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At least 19 recordsLinked to original sources

PubMed2004

Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein.

Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogen-activated protein kinase kinase, components of the extracellular signal-regulated protein kinase (ERK) pathway, thereby inhibiting the pathway and resulting in the suppression of cell proliferation. In the present study, we examined whether PEBP is involved in differentiation induction of human keratinocytes. PEBP expression was immunohistochemically examined in normal human skin and skin cancers with different differentiation properties. PEBP was not expressed in the basal layer of the epidermis but was expressed in the spinous and granular layers of normal skin. The protein was expressed in differentiated but not in undifferentiated carcinoma. PEBP expression was also examined in cultured normal human epidermal keratinocytes in which differentiation was induced by calcium treatment. Involucrin was used as a differentiation marker for spinous and granular cells. Northern blotting analysis indicated that both PEBP and involucrin mRNAs were enhanced 6 h after treatment with 2.0 mM CaCl(2). The protein amount of PEBP was also increased by this treatment. To investigate whether PEBP is involved in differentiation induction of keratinocytes, HaCaT keratinocytes were transfected with an expression vector. Fluorescent immunostain revealed that cells expressing PEBP exhibited enlarged and flattened cell shape, and induction of involucrin expression was demonstrated by immunoblot analysis. Although the protein amount of ERK was not altered, phosphorylated ERK levels were decreased and cell proliferation was partly inhibited by PEBP expression. These results indicate that PEBP not only inhibits cell proliferation but also induces differentiation of human keratinocytes.

Androgen-Binding Protein↗

PubMed2007

Promotion of cellular migration and apoptosis resistance by a mouse eye-specific phosphatidylethanolamine-binding protein.

The phosphatidylethanolamine (PE)-binding protein (PEBP) family is an evolutionary conserved group of proteins found in various species with multiple functions. We identified human PE-binding protein 4 (hPEBP4), a novel member of the PEBP family, as an anti-apoptotic molecule. Here we describe the cloning and functional characterization of the mouse homolog of hPEBP4 (mouse PEBP4, mPEBP4). Full-length cDNA of mPEBP4 contains 1048 bp with an open reading frame (ORF) of 729 bp, which is predicted to encode a 242-aa protein. mPEBP4 contains a PE-binding domain, in this case between amino acids 106 and 213. mPEBP4 localizes primarily to endoplasmic reticulum/Golgi apparatus. Interestingly, RT-PCR and in situ hybridization analyses indicate that mPEBP4 is specifically expressed in mouse eye tissue. We demonstrate that mPEBP4 promotes cellular migration and invasion by inhibiting ERK1/2 and JNK activation and up-regulating the expression of COX-2. In addition, mPEBP4 overexpression inhibits Epirubicin-induced cellular apoptosis. Considering that mPEBP4 is specifically expressed in retinal ganglion cells, whether mPEBP4 is an important molecule involved in visual function needs to be further investigated.

Amino Acid Sequence↗

PubMed1998

Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins.

BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function in vivo is not yet known. The expression of PEBP and related proteins seems to be correlated with development and cell morphogenesis, however. To obtain new insights into the PEBP family and its potential functions, we initiated a crystallographic study of bovine brain PEPB. RESULTS: The X-ray crystal structure of bovine brain PEBP has been solved using multiple isomorphous replacement methods, and refined to 1.84 A resolution. The structure displays a beta fold and exhibits one nonprolyl cis peptide bond. Analysis of cavities within the structure and sequence alignments were used to identify a putative ligand-binding site. This binding site is defined by residues of the C-terminal helix and the residues His85, Asp69, Gly109 and Tyr119. This site also corresponds to the binding site of phosphorylethanolamine, the polar head group of phosphatidylethanolamine. CONCLUSIONS: This study shows that PEBP is not related to the G-protein family nor to known lipid-binding proteins, and therefore defines a novel structural family of phospholipid-binding proteins. The PEBP structure contains no internal hydrophobic pocket, as described for lipocalins or small phospholipid-transfer proteins. Nevertheless, in PEBP, a small cavity close to the protein surface has a high affinity for anions, such as phosphate and acetate, and also phosphorylethanolamine. We suggest that this cavity corresponds to the binding site of the polar head group of phosphatidylethanolamine.

