Carboxypeptidase Research Articles

Detection of alpha toxin and enterotoxins of Clostridium perfringens isolated from minced meat by real time polymerase chain reaction (PCR)

Clostridium perfringens is one of the most widespread pathogen producing toxins related to variable pathogenic conditions, particularly food poisoning in humans. Thus, this study described the prevalence, enumeration, toxigenic types and antibiotic susceptibility of C. perfringens strains isolated from minced meat in Egypt as well as the validation of a real-time polymerase chain reaction (PCR) test for the identification of C. perfringens toxin genes. A high prevalence of C. perfringens (98/105, 93.3%) was detected in minced meat samples. The total count of viable C. perfringens in 23 samples was 2.0 × 102 to 4.5 × 102 with a mean value 3.7 × 102 ± 1.07 × 102 CFU/g. The toxin typing of C. perfringens based on lecithinase activity and dermonecrotic reactions in albino guinea pig exhibited 33 (33.7%) as toxigenic strains of C. perfringens type A and 65 (66.3%) as non-toxigenic strains. Antibiotic susceptibility testing of isolates against 15 different antimicrobial agents indicated that C. perfringens was extremely sensitive to penicillin, followed by erythromycin, tetracycline, doxycycline and amoxicillin. All the other drugs were relatively less effective against the isolates. The real time PCR (RT-PCR) was performed for screening of alpha (cpa), beta, epsilon, iota toxins and enterotoxin (cpe) genes in toxigenic isolates of C. perfringens type A. All toxigenic strains of C. perfringens type A (33, 33.7%) were positive for alpha toxin (cpa) and enterotoxin (cpe) genes, while none of these isolates carried beta, epsilon and iota toxin genes. To the best of the authors’ knowledge, this is one of the studies that used RT-PCR for the determination of toxigenic strains of C. perfringens in Egypt. It is suggested that RT-PCR could be used instead of the conventional culture procedures for identification of C. perfringens in minced meat in Egypt. Key words: Clostridium perfringens, minced meat, alpha toxin (cpa) gene, enterotoxin (cpe) gene, real time-polymerase chain reaction (RT-PCR).

Establishment of Molecular Design Strategy To Obtain Activatable Fluorescent Probes for Carboxypeptidases.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

Clostridium perfringens Contamination in Retail Meat and Meat-Based Products in Bursa, Turkey.

This study examined the incidence of Clostridium perfringens in raw, ready-to-cook (RTC), and ready-to-eat (RTE) meat and meat-based products (N = 306) collected from restaurants, supermarkets, and butcher shops in Bursa, Turkey. In addition, we investigated the presence of the C. perfringens enterotoxin (CPE), as well as cpe genes and their source (chromosomal or plasmid borne). In this study, tryptose sulfite cycloserine (TSC) agar for classic culture isolation and API and real-time polymerase chain reaction (RT-PCR) techniques were used to identify C. perfringens and detect cpa and cpe genes from these products, respectively. Seventeen C. perfringens isolates (5.6%) were isolated and identified with API 20A. In addition, 42 of 81 suspicious isolates (51.9%) were identified as C. perfringens using RT-PCR. Of the 81 suspicious isolates tested by RT-PCR, 22 (27.2%) carried the cpe gene either on the plasmid or chromosome. Twenty-one isolates were positive for chromosomal cpe (C-cpe), and one was positive for plasmid-borne cpe (P-cpe). CPE was detected in 31.8% (7/22) of the cpe positive isolates by the PET-RPLA test. In conclusion, C. perfringens and their CPEs were present in raw, RTC, and RTE meat and meat-based foods in this study. It is emphasized that the presence of C. perfringens and the cpe gene in these foods may be a potential risk for human health.

Affinity Purification of Neuropeptide Precursors from Mice Lacking Carboxypeptidase E Activity.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide-processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. Not all peptides with basic C-terminal residues within a cell are CPE substrates, and these other peptides will also be purified on the anhydrotrypsin affinity column. However, a comparison of peptides purified from wild-type mice and from mice lacking CPE allows for the rapid identification of CPE substrates based on their large increase in the absence of CPE.

