Mass spectrometry‐based secretome analysis of non‐small cell lung cancer cell lines

Tyrosine kinase inhibitors, such as erlotinib, display reliable responses and survival benefits for the treatment of human non‐small cell lung cancer (NSCLC) patients. However, primary or acquired resistance limits their therapeutic success. In this study, we conducted in‐depth mass spectrometric analyses of NSCLC cell secretomes. To identify secreted proteins that are differentially regulated in erlotinib‐sensitive (PC‐9) and ‐resistant (PC‐9ER) NSCLC cell lines, SILAC experiments were performed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins. Fourteen proteins were found to be differently regulated among erlotinib‐sensitive and ‐resistant NSCLC cell lines, with five proteins (tissue‐type plasminogen activator, epidermal growth factor receptor, urokinase‐type plasminogen activator, platelet‐derived growth factor D, and myeloid‐derived growth factor) showing the most prominent regulation. Tissue‐type plasminogen activator (t‐PA) was up to 10‐times upregulated in erlotinib‐resistant NSCLC cells compared with erlotinib‐sensitive cells. T‐PA is an established tumor marker for various cancer types and seems to be a promising prognostic marker to differentiate erlotinib‐sensitive from erlotinib‐resistant NSCLC cells. To gain further insights into t‐PA‐regulated pathways, a t‐PA variant was expressed in E. coli cells and its interactions with proteins secreted from erlotinib‐sensitive and ‐resistant NCSLC cells were studied by a combined affinity enrichment chemical cross‐linking/mass spectrometry (MS) approach. Fourteen proteins were identified as potential t‐PA interaction partners, deserving a closer inspection to unravel the mechanisms underlying erlotinib resistance in NSCLC cells.     

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry

This study aimed at establishing a sensitive multiple reaction monitoring‐mass spectrometry (MRM‐MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM‐MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM‐MS measurement were also determined. The method was applied to liver samples from ten non‐cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM‐based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM‐MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.     

Type 1 diabetes cadaveric human pancreata exhibit a unique exocrine tissue proteomic profile

Type 1 diabetes (T1D) is an autoimmune disorder resulting from a self‐destruction of pancreatic islet beta cells. The complete proteome of the human pancreas, where both the dysfunctional beta cells and their proximal environment co‐exist, remains unknown. Here, we used TMT10‐based isobaric labeling and multidimensional LC‐MS/MS to quantitatively profile the differences between pancreatic head region tissues from T1D (N = 5) and healthy subjects (N = 5). Among the 5357 (1% false discovery rate) confidently identified proteins, 145 showed statistically significant dysregulation between T1D and healthy subjects. The differentially expressed pancreatic proteome supports the growing notion of a potential role for exocrine pancreas involvement in T1D. This study also demonstrates the utility for this approach to analyze dysregulated proteins as a means to investigate islet biology, pancreatic pathology and T1D pathogenesis.     

Identification of lysosomal Npc1‐binding proteins: Cathepsin D activity is regulated by NPC1

Niemann–Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1‐binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032–1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC‐MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.     

Exposure of CCRF‐CEM cells to acridone derivative 8a triggers tumor death via multiple mechanisms

A newly synthesized acridone derivative 8a shows potent antitumor activity against CCRF‐CEM leukemia cells. Herein, the first proteomic study of 8a effects in CCRF‐CEM cells was performed by 2D nano‐LC‐ESI‐MS/MS to better understand the mechanisms of action of 8a . Data analyses based on PLGS, STRING, Cytoscape, and database for annotation, visualization, and integrated discovery identified 55 proteins that were differentially expressed in response to 8a exposure. Multiple cellular pathways were affected, including chromatin organization, energy metabolism, DNA repair, oxidative‐stress, and apoptosis. The changes in protein expression were further verified for PKM2. Moreover, 8a lowered down the expression of HEX and PFK‐1. Lactate production was decreased in 8a‐ treated cells, indicating suppression of glycolysis. The elevated XRCC6 and decreased histone expression levels suggested increased DNA damage in 8a‐ treated cells, which was confirmed by the increased γ‐H2AX foci. Molecular docking of 8a with DNA demonstrated direct interactions of 8a with DNA through three hydrogen bonds and four π–π interactions, potentially explaining the mode of action that 8a damaged to DNA. The differential protein profiling and dysfunction of metabolic pathways induced by 8a provide novel insights into the potential action mechanisms of 8a .     

Quantitative proteomics suggests metabolic reprogramming during ETHE1 deficiency

Deficiency of mitochondrial sulfur dioxygenase (ETHE1) causes the severe metabolic disorder ethylmalonic encephalopathy, which is characterized by early‐onset encephalopathy and defective cytochrome C oxidase because of hydrogen sulfide accumulation. Although the severe systemic consequences of the disorder are becoming clear, the molecular effects are not well defined. Therefore, for further elucidating the effects of ETHE1‐deficiency, we performed a large scale quantitative proteomics study on liver tissue from ETHE1‐deficient mice. Our results demonstrated a clear link between ETHE1‐deficiency and redox active proteins, as reflected by downregulation of several proteins related to oxidation‐reduction, such as different dehydrogenases and cytochrome P450 (CYP450) members. Furthermore, the protein data indicated impact of the ETHE1‐deficiency on metabolic reprogramming through upregulation of glycolytic enzymes and by altering several heterogeneous ribonucleoproteins, indicating novel link between ETHE1 and gene expression regulation. We also found increase in total protein acetylation level, pointing out the link between ETHE1 and acetylation, which is likely controlled by both redox state and cellular metabolites. These findings are relevant for understanding the complexity of the disease and may shed light on important functions influenced by ETHE1 deficiency and by the concomitant increase in the gaseous mediator hydrogen sulfide. All MS data have been deposited in the ProteomeXchange with the dataset identifiers PXD002741 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ) and PXD002742 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ).     

Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams‐per‐litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial‐scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so‐called quality control proteases appears to influence cell‐wall synthesis, resulting in the induction of the cell‐wall stress regulon that encodes another quality control protease.