Endothelin-converting Enzyme Research Articles

Chronic Sleep Deprivation Altered the Expression of Memory-Related Genes and Caused Cognitive Memory Dysfunction in Mice.

Lack of sleep, whether acute or chronic, is quite common and negatively affects an individual's memory and cognitive function. The question of whether chronic sleep deprivation (CSD) causes cognitive impairment to arise and progress is not well studied. To investigate the effects of CSD on memory and cognition, this study began by establishing a CSD mouse model. Behavioral experiments on animals revealed that CSD induced cognitive behavioral abnormalities reminiscent of Alzheimer's disease. Western blot experiments further demonstrated a considerable increase in amyloid-β (Aβ) expression in the mouse brain following CSD. Meanwhile, the hub gene Prkcg was searched for in the cerebellum using RNA-seq and bioinformatics analysis. PKCγ (Prkcg) expression was significantly reduced, as demonstrated by RT-qPCR and Western blot validations. Additionally, CSD was associated with downregulated CREB expression, decreased expression of the endothelin-converting enzyme (ECE1), and increased phosphorylation of ERK1/2 downstream of PKCγ. These findings suggested that CSD down-regulated PKCγ expression, decreased ECE1 expression, impaired Aβ degradation, and affected the PKCγ/ERK/CREB pathway and the synthesis of memory-related proteins. Overall, this study highlighted how CSD modulated PKCγ-related metabolism, impacting Aβ clearance and the production of memory-related proteins. Such insights are crucial for understanding and preventing sporadic Alzheimer's disease (sAD) associated with CSD.

Cisplatin-resistance and aggressiveness are enhanced by a highly stable endothelin-converting enzyme-1c in lung cancer cells

BackgroundLung cancer constitutes the leading cause of cancer mortality. High levels of endothelin-1 (ET-1), its cognate receptor ETAR and its activating enzyme, the endothelin-converting enzyme-1 (ECE-1), have been reported in several cancer types, including lung cancer. ECE-1 comprises four isoforms, which only differ in their cytoplasmic N-terminus. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE-1c, increasing its stability and leading to enhanced invasiveness in glioblastoma and colorectal cancer cells, which is believed to be mediated by the amino acid residue Lys-6, a conserved putative ubiquitination site neighboring the CK2-phosphorylated residues Ser-18 and Ser-20. Whether Lys-6 is linked to the acquisition of a cancer stem cell (CSC)-like phenotype and aggressiveness in human non–small cell lung cancer (NSCLC) cells has not been studied.MethodsIn order to establish the role of Lys-6 in the stability of ECE-1c and its involvement in lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine and constitutively expressed the wild-type (ECE-1cWT) and mutant (ECE-1cK6R) proteins in A549 and H1299 human NSCLC cells by lentiviral transduction. We determined the protein stability of these clones alone or in the presence of the CK2 inhibitor silmitasertib, compared to ECE-1cWT and mock-transduced cells. In addition, the concentration of secreted ET-1 in the growth media was determined by ELISA. Expression of stemness genes were determined by Western blot and RT-qPCR. Chemoresistance to cisplatin was studied by MTS viability assay. Migration and invasion were measured through transwell and Matrigel assays, respectively, and the side-population was determined using flow cytometry.ResultsECE-1cK6R displayed higher stability in NSCLC cells compared to ECE-1cWT-expressing cells, but ET-1 secreted levels showed no difference up to 48 h. Most importantly, ECE-1cK6R promoted expression of the stemness genes c-Myc, Sox-2, Oct-4, CD44 and CD133, which enhance cellular self-renewal capability. Also, the ECE-1cK6R-expressing cells showed higher cisplatin chemoresistance, correlating with an augmented side-population abundance due to the increased expression of the ABCG2 efflux pump. Finally, the ECE-1cK6R-expressing cells showed enhanced invasiveness, which correlated with the regulated expression of known EMT markers.ConclusionsOur findings suggest an important role of ECE-1c in lung cancer. ECE-1c is key in a non-canonical ET-1-independent mechanism which triggers a CSC-like phenotype, leading to enhanced lung cancer aggressiveness. Underlying this mechanism, ECE-1c is stabilized upon phosphorylation by CK2, which is upregulated in many cancers. Thus, phospho-ECE-1c may be considered as a novel prognostic biomarker of recurrence, as well as the CK2 inhibitor silmitasertib as a potential therapy for lung cancer patients.

