This study shows that caspase-1 autocleavage, ASC speck assembly, and mature IL-1β and IL-18 secretion accompany rapid DPP8/DPP9-regulated pyroptosis induction in macrophages expressing a LeTx-responsive Nlrp1b allele.

BackgroundProlyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight.MethodsPRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0–29.9; 30.0–39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group.ResultsWhite blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found.ConclusionOur novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases.

The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTAplasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research.

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The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.

Coronavirus disease 2019 (COVID-19) is a viral lower respiratory tract infection caused by the highly transmissible and pathogenic SARS-CoV-2 (severe acute respiratory-syndrome coronavirus-2). Besides respiratory failure, systemic thromboembolic complications are frequent in COVID-19 patients and suggested to be the result of a dysregulation of the hemostatic balance. Although several markers of coagulation and fibrinolysis have been studied extensively, little is known about the effect of SARS-CoV-2 infection on the potent antifibrinolytic enzyme carboxypeptidase U (CPU). Blood was collected longitudinally from 56 hospitalized COVID-19 patients and 32 healthy controls. Procarboxypeptidase U (proCPU) levels and total active and inactivated CPU (CPU+CPUi) antigen levels were measured. At study inclusion (shortly after hospital admission), proCPU levels were significantly lower and CPU+CPUi antigen levels significantly higher in COVID-19 patients compared to controls. Both proCPU and CPU+CPUi antigen levels showed a subsequent progressive increase in these patients. Hereafter, proCPU levels decreased and patients were, at discharge, comparable to the controls. CPU+CPUi antigen levels at discharge were still higher compared to controls. Baseline CPU+CPUi antigen levels (shortly after hospital admission) correlated with disease severity and the duration of hospitalization. In conclusion, CPU generation with concomitant proCPU consumption during early SARS-CoV-2 infection will (at least partly) contribute to the hypofibrinolytic state observed in COVID-19 patients, thus enlarging their risk for thrombosis. Moreover, given the association between CPU+CPUi antigen levels and both disease severity and duration of hospitalization, this parameter may be a potential biomarker with prognostic value in SARS-CoV-2 infection.

Prolyl carboxypeptidase (PRCP) is involved in metabolic disorders by hydrolyzing anorexigenic peptides. A link between serum PRCP activity and obesity has been reported, but its origin/source is still unclear. Previously proven correlations between human serum PRCP activity and the amount of adipose tissue may suggest that adipose tissue is an important source of circulating PRCP. We investigated PRCP activity in visceral, subcutaneous adipose tissue (VAT and SCAT), skeletal muscle tissue and serum of lean and obese men with or without type 2 diabetes (T2D). Correlations between PRCP activity, metabolic and biochemical parameters and immune cell populations were assessed. PRCP activity was the highest in VAT, compared to SCAT, and was very low in skeletal muscle tissue in the overall group. Serum PRCP activity was significantly higher in T2-diabetic obese men, compared to lean and obese non-diabetic men, and was positively correlated with glycemic control. A positive correlation was observed between serum PRCP activity and VAT immune cell populations, which might indicate that circulating PRCP activity is deriving rather from the immune fraction than from adipocytes. In conclusion, PRCP activity was observed in human adipose tissue for the first time and serum PRCP activity is correlated with T2D in obese men.