as a costimulatory signal to generate alloreactive Th1 cells in graft-versus-host disease.

Antigen-presenting cells (APCs) integrate signals emanating from local pathology and program appropriate T cell responses. In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases damage-associated molecular patterns (DAMPs) that generate proinflammatory APCs that secrete IL-12, which is a driver of donor Th1 responses, causing graft-versus-host disease (GVHD). Nevertheless, other mechanisms exist to initiate alloreactive T cell responses, as recipients with disrupted DAMP signaling or lacking IL-12 develop GVHD. We established that tissue damage signals are perceived directly by donor CD4+ T cells and promoted T cell expansion and differentiation. Specifically, the fibroblastic reticular cell-derived DAMP IL-33 is increased by recipient conditioning and is critical for the initial activation, proliferation, and differentiation of alloreactive Th1 cells. IL-33 stimulation of CD4+ T cells was not required for lymphopenia-induced expansion, however. IL-33 promoted IL-12-independent expression of Tbet and generation of Th1 cells that infiltrated GVHD target tissues. Mechanistically, IL-33 augmented CD4+ T cell TCR-associated signaling pathways in response to alloantigen. This enhanced T cell expansion and Th1 polarization, but inhibited the expression of regulatory molecules such as IL-10 and Foxp3. These data establish an unappreciated role for IL-33 as a costimulatory signal for donor Th1 generation after alloHCT

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

International audienc

Extensive analysis of the cytoplasmic proteome of human erythrocytes using the Peptide ligand library technology and advanced mass spectrometry

The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. Here we used a combinatorial library of hexameric peptides as a capturing agent to lower the signal of hemoglobin and amplify the signal of low to very low abundance proteins in the cytoplasm of human red blood cells (RBCs). Two types of hexapeptide library beads have been adopted: amino-terminal hexapeptide beads and beads in which the peptides have been further derivatized by carboxylation. The amplification of the signal of low abundance and suppression of the signal of high abundance species were fully demonstrated by two-dimensional gel maps and nano-LC-MSMS analysis. The effect of this new methodology on quantitative information also was explored. Moreover using this approach on an LTQ-Orbitrap mass spectrometer, we could identify with high confidence as many as 1578 proteins in the cytoplasmic fraction of a highly purified preparation of RBCs, allowing a deep exploration of the classical RBC pathways as well as the identification of unexpected minor proteins. In addition, we were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains. Thus, this extensive study provides a huge data set of proteins that are present in the RBC cytoplasm that may help to better understand the biology of this simplified cell and may open the way to further studies on blood pathologies using targeted approache

Themis2 lowers the threshold for B cell activation during positive selection

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is incompletely understood. Here we report that Themis2 increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. We found that Themis2 lowered the threshold for B cell activation by low but not high avidity antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer PLCγ2, and increased activation of PLCγ2 and its downstream pathways following B cell receptor stimulation. These findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality