Top-Down Proteomics of Mouse Islets With Beta Cell CPE Deletion Reveals Molecular Details in Prohormone Processing.

Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.

Carboxypeptidase E is a prognostic biomarker co-expressed with osteoblastic genes in osteosarcoma.

Osteosarcoma (OS) is a rare primary malignant bone tumor in adolescents and children with a poor prognosis. The identification of prognostic genes lags far behind advancements in treatment. In this study, we identified differential genes using mRNA microarray analysis of five paired OS tissues. Hub genes, gene set enrichment analysis, and pathway analysis were performed to gain insight into the pathway alterations of OS. Prognostic genes were screened using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) dataset, then overlapped with the differential gene dataset. The carboxypeptidase E (CPE) gene, found to be an independent risk factor, was further validated using RT-PCR and Gene Expression Omnibus (GEO) datasets. Additionally, we explored the specific expression of CPE in OS tissues by reanalyzing single-cell genomics. Interestingly, CPE was found to be co-expressed with osteoblast lineage cell clusters that expressed RUNX2, SP7, SPP1, and IBSP marker genes in OS. These results suggest that CPE could serve as a prognostic factor in osteoblastic OS and should be further investigated as a potential therapeutic target.

The C-terminal Amphipathic Helix of Carboxypeptidase E Mediates Export from the ER and Secretion via Lysosomes.

Carboxypeptidase E (CPE), an essential enzyme in the biosynthetic production line of most peptide hormones and neuropeptides, is predominantly expressed in endocrine tissues and in the nervous system. CPE is active in acidic environments where it cleaves the C'-terminal basic residues of peptide precursors to generate their bioactive form. Consequently, this highly conserved enzyme regulates numerous fundamental biological processes. Here, we combined live-cell microscopy and molecular analysis to examine the intracellular distribution and secretion dynamics of fluorescently tagged CPE. We show that, in non-endocrine cells, tagged-CPE is a soluble luminal protein that is efficiently exported from the ER via the Golgi apparatus to lysosomes. The C'-terminal conserved amphipathic helix serves as a lysosomal and secretory granule targeting and a secretion motif. Following secretion, CPE may be reinternalized into the lysosomes of neighboring cells.

Hippocampal delivery of neurotrophic factor-α1/carboxypeptidase E gene prevents neurodegeneration, amyloidosis, memory loss in Alzheimer's Disease male mice.

Alzheimer's Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aß1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aß1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aß1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation.

Deletion of Carboxypeptidase E in ß-Cells Disrupts Proinsulin Processing but Does Not Lead to Spontaneous Development of Diabetes in Mice.

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic ß-cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic ß-cell-specific Cpe knockout mice (ßCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in ßCpeKO islets remained intact. High-fat diet-fed ßCpeKO mice showed weight gain and glucose tolerance comparable with those of Wt littermates. Notably, ß-cell area was increased in chow-fed ßCpeKO mice and ß-cell replication was elevated in ßCpeKO islets. Transcriptomic analysis of ßCpeKO ß-cells revealed elevated glycolysis and Hif1α-target gene expression. On high glucose challenge, ß-cells from ßCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin injections, ßCpeKO mice had accelerated development of hyperglycemia with reduced ß-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining ß-cell function during the development of hyperglycemia. ARTICLE HIGHLIGHTS: Carboxypeptidase E (Cpe) is an enzyme that removes the carboxy-terminal arginine and lysine residues from peptide precursors. Mutations in CPE lead to obesity and type 2 diabetes in humans, and whole-body Cpe knockout or mutant mice are obese and hyperglycemic and fail to convert proinsulin to insulin. We show that ß-cell-specific Cpe deletion in mice (ßCpeKO) does not lead to the development of obesity or hyperglycemia, even after prolonged high-fat diet treatment. However, ß-cell proliferation rate and ß-cell area are increased, and the development of hyperglycemia induced by multiple low-dose streptozotocin injections is accelerated in ßCpeKO mice.

Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration.

