Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via ß-arrestin/ERK signaling.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in ß-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was ß-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited ß-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of ß-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the ß-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen.

Carboxypeptidase E (CPE) cleaves basic amino acid residues at the C-terminal end and involves in the biosynthesis of numerous peptide hormones and neurotransmitters. It was purified from human seminal plasma by ion exchange, heparin affinity and gel filtration chromatography followed by identification through SDS-PAGE and MALDI-TOF/MS analysis, which was further confirmed by western blotting. CPE was characterized as glycoprotein by Periodic Acid Schiff (PAS) staining and treating with deglycosylating enzyme N-glycosidase F. The interaction of CPE with heparin was illustrated by surface plasmon resonance (SPR) and in silico interaction analysis. The association constant (KA) and dissociation constant (KD) of CPE with heparin was determined by SPR and found to be 1.06 × 10(5)M and 9.46 × 10(-6)M, respectively. It was detected in human spermatozoa also by western blotting using mouse anti-CPE primary antibody. 20-100 µg/ml concentration of CPE was observed as highly effective in killing Escherichia coli by colony forming unit (CFU) assay. We suggest that CPE might act not only in the innate immunity of male reproductive tract but also regulate sperm fertilization process by interacting heparin.

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers.

Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9)--which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients.

Luminal interaction of phogrin with carboxypeptidase E for effective targeting to secretory granules.

Phogrin, a receptor tyrosine phosphatase-like protein, is localized to dense-core secretory granules (SGs) in various neuroendocrine cells. A previous report showed that the N-terminal luminal domain mediates targeting of this protein to SGs in AtT-20 cells. Here, we show that the luminal domain specifically interacts with carboxypeptidase E (CPE), one of the key proteins involved in peptide hormone sorting, in a weakly acidic condition. The luminal domain consists of pro-sequence domain (pro) and subsequent N-side mature domain and the pro domain was preferentially required for phogrin interaction with CPE and for its targeting to SGs. Small interfering RNA-directed reduction of the CPE protein level resulted in an improper accumulation of phogrin at the trans-Golgi network in AtT-20 cells. This finding indicates that CPE is involved in the sorting process of phogrin to SGs. However, SG localization of CPE was hindered by overexpression of the phogrin mutants that lack the transport motif of binding to clathrin adaptor complexes. Phogrin-depleted AtT-20 cells also exhibited reduced CPE targeting and increased CPE degradation. Our results suggest that the luminal interaction between phogrin and CPE contributes to their targeting to SGs in a cooperative manner in neuroendocrine cells.

Carboxypeptidase E cytoplasmic tail mediates localization of synaptic vesicles to the pre-active zone in hypothalamic pre-synaptic terminals.

How synaptic vesicles (SVs) are localized to the pre-active zone (5-200 nm beneath the active zone) in the nerve terminal, which may represent the slow response SV pool, is not fully understood. Electron microscopy revealed the number of SVs located in the pre-active zone, was significantly decreased in hypothalamic neurons of carboxypeptidase E knockout (CPE-KO) mice compared with wild-type mice. Additionally, we found K(+)-stimulated glutamate secretion from hypothalamic embryonic neurons was impaired in CPE-KO mice. Biochemical studies indicate that SVs from the hypothalamus of wild-type mice and synaptic-like microvesicles from PC12 cells contain a transmembrane form of CPE, with a cytoplasmic tail (CPE(C10)), maybe involved in synaptic function. Yeast two-hybrid and pull-down experiments showed that the CPE cytoplasmic tail interacted with gamma-adducin, which binds actin enriched at the nerve terminal. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that expression of GFP-CPE(C15) reduced the steady-state level of synaptophysin-mRFP containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings identify the CPE cytoplasmic tail, as a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells.

A bi-directional carboxypeptidase E-driven transport mechanism controls BDNF vesicle homeostasis in hippocampal neurons.

Anterograde transport of brain-derived neurotrophic factor (BDNF) vesicles from the soma to neurite terminals is necessary for activity-dependent secretion of BDNF to mediate synaptic plasticity, memory and learning, and retrograde BDNF transport back to the soma for recycling. In our study, overexpression of the cytoplasmic tail of the carboxypeptidase E (CPE) found in BDNF vesicles significantly reduced localization of BDNF in neurites of hippocampal neurons. Live-cell imaging showed that the velocity and distance of movement of fluorescent protein-tagged CPE- or BDNF-containing vesicles were reduced in both directions. In pulldown assays, the CPE tail interacted with dynactin along with kinesin-2 and kinesin-3, and cytoplasmic dynein. Competition assays using a CPE tail peptide verified specific interaction between the CPE tail and dynactin. Thus, the CPE cytoplasmic tail binds dynactin that recruits kinesins or dynein for driving bi-directional transport of BDNF vesicle to maintain vesicle homeostasis and secretion in hippocampal neurons.

Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules.

Secretogranin III (SgIII) and carboxypeptidase E (CPE) bind specifically to cholesterol-rich secretory granule (SG) membranes. We previously showed that SgIII binds chromogranin A (CgA) and targets CgA to the SGs in endocrine cells. We investigated the binding of SgIII and CPE because they frequently localize close to the periphery of SGs, and they bind each other in mouse corticotrope-derived AtT-20 cells. In Cpe fat mouse corticotropes, which have defective CPE, proopiomelanocortin (POMC)-derived adrenocorticotrophin hormone (ACTH)-containing peptides were distributed over the entire surface of the SGs, and displayed a regulated secretion by secretagogues. The Cpe fat pituitary exhibited elevated levels of SgIII and CgA, which suggests that they compensate for a sorting function of CPE for POMC and its intermediates to ACTH. Indeed, both SgIII and CgA were able to bind POMC-derived intermediates. In a competitive pull-down assay, excessive SgIII led to a decrease in CPE-bound POMC-derived intermediate molecules, and SgIII pulled-down by anti-ACTH antibody increased proportionately. We suggest that SgIII and CPE form the separate functional sorting complex by anchoring to cholesterol-rich SG membranes, and POMC-derived peptides are transferred from CPE to SgIII, and subsequently to CgA.

ProSAAS and prohormone convertase 1 are broadly expressed during mouse development.

ProSAAS (encoded by mouse gene Pcsk1n) is a recently described neuroendocrine secretory pathway protein that is cleaved into smaller peptides that may function in cell-cell signalling. ProSAAS and its processing intermediates are also potent inhibitors of prohormone convertase 1 (PC1), which is encoded by mouse gene Pcsk1. In order to gain insight into the function of proSAAS, we have examined the distribution of several proSAAS-derived peptides and PC1 by immunohistochemistry throughout mouse development. The distribution patterns of both SAAS and PC1 are broad from E9 to E11, with some enrichment in neural tube-derived tissues. By E15, the expression of SAAS is largely restricted to neuroendocrine tissues known to produce bioactive peptides. In general, the expression pattern of PC1 overlaps with that of SAAS and other proSAAS-derived peptides, consistent with the hypothesis that proSAAS functions as an endogenous PC1 inhibitor.