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Translational Psychiatry Apr 2023Carboxypeptidase E (CPE) is a multifunctional protein with many nonenzymatic functions in various systems. Previous studies using CPE knock-out mice have shown that CPE...
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, CPE, and CPE 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 CPE mice had normal body weight and glucose metabolism. The behavioral tests showed that CPE mice had impaired learning and memory compared with wild-type and CPE mice. Surprisingly, the subiculum (Sub) region of CPE 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 CPE mice. Interestingly, TrkB phosphorylation in the hippocampus was downregulated in CPE 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 CPE 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.
Topics: Mice; Animals; Mice, Knockout; Carboxypeptidase H; Hippocampus; Learning; Memory Disorders; Maze Learning
PubMed: 37100779
DOI: 10.1038/s41398-023-02429-y -
Cancer Letters Nov 2022Mechanisms 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... (Review)
Review
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.
Topics: Biomarkers; Carboxypeptidase H; Cell Line, Tumor; Humans; Male; Neoplasms; RNA, Messenger; RNA, Small Interfering
PubMed: 35988818
DOI: 10.1016/j.canlet.2022.215882 -
Endocrine Reviews Apr 2012Carboxypeptidase E (CPE) or carboxypeptidase H was first discovered in 1982 as an enkephalin-convertase that cleaved a C-terminal basic residue from enkephalin... (Review)
Review
Carboxypeptidase E (CPE) or carboxypeptidase H was first discovered in 1982 as an enkephalin-convertase that cleaved a C-terminal basic residue from enkephalin precursors to generate enkephalin. Since then, CPE has been shown to be a multifunctional protein that subserves many essential nonenzymatic roles in the endocrine and nervous systems. Here, we review the phylogeny, structure, and function of CPE in hormone and neuropeptide sorting and vesicle transport for secretion, alternative splicing of the CPE transcript, and single nucleotide polymorphisms in humans. With this and the analysis of mutant and knockout mice, the data collectively support important roles for CPE in the modulation of metabolic and glucose homeostasis, bone remodeling, obesity, fertility, neuroprotection, stress, sexual behavior, mood and emotional responses, learning, and memory. Recently, a splice variant form of CPE has been found to be an inducer of tumor growth and metastasis and a prognostic biomarker for metastasis in endocrine and nonendocrine tumors.
Topics: Animals; Carboxypeptidase H; Central Nervous System; Endocrine System; Humans; Models, Animal; Neoplasms
PubMed: 22402194
DOI: 10.1210/er.2011-1039 -
F1000Research 2021Pancreatic 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...
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. 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. 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. Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.
Topics: Carboxypeptidase H; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; RNA, Messenger; RNA, Small Interfering
PubMed: 35528956
DOI: 10.12688/f1000research.53737.2 -
Advances in Pharmacology (San Diego,... 2018Peptides and small molecules that bind to peptide receptors are important classes of drugs that are used for a wide variety of different applications. The search for... (Review)
Review
Peptides and small molecules that bind to peptide receptors are important classes of drugs that are used for a wide variety of different applications. The search for novel neuropeptides traditionally involved a time-consuming approach to purify each peptide to homogeneity and determine its amino acid sequence. The discovery in the 1980s of enkephalin convertase/carboxypeptidase E (CPE), and the observation that this enzyme was involved in the production of nearly every known neuropeptide led to the idea for a one-step affinity purification of CPE substrates. This approach was successfully used to isolate hundreds of known neuropeptides in mouse brain, as well as over a dozen novel peptides. Some of the novel peptides found using this approach are among the most abundant peptides present in brain, but had not been previously identified by traditional approaches. Recently, receptors for two of the novel peptides have been identified, confirming their role as neuropeptides that function in cell-cell signaling. Small molecules that bind to one of these receptors have been developed and found to significantly reduce food intake and anxiety-like behavior in an animal model. This review describes the entire project, from discovery of CPE to the novel peptides and their receptors.
Topics: Animals; Carboxypeptidase H; Humans; Molecular Targeted Therapy; Neuropeptides; Receptors, Cell Surface
PubMed: 29413529
DOI: 10.1016/bs.apha.2017.09.001 -
Journal of Healthcare Engineering 2022To observe the effect of carboxypeptidase E (CPE) on the ischemia and hypoxia (I/H) injury of primary cardiomyocytes.
OBJECTIVE
To observe the effect of carboxypeptidase E (CPE) on the ischemia and hypoxia (I/H) injury of primary cardiomyocytes.
METHODS
Quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to detect the expression of CPE in sham and myocardial infarction (MI) rat heart tissue, and the plasmid was transferred into primary cardiomyocytes by transfection technology. The apoptosis rate of cardiomyocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, Annexin V-PI staining, and Cell Counting Kit-8 (CCK-8) assay. In addition, Caspase kit and qRT-PCR technology were used to detect the expression of apoptosis-related factors. The cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, and qRT-PCR technology. In addition, Western blotting (WB) and qRT-PCR techniques were used to detect the Wnt/-catenin pathway.
RESULTS
First, we found that the expression of CPE in the marginal zone of MI was obviously reduced. Overexpression of CPE in primary cardiomyocytes can effectively inhibit ischemia/hypoxia (I/H)-induced apoptosis and decreased cell activity. In addition, CPE can promote cell proliferation and relieve the inhibitory effect of I/H on cardiomyocytes. At the same time, CPE can promote the expression of -catenin and c-myc.
