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Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC 16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABC C4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

It is a common belief that reduction of disulfide bridges and alkylation of thiols in proteins are indispensable steps in proteomic sample preparation. Since this chemical procedure is often incomplete and prone to side reactions we reexamined its importance. We found that reduction and alkylation do not increase the depth of analysis and quality of proteomic quantification and therefore these steps are not essential in 'shotgun'-type investigations of proteomes. Moreover, we found that compared to a standard procedure using iodoacetamide for thiol-alkylation, sample preparation under conditions protecting thiols from oxidation improves quality of peptides and allows identifying of 10-20% more peptides and proteins. Excluding thiol-alkylation from proteomic sample preparation shortens the workflows and decreases the probability of biases resulting from occurrence of artificially modified peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Jusvinza is an immunomodulatory drug composed of an altered peptide ligand (APL) designed from a novel CD4+ T cell epitope of human heat shock protein 60 (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). The peptide induces regulatory T cells and decreases levels of TNF-α and IL-17; pre-clinical and phase I clinical studies support its use for the treatment of RA. This peptide was repositioned for the treatment of COVID-19 patients with signs of hyperinflammation. Neutrophils play a pathogenic role in both RA and severe forms of COVID-19. To add novel evidence about the mechanism of action of Jusvinza, the proteomic profile regulated by this peptide of neutrophils isolated from four RA patients was investigated using LC-MS/MS and bioinformatics analysis. A total of 149 proteins were found to be differentially modulated in neutrophils treated with Jusvinza. The proteomic profile regulated by Jusvinza is characterized by the presence of proteins related to RNA splicing, phagocytosis, endocytosis, and immune functions. In response to Jusvinza treatment, several proteins that regulate the NF-κB signaling pathway were differentially modulated, supporting the peptide's anti-inflammatory effect. Proteins related to metabolic pathways that supply ATP for cellular functions or lipid metabolites with immunoregulatory properties were also identified. Additionally, several structural components of neutrophil extracellular traps (NETs) were decreased in Jusvinza-treated cells, supporting its impairment of this biological process. Of note, these findings were validated by in vitro experiments which confirmed that Jusvinza decreased NET formation. Such results provide evidence of the molecular mechanism of action and support the therapeutic potentialities of Jusvinza to treat other diseases characterized by hyperinflammation besides RA and COVID-19. [ABSTRACT FROM AUTHOR]

Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70. [Display omitted] • Proteomics unveils molecular pathways that compensate for proteasome inhibition • HSP70 family chaperones control the major compensatory pathways • The proteasome transcriptional bounce-back, induced by NRF1/2, is independent of HSP70 • Combining proteasome, HSP70, and NRF1 inhibitors is a promising anti-cancer strategy Cancer cells are addicted to the proteasome. They activate compensatory molecular pathways upon a proteasome inhibition. Oroń et al. find that HSP70 family chaperones control the most prominent of these pathways except for the proteasome transcriptional bounce-back, induced by NRF1/2. Thus, the combination of the proteasome, HSP70, and NRF1 inhibitors is a promising anti-cancer therapeutic strategy. [ABSTRACT FROM AUTHOR]

It has been reported that 476 proteins can be detected in plasma fibrin clots from patients with venous thromboembolism. Plasma fibrin clots proteomic composition in relation to their properties has not been studied in acute pulmonary embolism (PE). Clots generated from plasma of 20 PE patients and 20 healthy controls were assessed using mass spectrometry, clot permeability (K s), and clot lysis time (CLT). The proteomic composition of plasma fibrin clots from acute PE patients differed from that of control subjects in regard to 198 clot-bound proteins. In the acute PE group, we observed increased clot-bound fibrinogen, apolipoprotein B-100, platelet glycoprotein Ib, lipopolysaccharide-binding protein, and histones H3 + 4 and reduced fibronectin, α2-antiplasmin, α2-macroglobulin, factor (F)XIII, histidine-rich glycoprotein, antithrombin, von Willebrand Factor, plasminogen, and prothrombin. Among PE patients, low K s (≤3.83 × 10−9 cm2) was associated with increased clot-bound C-reactive protein, kininogen-1, protein S, β-2-microglobulin, and thromboxane-A synthase when compared with patients having K s > 3.83 × 10−9 cm2. K s correlated inversely with FIX and FV, thrombin-activatable fibrinolysis inhibitor, complement C1s, C7, C8, and apolipoprotein A-I. The specific protein composition in plasma fibrin clots from acute PE patients is associated with denser clot formation. Several proteins unrelated to the coagulation system can modulate fibrin phenotype in acute thrombotic states. Our study significantly advances the field of thrombosis and hemostasis. The plasma fibrin clot proteomics findings fill the gap of knowledge about the presence and the role of other proteins to the plasma fibrin clot in the acute phase of pulmonary embolism, aside fibrinogen, which is the main component of fibrin. The reported methodology, which involves the sample preparation using Multienzyme Digestion-Filter Aided Sample Preparation (MED FASP), data acquisition with the Quadrupole-Orbitrap mass spectrometer, and data analysis using the advanced tools such as MaxQuant, Total Protein Approach and Perseus, allows to gain not only the qualitative, but also the quantitative insights into the microworld of proteins entangled among the fibrin network. By comparing the clots formed from plasma of patients with acute pulmonary embolism with the clots from healthy control, we provide the specific protein composition associated with unfavorable clot properties observed in this disease. Moreover, our findings emphasize that several proteins unrelated to the coagulation system, can modulate fibrin phenotype in acute thrombotic states. Unlabelled Image • Fibrin clot proteome differs between pulmonary embolism patients and healthy controls. • In pulmonary embolism, clots proteome is associated with fibrin clot properties. • Proteins unrelated to coagulation modulate fibrin phenotype in acute thrombosis. [ABSTRACT FROM AUTHOR]