Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

International audienceIn Brief 20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled pro-teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFN␥ or amplified in a 20% O 2 environment. Graphical Abstract Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O 2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity

Portrait of blood-derived extracellular vesicles in patients with Parkinson's disease.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) - for comparative purposes in individuals with another chronic neurodegenerative condition - and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD

Vitamin C alters the amount of specific endoplasmic reticulum associated proteins involved in lipid metabolism in the liver of mice synthesizing a nonfunctional Werner syndrome (Wrn) mutant protein

<div><p>Werner syndrome (WS) is a premature aging disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domain. Mice lacking the helicase domain of the Wrn protein orthologue exhibit transcriptomic and metabolic alterations, some of which are reversed by vitamin C. Recent studies on these animals indicated that the mutant protein is associated with enriched endoplasmic reticulum (ER) fractions of tissues resulting in an ER stress response. In this study, we identified proteins that exhibit actual level differences in the ER enriched fraction between the liver of wild type and Wrn mutant mice using quantitative proteomic profiling with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Multiple Reaction Monitoring (MRM) and immunoblotting were performed to validate findings in a secondary independent cohort of wild type and Wrn mutant mice. DAVID 6.7 (NIH) was used for functional annotation analysis and indicated that the identified proteins exhibiting level changes between untreated wild type, Wrn mutant, and vitamin C treated Wrn mutant mice (ANOVA <i>P</i>–value < 0.05) were involved in fatty acid and steroid metabolism pathways (Bonferroni <i>P</i>-value = 0.0137). Finally, when we compared the transcriptomic and the proteomic data of our mouse cohorts only ~7% of the altered mRNA profiles encoding for ER gene products were consistent with their corresponding protein profiles measured by the label-free quantification methods. These results suggest that a great number of ER gene products are regulated at the post-transcriptional level in the liver of Wrn mutant mice exhibiting an ER stress response.</p></div

List of proteins showing significant differential expression in our different mouse cohorts (or groups).

<p>Heatmap depicting the Z-score of log base ten of normalized intensities from the LFQ data for each protein (rows) between individual (columns) wild type and <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice treated with or without vitamin C. Columns and rows are reordered by hierarchical clustering using the genotype and vitamin C treatments. The protein names are indicated on the right with their protein ID numbers in parentheses.</p

Schematic representation of the different steps undertaken to obtain a list of protein differentially expressed between our different mouse cohorts at a statistical significant level.

<p>The number of proteins identified in each step is indicated in parentheses. WT = wild type mice; Wrn = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice; Wrn+VitC = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice treated with 0.4% vitamin C (w/v) in drinking water since weaning.</p

Validation of protein levels of various gene products in our different mouse cohorts by immunoblotting.

<p>(A) Example of western blots showing protein levels of Csad, Egfr, Fasn, and calreticulin in our mouse cohorts. (B) Signal intensity of ER marker calreticulin. (C) Ratio of Csad over calreticulin signal from the western blots. (Tukey post ANOVA test: †<i>P</i> < 0.05 compared to wild type mice). (D) Ratio of Egfr signal over calreticulin signal from the western blots. (ANOVA <i>P</i>-value = 0.07). (E) Ratio of Fasn signal over calreticulin signal from the western blots (ANOVA <i>P</i>-value = 0.33).</p

Impact of vitamin C on the levels of different ER stress markers in liver total lysates from <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice.

<p>(A) Example of western blots showing protein levels of total PERK, total IRE1α, and calreticulin in three mice of each group. β-actin was used as loading controls. (B) Ratio of PERK signal over β-actin signal from the western blots. (C) Ratio of total IRE1α signal over β-actin signal from the western blots. (ANOVA: <i>P</i> > 0.05; but student <i>t</i>-test: *<i>P</i> = 0.039 for <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice versus wild type mice). (D) Ratio of calreticulin signal over β-actin signal from the western blots. (E) Western blots showing protein levels of total eIF2α and its phosphorylated form in six animals of each group. (F) Ratio of phosphorylated eIF2α signal over total eIF2α signal. (Tukey post ANOVA test: **<i>P</i> = 0.016 compared to all other groups of mice). (G) Western blot showing protein levels of total GRP78 in three mice of each group. β-actin was used as loading controls. (H) Ratio of GRP78 signal over β-actin signal from the western blots. (I) Western blot showing protein levels of total HSC70 in three mice of each group. β-actin was used as loading controls. (J) Ratio of HSC70 signal over β-actin signal from the western blots. Bars in all histograms represent SEM.</p

List of proteins correlating with mRNA expression alteration in <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice versus wild type animals^*.

<p>List of proteins correlating with mRNA expression alteration in <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice versus wild type animals^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193170#t002fn001" target="_blank">*</a>}.</p

Efficiency of the liver ER enriched fractionation procedure on the different groups of mice.

<p>(A) Schematic representation of the different steps undertaken to obtain different cellular fractions. (B) Example of western blots showing protein levels of GRP78, HSC70, calreticulin, MnSOD, catalase, topoisomerase I, and SVCT1 in the different liver fractions. Each lane contains 15 μg of proteins. (H = whole cell homogenate; P = pellet fraction from the first step of the procedure; S = supernatant fraction of the last step of the procedure; ER = ER enriched fraction). (C) Principal component analysis (PCA) graph showing the consistency of the fractionation procedure when we measured the protein levels from the immunoblots shown in Fig 2B in the different fractions of vitamin C treated and untreated <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice compared to wild type mice. WT = wild type mice; Wrn = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice; Wrn+VitC = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice treated with 0.4% vitamin C (w/v) in drinking water since weaning. X and Y axis show principal component 1 and principal component 2 that explain 48.7% and 33.6% of the total variance, respectively. Prediction ellipses are such that a new observation from the same group will fall inside the ellipse with a probability of 0.95.</p