Endothelin-1 down-regulates nuclear factor erythroid 2-related factor-2 and contributes to perivascular adipose tissue dysfunction in obesity.

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3β and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.

Comprehensive analysis of the endothelin system in the kidneys of mice, rats, and humans.

The intrarenal endothelin (ET) system is an established moderator of kidney physiology and mechanistic contributor to the pathophysiology and progression of chronic kidney disease in humans and rodents. The aim of the present study was to characterize ET system by combining single cell RNA sequencing (scRNA-seq) data with immunolocalization in human and rodent kidneys of both sexes. Using publicly available scRNA-seq data, we assessed sex and kidney disease status (human), age and sex (rats), and diurnal expression (mice) on the kidney ET system expression. In normal human biopsies of both sexes and in rodent kidney samples, the endothelin-converting enzyme-1 (ECE1) and ET-1 were prominent in the glomeruli and endothelium. These data agreed with the scRNA-seq data from these three species, with ECE1/Ece1 mRNA enriched in the endothelium. However, the EDN1/Edn1 gene (encodes ET-1) was rarely detected, even though it was immunolocalized within the kidneys, and plasma and urinary ET-1 excretion are easily measured. Within each species, there were some sex-specific differences. For example, in kidney biopsies from living donors, men had a greater glomerular endothelial cell endothelin receptor B (Ednrb) compared with women. In mice, females had greater kidney endothelial cell Ednrb than male mice. As commercially available antibodies did not work in all species, and RNA expression did not always correlate with protein levels, multiple approaches should be considered to maintain required rigor and reproducibility of the pre- and clinical studies evaluating the intrarenal ET system.

The Analysis of ECE1 and PPARG Variants in the Development of Osteopenia and Osteoporosis in Postmenopausal Women.

Osteoporosis is a multifactorial systemic skeletal disease that is characterized by a low bone mineral density (BMD) and the microarchitectural deterioration of bone tissue, leading to bone fragility. The search for new genes that may play an important role in the regulation of bone mass and the development of osteoporosis is ongoing. Recently, it was found that altering the activity of the endothelin-1-converting enzyme encoded by the ECE1 gene may affect bone mineral density (BMD). Another gene involved in the process of osteoblast differentiation and maturation is believed to be PPARG (peroxisome proliferator-activated receptor gamma). This participates in regulating the transformation of stem cells and affects the process of bone formation and resorption. Therefore, we analyzed the association of the ECE1 and PPARG variants with osteopenia and osteoporosis risk in the Polish population. This study included a group (n = 608) of unrelated Polish women (245 individuals with osteoporosis (aged: 57 ± 9), 109 individuals with osteopenia (aged: 53 ± 8) and 254 healthy controls (aged: 54 ± 8)). The real-time PCR technique was used to determine the genetic variants for rs213045 (-338G>T) and rs213046 (-839A>C) of the ECE1 gene and rs1801282 (Pro12Ala, C>G) of the PPARG gene. Analysis of the PPARG rs1801282 variants did not show any association with the risk of osteoporosis and osteopenia. However, in the densitometric results, lower median Z-score values were observed for the T allele compared to the G allele for the rs213045 variant of the ECE1 gene (-1.11 ± 1.07 vs. -0.78 ± 1.21, p = 0.021). Moreover, the TT genotype for the rs213045 variant was more common in women with osteopenia (13.8%, OR = 2.82, p < 0.05) and osteoporosis (7.8%, OR = 1.38, p > 0.05) compared to the control group (5.5%). Additionally, our results suggested that the T allele of rs213045 was more common in women with osteopenia compared to the controls. We further observed that the haplotype containing two major GA alleles of ECE1 (rs213045, rs213046) could reduce the risk of osteopenia in our population. Finally, we found that women with osteoporosis had statistically significantly lower body mass and BMI values compared to the control group. Our results suggest that the ECE1 rs213045 variant may increase the risk of osteopenia. However, the data obtained require confirmation in further studies.