Amino Acid Sequence↗

PubMed2004

A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization.

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.

Amino Acid Sequence↗

PubMed1995

Sequence homology of rat and human HCNP precursor proteins, bovine phosphatidylethanolamine-binding protein and rat 23-kDa protein associated with the opioid-binding protein.

The hippocampal cholinergic neurostimulating peptide (HCNP) enhances acetylcholine synthesis in rat medial septal tissues. We have cloned the cDNAs of the precursor proteins of rat and human HCNP and deduced their respective amino acid sequences. The HCNP sequences aligned at the N-terminal regions of their precursors. The deduced amino acid sequences showed homology with those of the bovine brain phosphatidylethanolamine-binding protein and the rat protein associated with the opioid-binding protein. These observations suggest that the HCNP precursor proteins may have multiple functions.

Acetylcholine↗

PubMed1995

An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins.

A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.

Amino Acid Sequence↗

PubMed2002

Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2.

The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta-sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

Amino Acid Sequence↗

PubMed2001

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.

Amino Acid Sequence↗

PubMed2006

Specific membrane binding of the carboxypeptidase Y inhibitor I(C), a phosphatidylethanolamine-binding protein family member.

I(C), an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phosphatidylethanolamine-binding protein family. The binding of I(C) to phospholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements, which revealed that the affinity of this inhibitor was not for phosphatidylethanolamine but for anionic phospholipids, such as phosphatidylserine, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, with K(D) values below 100 nm. The liposome-binding assay and surface plasmon resonance analyses of I(C), when complexed with carboxypeptidase Y, and the mutant forms of I(C) further suggest that the N-terminal segment (Met1-His18) in its carboxypeptidase Y-binding sites is involved in the specific and efficient binding to anionic phospholipid membranes. The binding of I(C) to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged I(C), suggesting that I(C) is specifically targeted to vacuolar membranes rather than cytoplasmic membranes, during the stationary growth phase. The present findings provide novel insights into the membrane-targeting and biological functions of I(C) and phosphatidylethanolamine-binding proteins.

Binding Sites↗

PubMed2005

A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1.

Capacitation is a pivotal event for mammalian spermatozoa, involving the loss of surface proteins known as decapacitation factors (DF) and consequent acquisition of fertilizing ability. Earlier studies showed that a mouse sperm DF binds to a receptor, DF-R, whose attachment to the sperm plasma membrane appears to involve a glycosylphosphatidylinositol (GPI) anchor. In the present study, purification and subsequent sequencing of DF-R has identified this approximately 23 kDa protein as phosphatidylethanolamine-binding protein 1 (PEBP 1). To obtain functional evidence that supports sequence homology data, purified recombinant PEBP 1 and PEBP 2 were evaluated for biological activity. While PEBP 1 was able to remove DF activity in solution at concentrations above approximately 1 nmol/l, PEBP 2 was ineffective, even at 600 nmol/l; this confirmed that DF-R is PEBP 1. Anti-PEBP 1 antiserum recognized recombinant PEBP 1 and a approximately 23 kDa protein in both mouse and human sperm lysates. Immunolocalization studies revealed that DF-R/PEBP 1 is located on the acrosomal cap, the post-acrosomal region and the flagellum of both mouse and human spermatozoa, with epitope accessibility being capacitation state-dependent and reversible. Treatment of cells with a phospholipase able to cleave GPI anchors essentially abolished immunostaining, thus confirming the extracellular location of DF-R/PEBP 1. We suggest that DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum, with functional consequences for membrane-associated proteins. Obtaining more detail about DF <--> DF-R interactions could lead to useful applications in both fertility treatments and new contraceptive approaches.