Isolation, molecular characterization and prevalence of Clostridium perfringens in sheep and goats of Kashmir Himalayas, India.

Aim:The study was conducted to report the occurrence of the Clostridium perfringens in sheep and goats of the Kashmir valley for the 1st time and to characterize them molecularly with respect to toxin genes to determine the prevalence of the various toxinotypes.Materials and Methods:A total of 177 samples (152 from sheep and 25 from goats) collected from healthy, diarrheic animals, and morbid material of animals suspected to have died of enterotoxaemia were screened for C. perfringens toxinotypes. The presumptive positive isolates were confirmed using 16S rRNA gene-based polymerase chain reaction (PCR). All the confirmed isolates were screened for six toxin genes, namely; cpa, cpb, etx, cpi, cpb2, and cpe using a multiplex PCR.Results:The PCR amplification of 16S rRNA gene revealed that out of 177 samples collected, 125 (70.62%) were found positive for C. perfringens, of which 110 (72.36%) were from sheep and 15 (60%) were from goats. The highest prevalence of C. perfringens toxinotype D was observed in lambs (56.16%) and kids (46.16%) followed by 3.84% in adult sheep while it was absent in samples obtained from adult goats. The multiplex PCR revealed that 67 (60.90%) isolates from sheep and 8 (53.33%) isolates from goats belonged to toxinotype A, while 43 (39.09%) isolates from sheep and 7 (46.66%) isolates from goats were detected as toxinotype D. None of the isolates was found to be toxinotype B, C, or E. All the C. perfringens toxinotype A isolates from sheep were negative for both cpb2 and cpe genes, however, 27.90% toxinotype D isolates from sheep carried cpb2 gene, and 6.97% possessed cpe gene. In contrast, 12.50% C. perfringens toxinotype A isolates from goats harbored cpb2 and cpe genes while 14.28% isolates belonging to toxinotype D carried cpb2 and cpe genes, respectively.Conclusion:The high prevalence of C. perfringens was observed, even in day-old lambs. The toxinotypes A and D are prevalent in both sheep and goats. The severity of disease and mortality may be associated with the presence of minor toxins in both the detected toxinotypes.

Detection and typing of Clostridium perfringens in some retail chicken meat products

Clostridium perfringens is considered as one of major food poisoning bacteria; which may refer to different lethal toxins production including C. perfringens enterotoxin. C. perfringens toxins have been contributed in many diseases in human being especially C. perfringens type A enterotoxin food poisoning. A total of 125 random raw and half cooked chicken meat samples represented by (breast, thigh, nuggets, panee and frankfurter “25 of each”) were collected from various retail stores and supermarkets in Qualyubia governorate to investigate the presence of C. perfringens bacteriologically and detect the cpa, etx, and cpe toxin genes by multiplex PCR. Results demonstrated that 6 out of 25 raw breast samples (24%), 8 out of 25 raw thigh samples (32%), 5 out of 25 nuggets samples (20%), 4 out of 25 panee samples (16%), and 4 out of 25 frankfurter samples (16%) were found to be contaminated with C. perfringens. Twenty-seven positive isolates obtained from these samples were identified as C. perfringens based on the microscopic examination and biochemical tests. It was detected that 8 (29.6%) out of 27 C. perfringens isolates carried only alpha toxin gene (type A), and only one isolate (3.7%) of them expressed both alpha and epsilon toxin genes (type D); while cpe gene never had been detected in any examined isolate, according to the multiplex PCR results.

Epidemiological studies on Clostridium perfringens food poisoning in retail foods.

Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. Meat and meat products are the most common vehicles of C. perfringens type A food poisoning. Contamination of meat by the intestinal contents of slaughtered animals may serve as an important source of this pathogen to the food supply. One hundred and fifty-five non-outbreak food samples were obtained from meat and retail food and examined for the presence of C. perfringens. Multiplex polymerase chain reaction assay to determine the toxin genotype of C. perfringens isolates, and extraction and purification of C. perfringens enterotoxin from enterotoxin gene (cpe)-positive isolates were carried out. The homogeneity of the purified enterotoxin was demonstrated by polyacrylamide gel electrophoresis. In addition, stool samples were collected from 150 persons who had been in contact with animals, and enzyme-linked immunosorbent assays were carried out for the qualitative determination of C. perfringens enterotoxin in the stool samples. The results demonstrated that approximately 2.6% of the tested meat and retail meat samples were contaminated with cpe-positive C. perfringens. The recommended laboratory criteria used to implicate C. perfringens in food-borne disease should involve the detection of C. perfringens enterotoxin production or the presence of the cpe gene in foods or faeces, or in the suspected C. perfringens isolates. In the present study some isolates such as tuna contained the enterotoxin gene although they had a low count of C. perfringens.

Characterization of enterotoxin producing Clostridium perfringens isolated from foods of animal origin

Screening of 105 samples comprising of milk, meat and their products for isolation of Clostridium perfringens revealed a total of 39 (37.14%) samples to be positive for C. perfringens, yielding an equal number of isolates. Among the total isolates, 11 (20.0%) belonged to milk and milk products, of which 10 were isolated from raw cow’s milk. The remaining isolate was from paneer sample. Similarly, 28 (56.0%) C. perfringens isolates were recovered from meat and meat products. Majority (37) of the isolates belonged to toxin type A while two isolates recovered each from raw beef and chicken meats were of toxin type C. Among the 39 isolates of C. perfringens, only 5(12.82%) isolates could reveal the presence of cpe (enterotoxin) gene. The protein profile (SDS-PAGE) of both the crude and partially purified enterotoxins prepared from cpe positive C. perfringens isolates (type A and C) revealed a very little difference. Both the partially purified enterotoxins of type A and C of C. perfringens displayed almost similar type of cytopathic effects (CPE) and death of cells in Vero cell line.

Characterization of enterotoxin producing Clostridium perfringens isolated from foods of animal origin

Screening of 105 samples comprising of milk, meat and their products for isolation of Clostridium perfringens revealed a total of 39 (37.14%) samples to be positive for C. perfringens, yielding an equal number of isolates. Among the total isolates, 11 (20.0%) belonged to milk and milk products, of which 10 were isolated from raw cow’s milk. The remaining isolate was from paneer sample. Similarly, 28 (56.0%) C. perfringens isolates were recovered from meat and meat products. Majority (37) of the isolates belonged to toxin type A while two isolates recovered each from raw beef and chicken meats were of toxin type C. Among the 39 isolates of C. perfringens, only 5(12.82%) isolates could reveal the presence of cpe (enterotoxin) gene. The protein profile (SDS-PAGE) of both the crude and partially purified enterotoxins prepared from cpe positive C. perfringens isolates (type A and C) revealed a very little difference. Both the partially purified enterotoxins of type A and C of C. perfringens displayed almost similar type of cytopathic effects (CPE) and death of cells in Vero cell line.

Bicarbonate and amino acids are co-germinants for spores of Clostridium perfringens type A isolates carrying plasmid-borne enterotoxin gene

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination.

Amyloid-β Impairs Vesicular Secretion in Neuronal and Astrocyte Peptidergic Transmission.