Urinary exosomal mRNAs as biomarkers for predicting the therapeutic effect of renin-angiotensin system inhibitors in IgA nephropathy patients

BackgroundRenin-angiotensin system inhibitors (RASi) treatment is the basic therapy for IgA nephropathy (IgAN) patients. However, there is few of biomarker that can predict the efficacy of RASi. This study aimed to find urinary exosomal mRNAs related to the therapeutic effect of RASi in the treatment of proteinuria in IgAN patients. Methods: We divided IgAN patients in screening cohort into A1 (proteinuria increase at 3 months), B1 (proteinuria decrease less than 50 % at 3 months), C1 (proteinuria decrease more than 50 % at 3 months) groups according to changes of proteinuria after treatment. The urinary exosomes were collected before biopsy, RNAs were extracted and analyzed with the microarray assay. The candidate genes were screened by differentially expressed genes (DEGs) analysis and then validated by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort. A receiver operating characteristic (ROC) curve was used to evaluate gene performance in predicting therapeutic effect on RASi reducing proteinuria in IgAN patients. Results: ECE1 and PDE1A mRNAs were significantly different among the three groups, and were gradually decreased among A1, B1 and C1 groups. In the validation cohort, the level of urinary exosomal ECE1 and PDE1A mRNAs were also significantly lower in A2 group compared with C2 group(ECE1, P < 0.001;PDE1A, P < 0.01). Besides, the level of ECE1 mRNA was also lower in B2 group compared with C2 group (P < 0.01). The ROC curve verified that urinary exosomal ECE1 and PDE1A gene level predicted RASi efficacy in IgAN patients with area under curve (AUC) 0.68 and 0.63 respectively. Conclusion: Urinary exosomal ECE1 and PDE1A mRNAs expression can serve as potential biomarkers for predicting the RASi efficacy to reduce proteinuria in IgAN patients.

Placental tissues from nonhypertensive black patients demonstrate lower placental leptin expression compared to white patients independent of BMI

Hypertensive disorders of pregnancy increases risk of maternal and fetal morbidity and mortality. In the US, clinical data indicates that black women are more likely to develop hypertensive pregnancy than white women for reasons largely unknown. The goal of this study was to determine if expression of factors associated with hypertensive pregnancies, in the absence of hypertension milieu, differed between placental tissues from black versus white patient deliveries. We hypothesized that placental tissues from black patient deliveries demonstrate increases in gene expression of these factors compared to white patient deliveries. We collected placentas at time of delivery from healthy, nonhypertensive black (n=13) and white (n=14) patients from AU Medical Center. Snap frozen tissue from maternal side of placenta was processed to isolate RNA, reverse transcriptase for cDNA generation and then subjected to digital droplet PCR analysis of known markers of preeclampsia: leptin (Lep), preproendothelin-1 (PPET1), endothelin converting enzyme-1 (ECE-1) and placental growth factor (PLGF). Data output was recorded for each expression in copies/ul. Body mass index (BMI) at delivery did not differ between black (32.0±2.1 kg/m2) and white patients (31.3±1.4). In addition, correlation analysis (Pearson’s Correlation Coeffcient) was performed for BMI vs expression of Lep, PPET1, ECE-1 and PLGF in all samples from black and white patients and no significant association was found (P&gt;0.05, R2&lt;0.5 for all comparisons). Comparisons of gene expression between white and black patients was performed by 2-tailed t test. Lep expression significantly decreased in black compared to white patient placenta (74±61 vs 590±173 copies/ul, *p&lt;0.05). However, placental expressions of PPET1 (844±185 black vs 626±164 white, copies/ul), ECE-1 (327±117 black vs 626±164 white, copies/ul), and PLGF (305±80 black vs 217±39 white, copies/ul) did not significantly increase or decrease in black compared to white patients. Collectively, these data surprisingly indicate that in nonhypertensive pregnancies, black patients demonstrate lower leptin expression compared to white patients. However, endothelin-1 associated ECE-1 and PPET1 expressions do not increase in nonhypertensive black patient placenta compared to white. As leptin expression is characteristically elevated in hypertensive pregnancy, future studies will determine if placentas collected from hypertensive black and white pregnancies demonstrate a discrepant gene expression in genes associated with increased risk of hypertensive pregnancy. NIH grants 1R01HL169576, 4R00HL146948. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circulating Extracellular Vesicles Are Elevated with Prediabetes and Adversely Affect Cerebral Endothelial Nitric Oxide and Endothelin-1 Production

