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Objectives: This study evaluated the plasma concentration of prolylcarboxypeptidase (PRCP) and its clinical relevance in patients with idiopathic acute optic neuritis (ON). Methods: We investigated the expression of PRCP in the optic nerves of experimental autoimmune optic neuritis (EAON)-induced mice. Peripheral blood samples were collected from ON patients (n = 20) and healthy controls (n = 20). ELISA was used to measure the plasma PRCP levels. We performed measurements of visual acuity and the mean thicknesses of the macular ganglion cell layer plus inner plexiform layer (GCL+IPL) at diagnosis and 6 months after diagnosis. Results: The PRCP mRNA expression in EAON-induced mice was markedly higher than that in naïve mice. The mean plasma PRCP level was significantly higher in patients with ON than in controls. Plasma PRCP levels were negatively correlated with logMAR visual acuity at 6 months after diagnosis and differences in macular GCL+IPL thickness during an ON attack. A plasma PRCP level of 49.98 (pg/mL) predicted the recurrence of ON with a 75% sensitivity and 87.5% specificity. Conclusions: Patients with idiopathic acute ON had higher plasma PRCP levels, and this was positively correlated with final visual outcome and well-preserved macular GCL+IPL thickness during an ON attack. The increase in plasma PRCP level may reflect its compensatory secretion to counteract neuroinflammation in ON patients.

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BACKGROUND: We examined how prekallikrein (PK) activation on human microvascular endothelial cells (HMVECs) is regulated by the ambient concentration of C1 inhibitor (C1INH) and prolylcarboxypeptidase (PRCP). OBJECTIVE: We sought to examine the specificity of PK activation on HMVECs by PRCP and the role of C1INH to regulate it, high-molecular-weight kininogen (HK) cleavage, and bradykinin (BK) liberation. METHODS: Investigations were performed on cultured HMVECs. Immunofluorescence, enzymatic activity assays, immunoblots, small interfering RNA knockdowns, and cell transfections were used to perform these studies. RESULTS: Cultured HMVECs constitutively coexpressed PK, HK, C1INH, and PRCP. PK activation on HMVECs was modulated by the ambient C1INH concentration. In the absence of C1INH, forming PKa on HMVECs cleaved 120-kDa HK completely to a 65-kDa H-chain and a 46-kDa L-chain in 60 minutes. In the presence of 2 µM C1INH, only 50% of the HK became cleaved. C1INH concentrations (0.0-2.5 µM) decreased but did not abolish BK liberated from HK by activated PK. Factor XII did not activate when incubated with HMVECs alone for 1 hour. However, if incubated in the presence of HK and PK, factor XII became activated. The specificity of PK activation on HMVECs by PRCP was shown by several inhibitors to each enzyme. Furthermore, PRCP small interfering RNA knockdowns magnified C1INH inhibitory activity on PK activation, and PRCP transfections reduced C1INH inhibition at any given concentration. CONCLUSIONS: These combined studies indicated that on HMVECs, PK activation and HK cleavage to liberate BK were modulated by the local concentrations of C1INH and PRCP.

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Emerging evidence demonstrates the vital role of synaptic transmission and structural remodeling in major depressive disorder. Activation of melanocortin receptors facilitates stress-induced emotional behavior. Prolylcarboxypeptidase (PRCP) is a serine protease, which splits the C-terminal amino acid of α-MSH and inactivates it. In this study, we asked whether PRCP, the endogenous enzyme of melanocortin system, might play a role in stress susceptibility via regulating synaptic adaptations. Mice were subjected to chronic social defeat stress (CSDS) or subthreshold social defeat stress (SSDS). Depressive-like behavior was assessed in SIT, SPT, TST and FST. Based on to behavioral assessments, mice were divided into the susceptible (SUS) and resilient (RES) groups. After social defeat stress, drug infusion or viral expression and behavioral tests, morphological and electrophysiological analysis were conducted in PFX-fixed and fresh brain slices containing the nucleus accumbens shell (NAcsh). We showed that PRCP was downregulated in NAcsh of susceptible mice. Administration of fluoxetine (20 mg·kg-1·d-1, i.p., for 2 weeks) ameliorated the depressive-like behavior, and restored the expression levels of PRCP in NAcsh of susceptible mice. Pharmacological or genetic inhibition of PRCP in NAcsh by microinjection of N-benzyloxycarbonyl-L-prolyl-L-prolinal (ZPP) or LV-shPRCP enhanced the excitatory synaptic transmission in NAcsh, facilitating stress susceptibility via central melanocortin receptors. On the contrary, overexpression of PRCP in NAcsh by microinjection of AAV-PRCP alleviated the depressive-like behavior and reversed the enhanced excitatory synaptic transmission, abnormal dendritogenesis and spinogenesis in NAcsh induced by chronic stress. Furthermore, chronic stress increased the level of CaMKIIα, a kinase closely related to synaptic plasticity, in NAcsh. The elevated level of CaMKIIα was reversed by overexpression of PRCP in NAcsh. Pharmacological inhibition of CaMKIIα in NAcsh alleviated stress susceptibility induced by PRCP knockdown. This study has revealed the essential role of PRCP in relieving stress susceptibility through melanocortin signaling-mediated synaptic plasticity in NAcsh.

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BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.

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TNBC is an aggressive cancer sub-type with limited treatment options and poor prognosis. New therapeutic targets are needed to improve outcomes in TNBC patients. PRCP is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. A role for PRCP in TNBC or other cancers, and its potential as a therapy target has not yet been tested. In the current study, we found high tumor expression of PRCP associates with worse outcome and earlier recurrence in TNBC patients. Knockdown of PRCP or treatment with a small molecule PRCP inhibitor blocked proliferation and survival in TNBC cell lines and inhibited growth of TNBC tumors in mice. Mechanistically, we found PRCP maintains signaling from multiple receptor tyrosine kinases (RTKs), potentially by promoting crosstalk between RTKs and G-protein coupled receptors (GPCRs). Lastly, we found that the PRCP inhibitor caused synergistic killing of TNBC cells when combined with the EGFR and ErbB2 inhibitor lapatinib. Our results suggest that PRCP is potential prognostic marker for TNBC patient outcome and a novel therapeutic target for TNBC treatment.

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The role of prolylcarboxypeptidase (PRCP) in myocardial ischemia/reperfusion (I/R) injury is unclear. Herein, we aimed to evaluate the protective effect of the PRCP-angiotensin-(1-7) [Ang-(1-7)]/bradykinin-(1-9) [BK-(1-9)] axis on myocardial I/R injury and identify the mechanisms involved. Plasma PRCP level and activity, as well as Ang-(1-7) and BK-(1-9) levels, were compared in healthy subjects, patients with unstable angina, and those with ST-segment-elevated acute myocardial infarction (AMI). Thereafter, the effects of PRCP overexpression and knockdown on left ventricular function, mitophagy, and levels of Ang-(1-7) and BK-(1-9) were examined in rats during myocardial I/R. Finally, the effects of Ang-(1-7) and BK-(1-9) on I/R-induced mitophagy and the signaling pathways involved were investigated in vitro in rat cardiomyocytes. AMI patients showed increased plasma level and activity of PRCP and levels of Ang-(1-7) and BK-(1-9) as compared with healthy subjects and those with unstable angina. PRCP protected against myocardial I/R injury in rats by paradoxical regulation of cardiomyocyte mitophagy during the ischemia and reperfusion phases, which was mediated by downstream Ang-(1-7) and BK-(1-9). We further depicted a possible role of activation of AMPK in mitophagy induction during ischemia and activation of Akt in mitophagy inhibition during reperfusion in the beneficial effects of Ang-(1-7) and BK-(1-9). Thus, the PRCP-Ang-(1-7)/BK-(1-9) axis may protect against myocardial I/R injury by paradoxical regulation of cardiomyocyte mitophagy during ischemia and reperfusion phases.

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INTRODUCTION: Prolylcarboxypeptidase (PrCP) is a serine protease that produces or degrades signaling proteins in several important pathways including the renin-angiotensin system (RAS), kallikrein-kinin system (KKS) and pro-opiomelanocortin (POMC) system. PrCP has the potential to be a therapeutic target for cardiovascular, inflammatory and metabolic diseases. Numerous classes of PrCP inhibitors have been developed by rational drug design and from high-throughput screening hits. These inhibitors have been tested in mouse models to assess their potential as new therapeutics. Areas Covered: This review covers the relevant studies that support PrCP as a target for drug discovery. All the significant patent applications and primary literature concerning the development of PrCP inhibitors are discussed. Expert Opinion: The pathways where PrCP is known to operate are complex and many aspects remain to be characterized. Many potent inhibitors of PrCP have been tested in vivo. The variable results obtained from in vivo studies with PrCP inhibitors suggest that additional understanding of the biochemistry and the required therapeutic inhibitor levels is necessary. Additional fundamental research into the signaling pathways is likely required before the true therapeutic potential of PrCP inhibition will be realized.