Carboxypeptidase E (CPE) is a multifunctional protein with many nonenzymatic functions in various systems. Previous studies using CPE knock-out mice have shown that CPE has neuroprotective effects against stress and is involved in learning and memory. However, the functions of CPE in neurons are still largely unknown. Here we used a Camk2a-Cre system to conditionally knockout CPE in neurons. The wild-type, CPEflox/-, and CPEflox/flox mice were weaned, ear-tagged, and tail clipped for genotyping at 3 weeks old, and they underwent open field, object recognition, Y-maze, and fear conditioning tests at 8 weeks old. The CPEflox/flox mice had normal body weight and glucose metabolism. The behavioral tests showed that CPEflox/flox mice had impaired learning and memory compared with wild-type and CPEflox/- mice. Surprisingly, the subiculum (Sub) region of CPEflox/flox mice was completely degenerated, unlike the CPE full knockout mice, which exhibit CA3 region neurodegeneration. In addition, doublecortin immunostaining suggested that neurogenesis in the dentate gyrus of the hippocampus was significantly reduced in CPEflox/flox mice. Interestingly, TrkB phosphorylation in the hippocampus was downregulated in CPEflox/flox mice, but brain-derived neurotrophic factor levels were not. In both the hippocampus and dorsal medial prefrontal cortex, we observed reduced MAP2 and GFAP expression in CPEflox/flox mice. Taken together, the results of this study demonstrate that specific neuronal CPE knockout leads to central nervous system dysfunction in mice, including learning and memory deficits, hippocampal Sub degeneration and impaired neurogenesis.

Carboxypeptidase E and its splice variants: Key regulators of growth and metastasis in multiple cancer types.

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis.

Mice heterozygous for a null mutation of CPE show reduced expression of carboxypeptidase e mRNA and enzyme activity but normal physiology, behavior, and levels of neuropeptides.

Carboxypeptidase E (CPE) is an essential enzyme that contributes to the biosynthesis of the vast majority of neuropeptides and peptide hormones. There are several reports claiming that small decreases in CPE activity cause physiological changes in animals and/or cultured cells, but these studies did not provide evidence that neuropeptide levels were affected by decreased CPE activity. In the present study, we tested if CPE is a rate-limiting enzyme in neuropeptide production using CpeNeo mice, which contain a neomycin cassette within the Cpe gene that eliminates enzyme expression. Homozygous CpeNeo/Neo mice show defects found in Cpefat/fat and/or Cpe global knockout (KO) mice, including greatly decreased levels of most neuropeptides, severely impaired fertility, depressive-like behavior, adult-onset obesity, and anxiety-like behavior. Removal of the neomycin cassette with Flp recombinase under a germline promoter restored expression of CPE activity and resulted in normal behavioral and physiological properties, including levels of neuropeptides. Mice heterozygous for the CpeNeo allele have greatly reduced levels of Cpe mRNA and CPE-like enzymatic activity. Despite the decreased levels of Cpe expression, heterozygous CpeNeo mice are behaviorally and physiologically identical to wild-type mice, with normal levels of most neuropeptides. These results indicate that CPE is not a rate-limiting enzyme in the production of most neuropeptides, casting doubt upon studies claiming small decreases in CPE activity contribute to obesity or other physiological effects.

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells.

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Carboxypeptidase E mRNA: Overexpression predicts recurrence and death in lung adenocarcinoma cancer patients.

BACKGROUND: Effective biomarkers for prediction of recurrence of lung adenocarcinoma cancer (LADC) patients are needed to determine treatment strategies post-surgery to improve outcome. OBJECTIVE: This study evaluates the efficacy of carboxypeptidase E (CPE) mRNA including its splice isoforms, CPE-ΔN, as a biomarker for predicting recurrence in adenocarcinoma patients. METHODS: RNA was extracted from resected tumors from 86 patients with different stages of non-small cell LADC. cDNA was synthesized and qRT-PCR carried out to determine the copy numbers of CPE/CPE-ΔN mRNA. Patients were followed for 7 years post-tumor resection to determine recurrence and death. RESULTS: ROC curve analysis showed the overall AUC for CPE/CPE-ΔN copy number was 0.563 in predicting recurrence and 0.562 in predicting death. Kaplan-Meier survival analysis showed statistical difference (p= 0.018), indicating that patients with high CPE/CPE-ΔN copy numbers had a shorter time of disease-free survival and also shorter time to death (p= 0.035). Subgroup analyses showed that association of disease-free survival time with CPE/CPE-ΔN copy number was stronger among stage I and II LADC patients (p= 0.047). CONCLUSIONS: CPE/CPE-ΔN mRNA is a potentially useful biomarker for predicting recurrence and death in LADC patients, especially in identifying patients at high risk of recurrence at early stages I and II.