Regulated secretion of neuropeptides and neurotrophic factors critically modulates function and plasticity of synapses and circuitries. It is believed that rising amyloid-β (Aβ) concentrations, synaptic dysfunction and network disorganization underlie early phases of Alzheimer’s disease (AD). Here, we analyze the impact of soluble Aβ1–42 assemblies on peptidergic secretion in cortical neurons and astrocytes. We show that neurons and astrocytes differentially produce and release carboxypeptidase E (CPE) and secretogranin III (SgIII), two dense-core vesicle (DCV) markers belonging to the regulated secretory pathway. Importantly, Aβ1–42, but not scrambled Aβ1–42, dramatically impairs basal and Ca2+-regulated secretions of endogenously produced CPE and SgIII in cultured neurons and astrocytes. Additionally, KCl-evoked secretion of the DCV cargo brain-derived neurotrophic factor (BDNF) is lowered by Aβ1–42 administration, whereas glutamate release from synaptic vesicle (SVs) remains unchanged. In agreement with cell culture results, Aβ1–42 effects on CPE and SgIII secretion are faithfully recapitulated in acute adult brain slices. These results demonstrate that neuronal and astrocyte secretion of DCV cargos is impaired by Aβ in vitro and in situ. Furthermore, Aβ-induced dysregulated peptidergic transmission could have an important role in the pathogenesis of AD and DCV cargos are possible candidates as cerebrospinal fluid (CSF) biomarkers.

Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux.

Carboxypeptidase E (CPE) has recently been described as a multifunctional protein that regulates proliferation, migration and survival in several tumor entities. In glioblastoma (GBM), the most malignant primary brain tumor, secreted CPE (sCPE) was shown to modulate tumor cell migration. In our current study, we aimed at clarifying the underlying molecular mechanisms regulating anti-migratory as well as novel metabolic effects of sCPE in GBM. Here we show that sCPE activates mTORC1 signaling in glioma cells detectable by phosphorylation of its downstream target RPS6. Additionally, sCPE diminishes glioma cell migration associated with a negative regulation of Rac1 signaling via RPS6, since both inhibition of mTOR and stimulation of Rac1 results in a reversed effect of sCPE on migration. Knockdown of CPE leads to a decrease of active RPS6 associated with increased GBM cell motility. Apart from this, we show that sCPE enhances glucose flux into the tricarboxylic acid cycle at the expense of lactate production, thereby decreasing aerobic glycolysis, which might as well contribute to a less invasive behavior of tumor cells. Our data contributes to a better understanding of the complexity of GBM cell migration and sheds new light on how tumor cell invasion and metabolic plasticity are interconnected.

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-β. 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.

Absence of Carboxypeptidase E/Neurotrophic Factor-Α1 in Knock-Out Mice Leads to Dysfunction of BDNF-TRKB Signaling in Hippocampus.

Carboxypeptidase E (CPE), first discovered as a prohormone processing enzyme, has also now been shown to be a secreted neurotrophic factor (neurotrophic factor-α1, NF-α1) that acts extracellularly as a signaling molecule to mediate neuroprotection, cortical stem cell differentiation, and antidepressive-like behavior in mice. Since brain-derived neurotrophic factor (BDNF) has very similar trophic functions, and its processing from pro-BDNF involves intracellular sorting of pro-BDNF to the regulated secretory pathway by CPE acting as a sorting receptor, we investigated whether the lack of CPE/NF-α1 would affect BDNF-TrkB signaling in mice. Previous studies have shown that CPE/NF-α1 knock-out (KO) mice exhibited severe neurodegeneration of the hippocampal CA3 region which raises the question of why other neurotrophic factors such as BDNF could not compensate for the deficiency of CPE. Here, we show that the expressions of pro-BDNF mRNA and protein in hippocampus of CPE-KO mice were similar to WT mice, but mature BDNF was ∼40% less in the CPE-KO mice, suggesting decreased intracellular processing of pro-BDNF. Furthermore, TrkB receptor levels were similar in both genotypes, but there was significantly decreased phosphorylation of TrkB receptor in the CPE-KO mice. Electrophysiological studies showed lack of formation of long-term potentiation in hippocampal slices of CPE-KO mice compared to WT mice, which was not rescued by application of BDNF, indicating dysfunction of the BDNF-TrkB signaling system. The CPE-KO mice showed normal postsynaptic AMPA response to kainate application in hippocampal slices and dissociated neurons. Our findings indicate that CPE/NF-α1 is essential for normal BDNF-TrkB signaling function in mouse hippocampus.

Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease

Since discovery in 1982, carboxypeptidase E (CPE) has been shown to be involved in the biosynthesis of a wide range of neuropeptides and peptide hormones in endocrine tissues, and in the nervous system. This protein is produced from pro-CPE and exists in soluble and membrane forms. Membrane CPE mediates the targeting of prohormones to the regulated secretory pathway, while soluble CPE acts as an exopeptidase and cleaves C-terminal basic residues from peptide intermediates to generate bioactive peptides. CPE also participates in protein internalization, vesicle transport and regulation of signaling pathways. Therefore, in two types of CPE mutant mice, Cpefat/Cpefat and Cpe knockout, loss of normal CPE leads to a lot of disorders, including diabetes, hyperproinsulinemia, low bone mineral density and deficits in learning and memory. In addition, the potential roles of CPE and ΔN-CPE, an N-terminal truncated form, in tumorigenesis and diagnosis were also addressed. Herein, we focus on dissecting the pathophysiological roles of CPE in the endocrine and nervous systems, and related diseases.

Three-dimensional structure of porcine pancreatic carboxypeptidase B with an acetate ion and two zinc atoms in the active site

Crystals of porcine pancreatic carboxypeptidase B (CPB) were grown by the capillary counter-diffusion method in the presence of polyethylene glycol and zinc acetate. The three-dimensional structure of CPB was determined at 1.40 A resolution using the X-ray diffraction data set collected from the crystals of the enzyme at the SPring 8 synchrotron facility and was refined to Rfact = 17.19%, Rfree = 19.78%. The structure contains five zinc atoms, two of which are present in the active site of the enzyme, and an acetate ion. The arrangement of an additional zinc atom in the active site and the acetate ion is different from that reported by Yoshimoto et al.

Toxinotyping and Antimicrobial Resistance of Clostridium Perfringens Isolated from Processed Chicken Meat Products.

IntroductionThe toxinotyping and antimicrobial susceptibility of Clostridium perfringens strains isolated from processed chicken meat were determined.Material and MethodsTwo hundred processed chicken meat samples from luncheon meats, nuggets, burgers, and sausages were screened for Clostridium perfringens by multiplex PCR assay for the presence of alpha (cpa), beta (cpb), epsilon (etx), iota (ia), and enterotoxin toxin (cpe) genes. The C. perfringens isolates were examined in vitro against eight antibiotics (streptomycin, amoxicillin, ampicillin, ciprofloxacin, lincomycin, cefotaxime, rifampicin, and trimethoprim-sulfamethoxazole)ResultsAn overall of 32 C. perfringens strains (16%) were isolated from 200 processed chicken meat samples tested. The prevalence of C. perfringens was significantly dependent on the type of toxin genes detected (P = 0.0), being the highest in sausages (32%), followed by luncheon meats (24%), burgers (6%), and nuggets (2%). C. perfringens type A was the most frequently present toxinotype (24/32; 75%), followed by type D (21.9 %) and type E (3.1%). Of the 32 C. perfringens strains tested, only 9 (28%) were enterotoxin gene carriers, with most representing type A (n = 6). C. perfringens strains differed in their resistance/susceptibility to commonly used antibiotics. Most of the strains tested were sensitive to ampicillin (97%) and amoxicillin (94%), with 100% of the strains being resistant to streptomycin and lincomycin. It is noteworthy that the nine isolates with enterotoxigenic potential had a higher resistance than the non-enterotoxigenic ones. ConclusionThe considerably high C. perfringens isolation rates from processed chicken meat samples and resistance to some of the commonly used antibiotics indicate a potential public health risk. Recent information about the isolation of enterotoxigenic C. perfringens type E from chicken sausage has been reported.

N-Glycosylation analysis of yeast Carboxypeptidase Y reveals the ultimate removal of phosphate from glycans at Asn368

Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y1 ions (peptide+HexNAc)+n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man11-15GlcNAc2 at Asn13, Man8-12GlcNAc2 at Asn87, Man9-14GlcNAc2 at Asn168 and phosphorylated Man12-17GlcNAc2 as well as Man11-16GlcNAc2 at Asn368. The presence of N-glycans with Man<18GlcNAc2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn13 or Asn168 followed by at Asn368. However, glycans at Asn87 which comprises Man8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn87 with the polypeptide chain implicating it in the folding of the protein.