Experimental and clinical studies indicate that a proatherogenic endothelial phenotype develops in the prediabetic, hyperglycemic state providing the platform for accelerated vascular disease with prediabetes and, in turn, type 2 diabetes. Prediabetes (fasting plasma glucose concentrations: 100-125 mg/dL) is associated with an increased risk of stroke. Circulating endothelial cell-derived microvesicles (EMVs) are now recognized as both a biomarker and mediator of vascular health and disease. In fact, EMVs have been shown to be involved in the pathogenesis of cerebrovascular dysfunction leading to increased stroke risk. Circulating EMVs are elevated in adults with prediabetes; whether EMVs associated with the prediabetic state promote endothelial abnormalities known to increase stroke risk is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with prediabetes on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 20 mid-life adults (age:44-69 yr): 10 normoglycemic (NG; 8M/2F; age: 55±2 yr; BMI: 25.7±0.8 kg/m2; fasting plasma glucose: 87±2 mg/dL) and 10 prediabetic (PreD: 8M/2F; 55±2 yr; 27.1±1.5 kg/m2; 107±2 mg/dL). Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Expression of intracellular proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~65%) in PreD vs NG (180±25 vs 110±13 EMV/μL) and significantly associated with plasma glucose concentrations (r=0.48; P=0.03). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (47.6±2.6 vs 58.6±3.5 AU; P=0.02) and p-eNOS (Thr495) higher (82.6±4.4 vs 31.1±2.5 AU; p&lt;0.001) in cells treated with EMVs from PreD vs NG. Concordantly, NO production was ~20% lower (6.0±0.2 vs 7.2±0.4 μmol/L; p&lt;0.05) in cells treated with EMVs from PreD adults. PreD-associated EMVs also significantly increased (~25-70%) the expression of Big ET-1 (804.4±47.7 vs 656.7±30.7 AU) and endothelin converting enzyme (290.0±24.6 vs 169.6±19.0 AU) as well as ET-1 production (30.2±4.3 vs 24.2±2.0 pg/mL) in hCMECs. In summary, EMVs harvested from adults with PreD reduced endothelial nitric oxide synthase (eNOS) activation and NO production and increased ET-1 synthesis and release in brain endothelial cells in vitro. Increased risk and incidence of stroke associated with prediabetes may be mediated by circulating EMVs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

Endothelin-converting Enzyme-1b Genetic Variants Increase the Risk of Coronary Artery Ectasia.