Animals↗

PubMed1998

Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction.

BACKGROUND: Proteins belonging to the phosphatidylethanolamine-binding protein (PEBP) family are highly conserved throughout nature and have no significant sequence homology with other proteins of known structure or function. A variety of biological roles have previously been described for members of this family, including lipid binding, roles as odorant effector molecules or opioids, interaction with the cell-signalling machinery, regulation of flowering plant stem architecture, and a function as a precursor protein of a bioactive brain neuropeptide. To date, no experimentally derived structural information has been available for this protein family. In this study we have used X-ray crystallography to determine the three-dimensional structure of human PEBP (hPEBP), in an attempt to clarify the biological role of this unique protein family. RESULTS: The crystal structures of two forms of hPEBP have been determined: one in the native state (at 2.05 A resolution) and one in complex with cacodylate (at 1.75 A resolution). The crystal structures reveal that hPEBP adopts a novel protein topology, dominated by the presence of a large central beta sheet, and is expected to represent the archaetypal fold for this family of proteins. Two potential functional sites have been identified from the structure: a putative ligand-binding site and a coupled cleavage site. hPEBP forms a dimer in the crystal with a distinctive dipole moment that may orient the oligomer for membrane binding. CONCLUSIONS: The crystal structure of hPEBP suggests that the ligand-binding site could accommodate the phosphate head groups of membrane lipids, therefore allowing the protein to adhere to the inner leaf of bilipid membranes where it would be ideally positioned to relay signals from the membrane to the cytoplasm. The structure also suggests that ligand binding may lead to coordinated release of the N-terminal region of the protein to form the hippocampal neurostimulatory peptide, which is known to be active in the development of the hippocampus. These studies are consistent with a primary biological role for hPEBP as a transducer of signals from the interior membrane surface.

Amino Acid Sequence↗

PubMed2005

Silencing of human phosphatidylethanolamine-binding protein 4 sensitizes breast cancer cells to tumor necrosis factor-alpha-induced apoptosis and cell growth arrest.

PURPOSE: The current therapeutic approach is not so effective in breast cancer patients. Alternative treatment protocols aimed at different targets need to be explored. We recently reported a novel phosphatidylethanolamine-binding protein, human phosphatidylethanolamine-binding protein 4 (hPEBP4), as an antiapoptotic molecule. The finding led us to explore a promising approach for breast cancer therapy via silencing the expression of hPEBP4. EXPERIMENTAL DESIGN: hPEBP4 expression in clinical breast specimens was examined by Tissue Microarrays. RNA interference was used to silence hPEBP4 expression in MCF-7 breast carcinoma cells and the effects on cell proliferation, cell cycle progression, apoptosis, as well as underlying mechanisms, were investigated. RESULTS: hPEBP4 was found to be expressed in up to 50% of breast cancers but in only <4% of normal breast tissues. Silencing of hPEBP4 potentiated tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis and cell cycle arrest in MCF-7 cells, which was due to the increased mitogen-activated protein kinase activation and the enhanced phosphatidylethanolamine externalization. Further investigation showed that silencing of hPEBP4 in MCF-7 cells promoted TNF-alpha-induced stability of p53, up-regulation of phospho-p53ser15, p21waf/cip, and Bax, and down-regulation of Bcl-2 and Bcl-xL, which were shown to depend on extracellular signal-regulated kinase 1/2 and c-jun NH2-terminal kinase activation by hPEBP4 silencing. Moreover, the increased proportion of cells in the G0-G1 phase of cell cycle was observed in hPEBP4-silenced MCF-7 cells on TNF-alpha treatment and the expression of cyclin A and cyclin E was down-regulated more significantly. CONCLUSIONS: The antiapoptotic effect and the preferential expression pattern in breast cancer tissues make hPEBP4 a new target for breast cancer therapy. Silencing of hPEBP4 expression may be a promising approach for the treatment of breast carcinoma.