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Prolylcarboxypeptidase (PRCP) is a carboxypeptidase that cleaves angiotensin II (AngII) forming Ang(1-7). The impact of genetic PRCP deficiency on AngII metabolism, blood pressure (BP), kidney histology, and cardiac phenotype was investigated in two lines of PRCP-deficient mice: KST302 derived in C57BL/6 background and GST090 derived in FVB/N background. The GST090 line had increased mean arterial pressure (MAP) (113.7 ± 2.07 vs. WT 105.0 ± 1.23 mmHg; p < 0.01) and left ventricular hypertrophy (LVH) (ratio of diastolic left ventricular posterior wall dimension to left ventricular diameter 0.239 ± 0.0163 vs. WT 0.193 ± 0.0049; p < 0.05). Mice in the KST302 line also had mild hypertension and LVH. Cardiac defects, increased glomerular size, and glomerular mesangial expansion were also observed. After infusion of AngII to mice in the KST302 line, both MAP and LVH increased, but the constitutive differences between the gene trap mice and controls were no longer observed. Plasma and cardiac AngII and Ang(1-7) were not significantly different between PRCP-deficient mice and controls. Thus, PRCP deficiency is associated with elevated blood pressure and cardiac alterations including LVH and cardiac defects independently of systemic or cardiac AngII and Ang(1-7). An ex vivo assay showed that recombinant PRCP, unlike recombinant ACE2, did not degrade AngII to form Ang(1-7) in plasma at pH 7.4. PRCP was localized in α-intercalated cells of the kidney collecting tubule. The low pH prevailing at this site and the acidic pH preference of PRCP suggest a role of this enzyme in regulating AngII degradation in the collecting tubule where this peptide increases sodium reabsorption and therfore BP. However, there are other potential mechanisms for increased BP in this model that need to be considered as well. PRCP converts AngII to Ang(1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP. KEY MESSAGE: Prolylcarboxypeptidase (PRCP) converts AngII to Ang (1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP.

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BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors (ACEis) are beneficial in patients with heart failure, yet their role after heart transplantation (HTx) remains ambiguous. Particularly, the effects of ACEis on plasma and cardiac metabolites of the "classical" and "alternative" renin-angiotensin system (RAS) in HTx patients are unknown. METHODS: This cross-sectional study used a novel mass spectrometry-based approach to analyze plasma and tissue RAS regulation in homogenates of heart biopsy specimens from 10 stable HTx patients without RAS blockade and in 15 patients with ACEi therapy. Angiotensin (Ang) levels in plasma and Ang formation rates in biopsy tissue homogenates were measured. RESULTS: Plasma Ang II formation is exclusively ACE dependent, whereas cardiac Ang II formation is primarily chymase dependent in HTx patients. ACEi therapy substantially increased plasma Ang-(1-7), the key effector of the alternative RAS, leaving plasma Ang II largely intact. Importantly, neprilysin and prolyl-carboxypeptidase but not angiotensin converting enzyme 2 are essential for cardiac tissue Ang-(1-7) formation. CONCLUSION: ACE is the key enzyme for the generation of plasma Ang II, whereas chymase is responsible for cardiac tissue production of Ang II. Furthermore, our findings reveal that neprilysin and prolyl-carboxypeptidase are the essential cardiac enzymes for the alternative RAS after HTx. These novel insights into the versatile regulation of the RAS in HTx patients might affect future therapeutic avenues, such as chymase and neprilysin inhibition, beyond classical Ang II blockade.