Coronary artery ectasia (CAE), defined as a 1.5-fold or greater enlargement of a coronary artery segment compared to the adjacent normal coronary artery, is frequently associated with atherosclerotic coronary artery disease (CAD). Membrane-bound endothelin converting enzyme-1 (ECE-1) is involved in the maturation process of the most potent vasoconstrictor ET-1. Polymorphisms in the endothelin (ET) gene family have been shown associated with the development of atherosclerosis. This study aims to investigate the effects of rs213045 and rs2038089 polymorphisms in the ECE-1 gene which have been previously shown to be associated with atherosclerosis and hypertension (HT), in CAE patients. Ninety-six CAE and 175 patients with normal coronary arteries were included in the study. ECE-1b gene variations rs213045 and rs2038089 were determined by real-time PCR. The frequencies of rs213045 C > A (C338A) CC genotype (60.4% vs. 35.4%, p < 0.001) and rs2038089 T > C T allele (64.58% vs. 35.42%, p = 0.017) were higher in the CAE group compared to the control group. The multivariate regression analysis showed that the ECE-1b rs213045 CC genotype (p = 0.001), rs2038089 T allele (p = 0.017), and hypercholesterolemia (HC) (p = 0.001) are risk factors for CAE. Moreover, in nondiabetic individuals of the CAE and control groups, it was observed that the rs213045 CC genotype (p < 0.001), and rs2038089 T allele (p = 0.003) were a risk factor for CAE, but this relationship was not found in the diabetic subgroups of the study groups (p > 0.05). These results show that ECE-1b polymorphisms may be associated with the risk of CAE and this relationship may change according to the presence of type II diabetes.

الكشف عن بعض عوامل الضراوة بين المبيضات المعزولة من المرضى وانتشار جين candidalysin ECE1

المبيضات البيضاء هي مسبب شائع لعدوى الجهاز التنفسي وداء المبيضات الفموي لدى الناس ، ومسببات أمراض الخميرة الانتهازية والأسباب الرئيسية للمرض والوفيات لدى الأشخاص الذين يعانون من نقص المناعة وتسبب التهابات سطحية على الأسطح المخاطية التي تصيب ملايين الأشخاص في جميع أنحاء العالم. كان الهدف الرئيسي من هذه الدراسة هو التحقق من انتشار بعض عوامل الضراوة التي لها القدرة على تكوين الأغشية الحيوية (Biofilm)، والبروتينيز (Proteinase)، وإنتاج الهيمولايسين( Hymolysine) بين عزلات C. albicans التي تشمل انتشار جين Candidalysin (ECE1). تم جمع العينات خلال الفترة من ايار 2022 الى اب 2022 من 280 عينة (مسحة) لمرضى عراقيين من مختلف الاعمار والاجناس غير مكررين كانوا يعانون من داء المبيضات الفموي وامراض الجهاز التنفسي في مستشفيات بغداد (مستشفى مدينه الطب و مستشفى اليرموك التعليمي ومستشفى مدينه الامامين الطبية التعليمية). تم زراعة العينات التي تم جمعها على وسط اكار السابرويد دكستروز (SDA) مضاف اليه كلورامفينيكول كمضاد للبكتيريا بتركيز 10 ميكجم / مل ، وتم حضن الاطباق الملقحة على وسط SDA عند 37 درجة مئوية لمدة تتراوح بين 24-48 ساعة.أظهرت نتائج الاستنبات أن 102 عينة كانت موجبه، منها58 (56.86%) من تجويف الفم و 44 (43.14%) من الجهاز التنفسي، بينما 178 منها كانت سالبة , تم الكشف عن عزلات المبيضات باستخدام الطرق التقليدية والتي تشمل (النمو على وسط HiCrome Candida ، إنتاج الأنبوب الجرثومي Germ tube ، تكوين الابواغ الكلاميديه Clamydospore وتم تأكيد نتئج التعريف باستخدام نضام VITEK-2 بينت النتائج ان من بين 102 عزلة C. albicans هي الاكثر شيوعا بين الانواع بنسبة 68.63 % ثم تلتها C. tropicalis بنسبة %10.78 ثم C. kruzei %5.88 وC. kefyr %5.88 C. parapsilosis % 4.9 بينما كانت C. glabrata % 3.9 وتمثل النسبة الاقل بين الانواع . تم فحص حساسية عزلات المبيضات للأدوية المضادة للفطريات بطريقة انتشار القرص ، والتي أجريت على النحو الموصى به بواسطة(CLSI) M44-A -وثيقة . أظهرت العزلات درجة عالية من الحساسية للأمفوتريسين-ب 93.14% وكلوتريمازول 90.20% ونسياتين 85.92% ،تم التحقق من انتشار جينCandidalysin (ECE1) بين 70 عزلة من C. albicans باستخدام تقنية تفاعل البلمرة المتسلسل PCR ، وأظهرت النتائج أن 41 58.57% تحتوي على جين Ece1 في تجويف الفم والجهاز التنفسي. و 34 عزلة 48.57% من C. albicans كانت منتجة قوية للغشاء الحيوي, بينما 30 عزلة 42.86% عزلة انتجت بروتيناز و 20 عزلة 28.57% عزلة لديها القدرة على تحلل الدم.