Apoptosis↗

PubMed2001

Human phosphatidylethanolamine-binding protein facilitates heterotrimeric G protein-dependent signaling.

In this study we report that human phosphatidylethanolamine-binding protein (hPBP) facilitates heterotrimeric G protein-coupled signaling. In Xenopus laevis oocytes, coexpression of hPBP with human mu opioid receptor, human delta opioid receptor, or human somatostatin receptor 2 evoked an agonist-induced increase in potassium conductance of G protein-activated inwardly rectifying potassium channels. This activation of heterotrimeric G protein signaling in oocytes could also be elicited by injection of bacterially overexpressed and purified hPBP. Stimulatory effect was pertussis toxin-sensitive and present even in the absence of coexpressed receptors. Additionally, an increase in G protein-mediated inhibition of adenylate cyclase activity, measured by the inhibition of forskolin-mediated cAMP accumulation, could be detected in HEK293 and NIH3T3 cells after expression of hPBP and in Xenopus oocytes after injection of hPBP. As [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to membranes prepared from hPBP-expressing cells was significantly elevated and recombinant hPBP dose-dependently stimulated [(35)S]GTPgammaS binding to native membranes, the results presented provide strong evidence that hPBP-induced effects are G protein-dependent. These data suggest a novel function of hPBP in regulating G protein and G protein-coupled receptor signaling in vivo.

3T3 Cells↗

PubMed1999

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.

Air↗

PubMed2001

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors.

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.

Amino Acid Sequence↗

PubMed1999

Localisation of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat.

Phosphatidylethanolamine-binding protein (PEBP) is a highly-conserved 21- to 23-kDa basic protein that shows preferential affinity in vitro for phosphatidylethanolamine. Previous studies have focussed on PEBP in the brain and male reproductive tract where it has been proposed to play a role in membrane biogenesis. In the present more comprehensive study, rat PEBP transcripts and protein have been found to be expressed in all tissues examined, although the levels vary considerably between tissues. However, at the cellular level, PEBP expression is enigmatic, being restricted to a diverse range of highly specialised neuronal and non-neuronal cell types. The nature of this diversity, ranging from oligodendrocytes to plasma cells, whilst not precluding a role for PEBP in membrane biogenesis in some cell types, would imply that this is not the major function in others.

Adrenal Glands↗

PubMed2004

The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology.

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Androgen-Binding Protein↗

PubMed1995

Phosphatidylethanolamine binding protein is an abundant secretory product of haploid testicular germ cells in the rat.

An abundant cellular and secretory product of isolated seminiferous tubules from adult rats was identified as having an apparent molecular weight of approximately 24,000 and a pI of 5.3 on autoradiographs of two-dimensional polyacrylamide gels. A protein with identical migration characteristics was identified as a major secretory product of isolated round spermatids. Microsequencing revealed that the protein had homology to phosphatidylethanolamine binding protein (PEBP) identified in rat brain. Primers were used in conjunction with RTPCR to amplify a partial cDNA which was used to probe a rat testis library to obtain full length clones. On Northern blots, PEBP mRNA was abundant in adult rat testis and epididymis and fractions enriched in germ cells but was very low/absent from fetal or immature rat testis or adult rat Sertoli cells. In situ hybridisation identified that abundant mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter at particularly high levels in round and elongating spermatids until step 14. Proteins with significant sequence homology to the rat testis PEBP have been identified previously in mouse testis and epididymis, in rat germ cell cultures and coating the surface of mature rat sperm. Differences in the timing of expression of the PEBP mRNA (first expressed in pachytene spermatocytes) and secretion of the PEBP protein (not a major secretory product until round spermatids) is consistent with PEBP mRNA undergoing delayed translation. The role(s) of secreted lipid binding proteins in spermatogenesis are discussed.

Amino Acid Sequence↗