BackgroundSerine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.MethodsEighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.ResultsEnzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.ConclusionsThis study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.
Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein-protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.
ObjectiveNeutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology.MethodsNETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed.ResultsWe found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers.ConclusionsOur results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.
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