Hypoxia-inducible factor-1α contributes to the proliferation of cholesteatoma keratinocytes through regulating endothelin converting enzyme 1 expression.

Cholesteatoma is a hyperproliferative, pseudoneoplastic lesion of the middle ear characterized by aggressive growth and bone destruction. Hypoxia-inducible factor-1α (HIF-1α, also known as HIF1A) is a key transcription factor that enters the nucleus and upregulates many genes involved in cancer progression in the oxygen-free environment. This study is designed to explore the role and mechanism of HIF1A in the progression of cholesteatoma. HIF1A and endothelin converting enzyme 1 (ECE1) levels were determined using real-time quantitative polymerase chain reaction. The protein levels of HIF1A, Cyclin D1, proliferating cell nuclear antigen, and ECE1 were measured using western blot. Cell viability, proliferation, and cell cycle progression were analyzed using cell counting kit-8, Colony formation, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays. Binding between HIF-1α and ECE1 promoter was predicted by Jaspar and verified using Chromatin immunoprecipitation and dual-luciferase reporter assays. HIF1A and ECE1 were highly expressed in cholesteatoma patients and keratinocytes. Moreover, HIF1A knockdown might suppress the cell viability, proliferation, and cycle progression of cholesteatoma keratinocytes. Furthermore, HIF1A upregulated the transcription of ECE1 through binding to its promoter region. HIF1A might expedite cholesteatoma keratinocyte proliferation partly by increasing ECE1 expression, providing a possible therapeutic target for the cholesteatoma treatment.

SiRNA Targeting ECE-1 Partially Reverses Pulmonary Arterial Hypertensionassociated Damage in a Monocrotaline Model.

The aim of this study was to develop a possible treatment for pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a rare disease characterised by a pulmonary arterial pressure greater than 20 mmHg. One of the factors that contribute to PAH is an increase in the production of endothelin-1, a polypeptide that increases vascular resistance in the pulmonary arteries, leading to increased pulmonary arterial pressure and right ventricular hypertrophy. The objective of this study was to design, synthesize, and evaluate two siRNAs directed against endothelin-1 in a rat model of PAH induced with monocrotaline. Wistar rats were administered monocrotaline (60 mg/kg) to induce a PAH model. Following two weeks of PAH evolution, the siRNAs were administered, and after two weeks, right ventricular hypertrophy was evaluated using the RV/LV+S ratio, blood pressure, weight, and relative expression of ECE-1 (Endothelin-converting enzyme-1) mRNA (messenger RNA) by RT-PCR (real-time PCR). The monocrotaline group showed an increase in the hypertrophy index and in ECE-1 mRNA, as well as a significant decrease in weight compared to the control group, while in the monocrotaline + siRNA group, a significant decrease was observed in the relative expression of ECE-1 mRNA, as well as in right ventricular hypertrophy. Based on the above information, we conclude that the administration of siRNAs directed to ECE-1 decreases the damage associated with PAH.

Unveiling the peptidases of parasites from the office chair - The endothelin-converting enzyme case study.