CONCLUSION
Overexpression of CPE in primary cardiomyocytes can effectively alleviate the decreased cell activity, increased apoptosis, and decreased proliferation caused by I/H and regulated by Wnt/-catenin pathway.
Topics: Animals; Apoptosis; Carboxypeptidase H; Cell Proliferation; Hypoxia; Myocardial Infarction; Myocytes, Cardiac; Rats; beta Catenin
PubMed: 35340224
DOI: 10.1155/2022/3155171 -
Molecules and Cells Nov 2020Hypoxia induces the expression of several genes through the activation of a master transcription factor, hypoxia-inducible factor (HIF)-1α. This study shows that...
Hypoxia induces the expression of several genes through the activation of a master transcription factor, hypoxia-inducible factor (HIF)-1α. This study shows that hypoxia strongly induced the expression of two carboxypeptidases (CP), CPA4 and CPE, in an HIF-1α-dependent manner. The hypoxic induction of and gene was accompanied by the recruitment of HIF-1α and upregulation in the active histone modification, H3K4me3, at their promoter regions. The hypoxic responsiveness of and genes was observed in human adipocytes, human adipose-derived stem cells, and human primary fibroblasts but not mouse primary adipocyte progenitor cells. CPA4 and CPE have been identified as secreted exopeptidases that degrade and process other secreted proteins and matrix proteins. This finding suggests that hypoxia changes the microenvironment of the obese hypoxic adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE.
Topics: Carboxypeptidase H; Carboxypeptidases A; Cell Hypoxia; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Stem Cells
PubMed: 33203807
DOI: 10.14348/molcells.2020.0100 -
The Journal of Neuroscience : the... Aug 2021Activity-dependent insertion of the tropomyosin-related kinase B (TrkB) receptor into the plasma membrane can explain, in part, the preferential effect of brain-derived...
Activity-dependent insertion of the tropomyosin-related kinase B (TrkB) receptor into the plasma membrane can explain, in part, the preferential effect of brain-derived neurotrophic factor (BDNF) on active neurons and synapses; however, the underlying molecular mechanisms remain obscure. Here, we report a novel function for carboxypeptidase E (CPE) in controlling chemical long-term potentiation stimuli-induced TrkB surface delivery in hippocampal neurons. Total internal reflection fluorescence assays and line plot assays showed that CPE facilitates TrkB transport from dendritic shafts to the plasma membrane. The Box2 domain in the juxtamembrane region of TrkB and the C terminus of CPE are critical for the activity-dependent plasma membrane insertion of TrkB. Moreover, the transactivator of transcription TAT-CPE, which could block the association between CPE and TrkB, significantly inhibited neuronal activity-enhanced BDNF signaling and dendritic spine morphologic plasticity in cultured hippocampal neurons. Microinfusion of TAT-CPE into the dorsal hippocampus of male C57BL/6 mice inhibited the endogenous interaction between TrkB and CPE and diminished fear-conditioning-induced TrkB phosphorylation, which might lead to an impairment in hippocampal memory acquisition and consolidation but not retrieval. These results suggest that CPE modulates activity-induced TrkB surface insertion and hippocampal-dependent memory and sheds light on our understanding of the role of CPE in TrkB-dependent synaptic plasticity and memory modulation. It is well known that BDNF acts preferentially on active neurons; however, the underlying molecular mechanism is not fully understood. In this study, we found that the cytoplasmic tail of CPE could interact with TrkB and facilitate the neuronal activity-dependent movement of TrkB vesicles to the plasma membrane. Blocking the association between CPE and TrkB decreased fear-conditioning-induced TrkB phosphorylation and led to hippocampal memory deficits. These findings provide novel insights into the role of CPE in TrkB intracellular trafficking as well as in mediating BDNF/TrkB function in synaptic plasticity and hippocampal memory.
Topics: Animals; Avoidance Learning; Biotinylation; Carboxypeptidase H; Fear; HEK293 Cells; Hippocampus; Humans; Intravital Microscopy; Male; Maze Learning; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Open Field Test; Protein Transport; Protein-Tyrosine Kinases; RNA Interference; RNA, Small Interfering; Rats, Sprague-Dawley; Recognition, Psychology; Signal Transduction; Rats
PubMed: 34266900
DOI: 10.1523/JNEUROSCI.0236-21.2021 -
The Biochemical Journal Jul 1987A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the...
A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.
Topics: Adenoma, Islet Cell; Amino Acids; Animals; Carboxypeptidase H; Carboxypeptidases; Chromatography, Gel; Chromatography, High Pressure Liquid; Cytoplasmic Granules; Electrophoresis, Polyacrylamide Gel; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Proinsulin; Rats; Substrate Specificity
PubMed: 2822027
DOI: 10.1042/bj2450575 -
International Journal of Molecular... Mar 2022Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the...
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 mRNA and protein. mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher 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 , 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.
Topics: Carboxypeptidase H; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Exosomes; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Liver Neoplasms; MicroRNAs; Phenotype; Pilot Projects; RNA, Messenger; RNA, Small Interfering
PubMed: 35328535
DOI: 10.3390/ijms23063113