Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicle biogenesis – the routes of vesicular export are adaptable.

Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well‐characterized “real life” sample that can be used as reference standard in urine clinical proteomics studies.Experimental design: We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE‐MS, LC‐MS, LC‐MS/MS, 1‐D gel analysis in combination with nano‐LC MS/MS (using LTQ‐FT ultra), and 2‐DE‐MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds.Results: The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in “inter‐laboratory” and “inter‐platform” data comparison is also demonstrated.Conclusions and clinical relevance: These well‐characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

Techniques in mass spectrometry (MS) combined with chemical cross‐linking have proven to be efficient tools for the rapid determination of low‐resolution three‐dimensional (3‐D) structures of proteins. The general procedure involves chemical cross‐linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross‐linked proteins makes it difficult to identify relevant intermolecular cross‐linked peptides from MS data. We present a method for mapping low‐resolution 3‐D protein structures by combining chemical cross‐linking with high‐resolution FTICR (Fourier transform ion‐cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo‐bifunctional, amine‐reactive cross‐linking reagents that bridge distances from 6 to 16 Å. The non‐digested cross‐linking reaction mixtures were monitored by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) to determine the extent of cross‐linking. Enzymatically digested reaction mixtures were separated by nano‐high‐performance liquid chromatography (nano‐HPLC) on reverse‐phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano‐HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano‐ESI (electrospray ionization) source. Cross‐linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross‐linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross‐linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme. Copyright © 2003 John Wiley & Sons, Ltd.

Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGFß1, AgII or PDGF stimulation of renal cells resulted in ER-stress followed by activation of the protective unfolded protein response pathway and high secretory level of protein disulfide isomerase ERP57. The TGFß1 impairment of ER-function could be reversed by BMP7 treatment suggesting a specific involvement in renal fibrosis. Clear correlation between the degree of fibrosis, ER-stress and ERP57 level could be evidenced in fibrosis animal models and in biopsies of renal fibrosis patient's. Protein interaction studies revealed that secreted ERP57 exhibit a strong interaction with ECM proteins. Knockdown of ERP57 or antibody targeted inhibition of the secreted form impaired significantly the secretion and accumulation of ECM. Moreover ERP57 was excreted in earlier stage of chronic kidney diseases and its level in urine correlated with the degree of renal fibrosis suggesting that the ERP57 secretion may represent one of the first signs of renal fibrosis onset and progression.

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-β–mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.

Malignant cells differ from benign ones in their metabolome and it is largely unknown whether this difference is reflected in the metabolic profile of their microvesicles (MV), which are secreted into the blood of cancer patients. Here, they are present together with MV from the various blood and endothelial cells. Harvesting MV from 78 breast cancer patients (BC) and 30 controls, we characterized the whole blood MV metabolome using targeted and untargeted mass spectrometry. Especially (lyso)-phosphatidylcholines and sphingomyelins were detected in a relevant abundance. Eight metabolites showed a significant discriminatory power between BC and controls. High concentrations of lysoPCaC26:0 and PCaaC38:5 were associated with shorter overall survival. Comparing BC subtype-specific metabolome profiles, 24 metabolites were differentially expressed between luminal A and luminal B. Pathway analysis revealed alterations in the glycerophospholipid metabolism for the whole cancer cohort and in the ether lipid metabolism for the molecular subtype luminal B. Although this mixture of blood-derived MV contains only a minor number of tumor MV, a combination of metabolites was identified that distinguished between BC and controls as well as between molecular subtypes, and was predictive for overall survival. This suggests that these metabolites represent promising biomarkers and, moreover, that they may be functionally relevant for tumor progression.