The emergence of high-throughput methodologies such as next-generation sequencing and proteomics has necessitated significant advancements in biological databases and bioinformatic tools, therefore reshaping the landscape of research into parasitic peptidases. In this review we outline the development of these resources along the -omics technologies and their transformative impact on the field. Apart from extensive summary of general and specific databases and tools, we provide a general pipeline on how to use these resources effectively to identify candidate peptidases from these large datasets and how to gain as much information about them as possible without leaving the office chair. This pipeline is then applied in an illustrative case study on the endothelin-converting enzyme 1 homologue from Schistosoma mansoni and attempts to highlight the contemporary capabilities of bioinformatics. The case study demonstrate how such approach can aid to hypothesize enzyme functions and interactions through computational analysis alone effectively and emphasizes how such virtual investigations can guide and optimize subsequent wet lab experiments therefore potentially saving precious time and resources. Finally, by showing what can be achieved without traditional wet laboratory methods, this review provides a compelling narrative on the use of bioinformatics to bridge the gap between big data and practical research applications, highlighting the key role of these technologies in furthering our understanding of parasitic diseases.

The CK2/ECE1c Partnership: An Unveiled Pathway to Aggressiveness in Cancer

The endothelin-1 (ET1) peptide has a pathological role in the activation of proliferation, survival and invasiveness pathways in different cancers. ET1’s effects rely on its activation by the endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms, differing only in their cytoplasmic N-terminuses. We already demonstrated in colorectal cancer, glioblastoma, and preliminarily lung cancer, that the isoform ECE1c heightens aggressiveness by promoting cancer stem cell traits. This is achieved through a non-canonical ET1-independent mechanism of enhancement of ECE1c’s stability upon CK2-dependent phosphorylation at S18 and S20. Here, a K6 residue is presumably responsible for ECE1c ubiquitination as its mutation to R impairs proteasomal degradation. However, how phosphorylation enhances ECE1c’s stability and how this translates into aggressiveness are still open questions. In this brief report, by swapping residues to either phospho-mimetic or phospho-resistant amino acids, we propose that the N-terminus may also be phosphorylated at Y5 and/or T9 by an unknown kinase(s). In addition, N-terminus phosphorylation may lead to a blockage of K6 ubiquitination, increasing ECE1c’s stability and presumably activating the Wnt/β-catenin signaling pathway. Thus, a novel CK2/ECE1c partnership may be emerging to promote aggressiveness and thus become a biomarker of poor prognosis and a potential therapeutic target for several cancers.

A non-canonical role of endothelin converting enzyme 1 (ECE1)in promoting lung cancer development via directly targetingprotein kinase B (AKT).

Lung cancer is the second most common malignancy in the world, and lung adenocarcinoma (LUAD) in particular is the leading cause of cancer death worldwide. Endothelin converting enzyme 1 (ECE1) is a membrane-bound metalloprotease involved in endothelin-1 (ET-1) processing and regulates vasoconstriction. However, very few studies have reported the involvement of ECE1 in regulating tumor cell proliferation, and the mechanism remains poorly understood. Therefore, we aimed to determine the role of ECE1 in lung cancer development. The Cancer Genome Atlas database and Kaplan-Meier plotter were used to assess the association between ECE1 and lung cancer. The expression of ECE1 was detected using immunohistochemistry staining and western blotting. A variety of in vitro assays were performed to evaluate the effects of ECE1 on the colony formation, proliferation, migration and invasion using ECE1 knockdown lung cancer cells. The gene expression profiles regulated by ECE1 were investigated by RNA sequencing. An immunoprecipitation assay and immunofluorescence assay were used to evaluate the mechanism underlying the regulatory effect of ECE1 on protein kinase B (AKT). The effect of ECE1 on tumor development was assessed by xenografted lung cancer cells in either C57BL/6 mice or nude mice. ECE1 was upregulated in LUAD and correlated with the poor prognosis of patients with LUAD. Functional studies showed that knockdown of ECE1 retarded the progression of tumors formed by lung cancer cells at least partly by inhibiting tumor cell proliferation. Moreover, ECE1 accelerated tumor cell proliferation through promoting AKT activation dispensable of its canonical target ET-1. Mechanically, ECE1 interacted with the pleckstrin homology (PH) domain of AKT and facilitated its translocation to the plasma membrane for activation. Furthermore, the inhibition of AKT activity counteracted the lung cancer cell growth inhibition observed both in vitro and in xenografts caused by ECE1 suppression. The present study reveals a non-canonical function of ECE1 in regulating AKT activation and cell proliferation, which provides the basis for the development of a novel strategy for the intervention of cancer including LUAD by abrogating ECE1-AKT signaling.