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BACKGROUND: Prolylcarboxypeptidase (PRCP, EC:3.4.16.2) is a cardioprotective protease. Plasma PRCP levels are elevated in type 2 diabetes (T2D) mellitus and cardiovascular diseases. OBJECTIVE: Since diabetic cardiomyopathy is a late complication of uncontrolled diabetes, we tested the hypothesis that glucose and free fatty acid related risk factors for T2D mellitus and cardiovascular disease may reduce the cardioprotective property of PRCP. METHOD: We examined the effects of glucose, saturated fatty acids, and unsaturated fatty acids on PRCP expression in cultured H9c2 cells as an in-vitro model for pharmacological studies. Selective inhibitors, known cardioprotective agents and saturating amounts of neutralizing antibodies were used to validate the effect of free fatty acids on the expression and function of PRCP. RESULTS: The palmitate-mediated reduction of PRCP was concentration and time-dependent. Next, we explored the cardioprotective potential of thyroxin (T4) and insulin. Both T4 and insulin were able to prevent the palmitate-mediated reduction of PRCP expression in H9c2 cells. Inhibition of NF-kB with its specific inhibitor Bay 11-7082 or blockade of palmitate with polyunsaturated fatty acids was ineffective in preventing palmitate-mediated decreases in PRCP expression. CONCLUSION: Our data indicate that elevated palmitate inhibits PRCP expression in rat cardiomyocyte. From this inference PRCP level should be monitored in obese or diabetic patients because this simple measure could identify individuals at high risk of developing health problems, such as heart failure.

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UNLABELLED: Aim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1-7) (Ang-(1-7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1-7). However, their relation with circulating Ang 2 levels after lung ischemia-reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI. MATERIALS AND METHODS: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue. RESULTS: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI. CONCLUSIONS: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

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Plasma kallikrein formed from prekallikrein (PK) produces bradykinin from kininogens and activates factor XII. Plasma PK is activated by factors αXIIa, ßXIIa, or prolylcarboxypeptidase (PRCP). A cross-sectional investigation determined if there is an association of PRCP and KLKB1 polymorphisms with cardiovascular disease (CVD). DNA was obtained from 2243 individuals from the Prevention of Events with Angiotensin Converting Enzyme trial. Two PRCP SNPs, rs7104980 and rs2298668, and two KLKB1 SNPs, rs3733402 and rs3087505, were genotyped. Logistic regression models were performed for history of diabetes, myocardial infarction, stroke, angina, angiographic coronary disease, CABG, intermittent claudication, percutaneous transluminal coronary angioplasty (PTCA), and transient ischemic attack. The PRCP SNP rs7104980 increased the odds of having a history of PTCA by 21% [odds ratio (OR) = 1.211; 95% confidence intervals (CI) = (1.008, 1.454)]; P = 0.041, but was non-significant after Bonferroni correction. Alternatively, having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% [OR = 0.759; 95% CI = (0.622, 0.927)]; P = 0.007 that was statistically significant (P < 0.01) after Bonferroni correction for multiple hypothesis testing. When the best-fit model based on the Akaike information criterion controlled for age, weight, gender, hypertension, and history of angina, the G allele of KLKB1 rs3733402 that is associated with less plasma kallikrein activity correlated with reduced history of CVD.

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Prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine peptidase that inactivates angiotensin II and activates pre-kallikrein, is thought to have anti-hypertensive and anti-proliferative roles in cardiovascular homeostasis. We hypothesized that PRCP function may be altered in heart tissue under conditions that predispose to left ventricle hypertrophy (LVH) in rats. We therefore used real-time PCR and western-blotting to examine the mRNA and protein expression of PRCP in the hearts of spontaneously hypertensive rats (SHR) at pre-hypertensive (5-week-old) and hypertensive (16-week-old) stages compared with age-matched hypertensive (2 kidney-1 clip; 2K-1C) rats and normotensive Wistar rats. PRCP mRNA expression was significantly reduced in hearts of 5- and 16-week-old SHR compared with age-matched Wistar controls, 2K-1C hypertensive rats and sham-operated normotensive rats. There were no significant differences in the PRCP mRNA and protein expression levels in hearts from hypertensive renovascular and sham-operated normotensive rats. Prolonged treatment of SHR with the AT1 receptor antagonist losartan (40 mg/kg, gavage for 8 weeks) reduced the left ventricular weight/body weight ratio (LVW/BW), as well as the mRNA expression of collagen type 1, collagen type 3 and MMP9 in left ventricular tissue, without affecting PRCP gene and protein expression. Our results suggest that diminished PRCP gene and protein expression might be constitutionally involved in the SHR phenotype. In addition, since neither the development of arterial hypertension in the 2K-1C model nor its successful treatment in SHR altered PRCP gene and protein expression in heart tissue, it appears unlikely that PRCP function is regulated by the renin-angiotensin system or by afterload conditions.

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Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.

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The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.

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