A Novel Single Nucleotide T980C Polymorphism in the Human Carboxypeptidase E Gene Results in Loss of Neuroprotective Function.

Report of a human with a homozygous truncating null mutation of the Carboxypeptidase E (CPE) gene with endocrinological and neurological deficits prompted us to search for other mutations in the human CPE gene that might be linked to disease. We searched an EST database and identified from a small population of patients, a novel T to C single nucleotide polymorphism (SNP) in the CPE gene at bp980 of exon 4, herein called TC-CPE. This introduces a tryptophan to arginine (W235R) mutation in the catalytic domain of human CPE protein. Over-expression of TC-CPE in N2A cells, a neuroendocrine cell line, showed that it was synthesized, but was found in lesser amounts compared to over-expressed WT-CPE in these cells. Furthermore, TC-CPE was secreted poorly from these N2A cells. The levels of TC-CPE were significantly increased after the N2A cells were treated with MG132 (a proteasome inhibitor), suggesting that TC-CPE was targeted to proteasomes for degradation in N2A cells. In addition, TC-CPE induced ER stress as demonstrated by the increased expression of CHOP in N2A cells. Double labeling of CPE and calnexin (and ER marker) suggested the accumulation of TC-CPE in the ER, and the accumulation appears to be enhanced by the treatment of MG132 in the cells. Moreover, the secreted levels of TC-CPE were not affected by the treatment of MG132 in the cells. Over-expression studies revealed that while N2A cells transfected with WT-CPE showed reduced cytotoxicity when challenged with H2O2 compared to cells expressing an empty vector, cells transfected with TC-CPE had no effect. Furthermore, WT-CPE condition medium showed protective effect against oxidative stress, but not TC-CPE condition medium. Although co-expression of WT-CPE and TC-CPE in N2A cells resulted in the reduction in secretion of WT-CPE, co-expression of WT-CPE and TC-CPE did not significantly affect the protective effect of WT-CPE. Taken together, we have identified a novel SNP in the CPE gene which results in the loss of its neuroprotective function in cells and may confer neurological disorders in humans.

A human carboxypeptidase E/NF-α1 gene mutation in an Alzheimer's disease patient leads to dementia and depression in mice.

Patients with Alzheimer's disease (AD), a common dementia among the aging population, often also suffer from depression. This comorbidity is poorly understood. Although most forms of AD are not genetically inherited, we have identified a new human mutation in the carboxypeptidase E (CPE)/neurotrophic factor-α1 (NF-α1) gene from an AD patient that caused memory deficit and depressive-like behavior in transgenic mice. This mutation consists of three adenosine inserts, introducing nine amino acids, including two glutamines into the mutant protein, herein called CPE-QQ. Expression of CPE-QQ in Neuro2a cells demonstrated that it was not secreted, but accumulated in the endoplasmic reticulum and was subsequently degraded by proteasomes. Expression of CPE-QQ in rat hippocampal neurons resulted in cell death, through increased ER stress and decreased expression of pro-survival protein, BCL-2. Transgenic mice expressing CPE-QQ did not show any difference in the processing enzyme activity of CPE compared with wild-type mice. However, the transgenic mice exhibited poor memory, depressive-like behavior, severely decreased dendrites in the hippocampal CA3 region and medial prefrontal cortex indicative of neurodegeneration, hyperphosphorylation of tau at Ser396, and diminished neurogenesis in the dentate gyrus at 50 weeks old. All these pathologies are associated with AD and the latter with depression and were observed in 50-week-old mice. Interestingly, the younger CPE-QQ mice (11 weeks old) did not show deficits in dendrite outgrowth and neurogenesis. This study has uncovered a human CPE/NF-α1 gene mutation that could lead to comorbidity of dementia and depression, emphasizing the importance of this gene in cognitive function.