Nascent Aβ42 Fibrillization in Synaptic Endosomes Precedes Plaque Formation in a Mouse Model of Alzheimer's-like β-Amyloidosis.

Accumulation of amyloid-β peptide (Aβ) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aβ aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aβ accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aβ-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aβ homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aβ accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aβ monomers, oligomers, and fibrils. Levels of all soluble Aβ species declined thereafter, as Aβ42 turned progressively insoluble and accumulated in Aβ-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aβ was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aβ42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aβ42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aβ accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aβ in endosomes and, in TgCRND8 mice, Aβ overproduction causes rapid loss of Aβ42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aβ in extracellular plaques is a defining feature of AD. Aβ aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aβ is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aβ may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aβ aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aβ proteostasis.

Involvement of the aryl hydrocarbon receptor (AhR) in the mechanism of action of elastin-derived peptide (VGVAPG) and its impact on neurosteroidogenesis

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor from the family of basic helix-loop-helix transcription factors. Several studies have indicated an important role of AhR signaling pathways in senescence, aging, and neurodegenerative diseases. During aging, elastin is degraded and elastin-derived peptides (EDPs) are formed. EDPs have been detected in human blood, serum, and cerebrospinal fluid. Literature data suggest a role of EDPs in the development of neurodegenerative diseases. However, the impact of EDPs on the AhR signaling pathway has never been investigated. Therefore, the aim of our paper was to study the role of AhR in the mechanism of action of the VGVAPG peptide (one of the EDPs) in mouse primary astrocytes in vitro. Our experiments have shown that AhR plays an important role in the EDP mechanism of action in a model of mouse primary astrocytes. Moreover, due to the involvement of Sirt3, Pparγ, AhR, Glb1, Nf-κb1, Ece1, Ide, and Nepr genes and the production and release of neurosteroids, VGVAPG can accelerate the development of neurodegenerative diseases in which the proper metabolism of astrocytes is crucial. Furthermore, our studies have proved that AhR is likely involved in the co-control of the Sirt1, Glb1, Nf-κb1, Ece1, and Nepr expression in astrocytes.

The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.

Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.

Molecular basis of selective amyloid-β degrading enzymes in Alzheimer's disease.

The accumulation of the small 42-residue long peptide amyloid-β (Aβ) has been proposed as a major trigger for the development of Alzheimer's disease (AD). Within the brain, the concentration of Aβ peptide is tightly controlled through production and clearance mechanisms. Substantial experimental evidence now shows that reduced levels of Aβ clearance are present in individuals living with AD. This accumulation of Aβ can lead to the formation of large aggregated amyloid plaques-one of two detectable hallmarks of the disease. Aβ-degrading enzymes (ADEs) are major players in the clearance of Aβ. Stimulating ADE activity or expression, in order to compensate for the decreased clearance in the AD phenotype, provides a promising therapeutic target. It has been reported in mice that upregulation of ADEs can reduce the levels of Aβ peptide and amyloid plaques-in some cases, this led to improved cognitive function. Among several known ADEs, neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), insulin degrading enzyme (IDE) and angiotensin-1 converting enzyme (ACE) from the zinc metalloprotease family have been identified as important. These ADEs have the capacity to digest soluble Aβ which, in turn, cannot form the toxic oligomeric species. While they are known for their amyloid degradation, they exhibit complexity through promiscuous nature and a broad range of substrates that they can degrade. This review highlights current structural and functional understanding of these key ADEs, giving some insight into the molecular interactions that leads to the hydrolysis of peptide substrates, the crucial tasks performed by them and the potential for therapeutic use in the future.