BDV Syndrome: An Emerging Syndrome With Profound Obesity and Neurodevelopmental Delay Resembling Prader-Willi Syndrome.

CONTEXT: CPE encodes carboxypeptidase E, an enzyme that converts proneuropeptides and propeptide hormones to bioactive forms. It is widely expressed in the endocrine and central nervous system. To date, 4 individuals from 2 families with core clinical features including morbid obesity, neurodevelopmental delay, and hypogonadotropic hypogonadism, harboring biallelic loss-of-function (LoF) CPE variants, have been reported. OBJECTIVE: We describe 4 affected individuals from 3 unrelated consanguineous families, 2 siblings of Syrian, 1 of Egyptian, and 1 of Pakistani descent, all harboring novel homozygous CPE LoF variants. METHODS: After excluding Prader-Willi syndrome (PWS), exome sequencing was performed in both Syrian siblings. The variants identified in the other 2 individuals were reported as research variants in a large-scale exome study and in the ClinVar database. Computational modeling of all possible missense alterations allowed assessing CPE tolerance to missense variants. RESULTS: All affected individuals were severely obese with neurodevelopmental delay and other endocrine anomalies. Three individuals from 2 families shared the same CPE homozygous truncating variant c.361C > T, p.(Arg121*), while the fourth carried the c.994del, p.(Ser333Alafs*22) variant. Comparison of clinical features with previously described cases and standardization according to the Human Phenotype Ontology terms indicated a recognizable clinical phenotype, which we termed Blakemore-Durmaz-Vasileiou (BDV) syndrome. Computational analysis indicated high conservation of CPE domains and intolerance to missense changes. CONCLUSION: Biallelic truncating CPE variants are associated with BDV syndrome, a clinically recognizable monogenic recessive syndrome with childhood-onset obesity, neurodevelopmental delay, hypogonadotropic hypogonadism, and hypothyroidism. BDV syndrome resembles PWS. Our findings suggest missense variants may also be clinically relevant.

Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells.

Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein.  Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.

A New Cause of Obesity Syndrome Associated with a Mutation in the Carboxypeptidase Gene Detected in Three Siblings with Obesity, Intellectual Disability and Hypogonadotropic Hypogonadism

Objective: Carboxypeptidase E (CPE) plays a critical role in the biosynthesis of peptide hormones and neuropeptides in the endocrine system and central nervous system. CPE knockout mice models exhibit disorders such as diabetes, hyperproinsulinaemia, low bone mineral density and neurodevelopmental disorders. Only one patient is described with morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotropic hypogonadism, which was associated with a homozygous frameshift deletion in CPE. Methods: Herein are described three siblings with obesity, intellectual disability and hypogonadotropic hypogonadism. Whole exome sequencing (WES) was performed in the index case. Candidate variants were prioritised and segregation of the variant, consistent with the phenotype of the index case, was assessed by Sanger sequencing in affected siblings and parents. Results: WES analysis revealed a homozygous nonsense c.405C>A (p.Y135*) mutation in CPE. Validation and segregation analysis confirmed the homozygous mutation in the index case and his affected siblings. The parents were phenotypically normal heterozygous mutation carriers. Conclusion: This study provides additional evidence of the association between a homozygous nonsense mutation in CPE and a clinical phenotype consisting of obesity, intellectual disability and hypogonadotropic hypogonadism, which may be considered as a new monogenic obesity syndrome.

Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary.

Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classic neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically defined cell type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues, which serve as tags for affinity purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell type.

Carboxypeptidase E down-regulation regulates transcriptional and epigenetic profiles in pancreatic cancer cell line: A network analysis.

BACKGROUND: Pancreatic cancer is a malignant tumor and its incidence has increased in recent years. Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme that has been shown to be associated with tumor growth and invasion in various cancers including pancreatic cancer. OBJECTIVE: To understand the molecular mechanism underlying the proliferative effects of CPE in cancer cells. METHODS: We down-regulated CPE gene expression in PANC-1 cell, a pancreatic cell line, and investigated mRNA, miRNA, circRNA and lncRNA expression profiling in PANC-1 cells from control group and CPE knock-down group by microarray analysis. We further validated the top 14 differentially expressed circRNAs by qRT-PCR. RESULTS: Our results showed that CPE down-regulation caused decreased cell proliferation. The microarray data showed 107, 15, 299 and 360 differentially expressed mRNAs, miRNAs, circRNAs, and lncRNAs, respectively between control group and CPE knock-down group. Of Which, 41 mRNAs, 12 miRNAs, 133 circRNAs, and 262 lncRNAs were down-regulated; 66 mRNAs, 3 miRNAs, 166 circRNAs, and 98 lncRNAs were up-regulated. Bioinformatics analysis showed that the top significantly enriched pathways for the differentially expressed RNAs were related to cancer onset and/or progression, these included p53 signaling pathway, ECM-receptor interaction, focal adhesion and Wnt signaling pathway. We further performed network analysis to assess the mRNA, miRNA, circRNA and lncRNA correlations, and showed that HUWE1, hsa-miR-6780b-5p, has_circ_0058208 and lnc-G3BP1-3:8 were in the core position of the network. CONCLUSIONS: Taken together, these results identified potential CPE regulated core genes and pathways for cell proliferation in pancreatic cancer cell, and therefore provide potential targets for the treatment of pancreatic cancer.

Carboxypeptidase E-∆N Promotes Proliferation and Invasion of Pancreatic Cancer Cells via Upregulation of CXCR2 Gene Expression.

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

EGL-3 and EGL-21 are required to trigger nocifensive response of Caenorhabditis elegans to noxious heat.

Caenorhabditis elegans (C. elegans) is a widely used model organism to examine nocifensive response to noxious stimuli, including heat avoidance. Recently, comprehensive analysis of the genome sequence revealed several pro-neuropeptide genes, encoding a series of bioactive neuropeptides. C. elegans neuropeptides are involved in the modulation of essentially all behaviors including locomotion, mechanosensation, thermosensation and chemosensation. The maturation of pro-neuropeptide to neuropeptide is performed by ortholog pro-protein convertases and carboxypeptidase E (e.g. EGL-3 and EGL-21). We hypothesized that C. elegans egl-3 or egl-21 mutants will have a significant decrease in mature neuropeptides and they will display an impaired heat avoidance behavior. Our data has shown that thermal avoidance behavior of egl-3 and egl-21 mutants was significantly hampered compared to WT(N2) C. elegans. Moreover, flp-18, flp-21 and npr-1 mutant C. elegans displayed a similar phenotype. EGL-3 pro-protein convertase and EGL-21 carboxypeptidase E are essential enzymes for the maturation of pro-neuropeptides to active neuropeptides in C. elegans. Quantitative mass spectrometry analyses with egl-3 and egl-21 mutant C. elegans homogenates demonstrated that proteolysis of ProFLP-18 and ProFLP-21 are severely impeded, leading to a lack of mature bioactive neuropeptides. Not only FLP-21 but also FLP-18 related mature neuropeptides, both are ligands of NPR-1 and are needed to trigger nocifensive response of C. elegans to noxious heat.

Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E transmits its anti-migratory function in glioma cells via transcriptional regulation of cell architecture and motility regulating factors.

Glioblastoma (GBM), the most frequent and aggressive malignant primary brain tumor, is characterized by a highly invasive growth. In our previous study we showed that overexpression of Carboxypeptidase E (CPE) mitigated glioma cell migration. In the present study we aimed at deciphering the regulatory mechanisms of the secreted form of CPE (sCPE). By transcriptome analysis and inhibition of signaling pathways involved in the regulation of cell growth and motility, we discovered that overexpression of sCPE was accompanied by differential regulation of mRNAs connected to the motility-associated networks, among others FAK, PAK, Cdc42, integrin, STAT3 as well as TGF-ß. Especially SLUG was downregulated in sCPE-overexpressing glioma cells, paralleled by reduced expression of matrix-metalloproteinases (MMP) and, in consequence, by decreased cell migration. Expression of SLUG was regulated by ERK since inhibition of ERK reverted sCPE-mediated SLUG downregulation and enhanced cell motility. In a mouse glioma model, overexpression of sCPE significantly prolonged survival. Our results implicate a novel role for sCPE that mainly affects the expression of motility-associated genes via several signal pathways.