Expression and Purification of Delta Sleep-Inducing Peptide Fused with Protein Transduction Domain and Human Serum Albumin in Pichia pastoris.

BACKGROUND: Sleep is a natural part of every individual's life. Delta sleep-inducing peptide (DSIP) is a nonapeptide that could promote sleep through the induction of slow wave sleep. However, little is known about the pharmacological effect of DSIP on insomnia. OBJECTIVES: The main objective of this study was to analyze the pharmacological effect of DSIP on insomnia. METHODS: We designed a fusion protein containing N-terminal TAT-based transduction domain followed by human serum albumin and DSIP and designated this protein as PHD fusion protein. The PHD fusion protein were expressed in Pichia pastoris and purified. Mice were administered single subcutaneous injections three concentrations of PHD fusion protein (0.5, 1, 2 mg/kg), and the pharmacological activity of PHD fusion protein was studied using classic pentobarbitalinduced sleep test. RESULTS: We expressed the PHD fusion protein in P. pastoris; furthermore, the PHD fused protein was purified to near homogeneity by DEAE Sepharose FF, Phenyl Sepharose HP and Blue Sepharose 6 FF. Our result showed that the increase of pentobarbital-induced hypnotic effect characterized by reducing sleep latency and prolonged sleep duration was observed for increasing concentrations of PHD fusion protein (P<0.05); moreover, different dose of PHD fusion protein could induce the mice to re-sleep in a dose-dependent manner, whereas higher doses of PHD fusion protein (1.0, 2.0 mg/kg) significantly increased the rate of sleep re-onset compared with the vehicle group of mice (P<0.05). CONCLUSION: PHD fusion protein increased the hypnotic effects of pentobarbital by reducing sleep latency and prolonged sleep duration. The present study suggested PHD fusion protein could be a new drug candidate for insomnia.

Delta-sleep inducing peptide entrapment in the charged macroporous matrices.

Various biomolecules, for example proteins, peptides etc., entrapped in polymer matrices, impact interactions between matrix and cells, including stimulation of cell adhesion and proliferation. Delta-sleep inducing peptide (DSIP) possesses numerous beneficial properties, including its abilities in burn treatment and neuronal protection. DSIP entrapment in two macroporous polymer matrices based on copolymer of dimethylaminoethyl methacrylate and methylen-bis-acrylamide (Co-DMAEMA-MBAA) and copolymer of acrylic acid and methylen-bis-acrylamide (Co-AA-MBAA) has been studied. Quite 100% of DSIP has been entrapped into positively charged Co-DMAEMA-MBAA matrix, while the quantity of DSIP adsorbed on negatively charged Co-AA-MBAA was only 2-6%. DSIP release from Co-DMAEMA-MBAA was observed in saline solutions (0.9% NaCl and PBS) while there was no DSIP release in water or 25% ethanol, thus ionic strength was a reason of this process.

[Delta-sleep inducing peptide entrapment and release from polymer hydrogels based on modified polyvinyl alcohol in vitro].

The aim of the study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to evaluate peptide release kinetics from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels on the basis of poly(vinyl alcohol) acrylic derivative (Acr-PVA) as well as macroporous hydogels containing epoxy groups which were synthesized by copolymerization of this monomer with glycidyl methacrylate. The isotropic hydrogels were fabricated at positive temperatures while the macroporous hydrogels (cryogels) were prepared at the temperatures below zero. The peptide was entrapped into macroporous modified PVA hydrogels by addition of a peptide solution on previously fabricated matrices, while into PVA-GMA hydrogels containing epoxy groups peptide immobilization was carried out by incubation of hydrogel matrices in the peptide solution. In the case of isotropic hydrogels the peptide was added into the polymer mixture at a hydrogel formation reaction. The peptide release kinetics was studied by incubation of hydrogels in PBS (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by phase-reverse HPLC. DSIP release from the macroporous PVA hydrogel after 30 min incubation was 74, 70 and 64% in water, PBS and 0.9% NaCl, relatively, and it was completed in 3 hs. From the isotropic hydrogel the release neither peptide nor products of its degradation was not observed even after 48 hs of incubation. For freshly prepared hydrogel the release kinetics was as follows: 27 and 78% in 30 and 33 hs, relatively. In the case of the lyophilized hydrogel samples the peptide release was 63% in 30 min incubation while drying patterns at room temperature for 3 days resulted in significant peptide loss because its structure damage.

[Olygopeptide KND as a putative endogenous prototype of delta sleep inducing peptide (DSIP). Comparative study of biological properties].

We have undertaken a comparative study on physiological activity of well known neuropeptide DSIP (WAGGDASG E) and new closely related peptide KND (WKGGNASGE) in vivo assays. The sequence of K2, N5-DSIP (KND) was found recently by the computer search for DSIP homologous sequences in available nucleotide and protein databases at 324-332 site of Lysine-specific demethylase 3 B (EC 1.14.11, Swiss-Prot: Q7LBC6.1, 1-1761aa). This human lysine-specific histone demethylase is a representative of the recently discovered family of so called JmjC-domain-containing histone demethylases encoded by JMJD1B gene and ubiquitously expressed in tissues of various mammalian species. Biological investigations performed in this work confirm our preliminary data that DSIP-related peptide KND exhibits the similar biological properties in comparison with DSIP. Assessed by us antioxidative, anticonvulsive and behavioral effects of KND were even more expressed than in DSIP case. These results provide the additional evidences to support our suggestion that KND can be a possible endogenous prototype of "real" DSIP.

Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis.

Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with (18)O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in (18)O-water to regenerate isoAsp. The specific replacement of (16)O with (18)O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the ß-delta sleep-inducing peptide (DSIP) are demonstrated.

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP.

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.

Delta sleep-inducing peptide (DSIP): a still unresolved riddle.

Delta sleep-inducing peptide (DSIP) was isolated from rabbit cerebral venous blood by Schoenenberger-Monnier group from Basel in 1977 and initially regarded as a candidate sleep-promoting factor. However, the link between DSIP and sleep has never been further characterized, in part because of the lack of isolation of the DSIP gene, protein and possible related receptor. Thus the hypothesis regarding DSIP as a sleep factor is extremely poorly documented and still weak. Although DSIP itself presented a focus of study for a number of researchers, its natural occurrence and biological activity still remains obscure. DSIP structure is different from any other known representative of the various peptide families. In this mini-review we hypothesize the existence of a DSIP-like peptide(s) that is responsible (at least partly) for DSIP-like immunoreactivity and DSIP biological activity. This assumption is based on: (i) a highly specific distribution of DSIP-like immunoreactivity in the neurosecretory hypothalamic nuclei of various vertebrate species that are not particularly relevant for sleep regulation, as revealed by the histochemical studies of the Geneva group (Charnay et al.); (ii) a large spectrum of DSIP biological activity revealed by biochemical and physiological studies in vitro; (iii) significant slow-wave sleep (SWS) promoting activity of certain artificial DSIP structural analogues (but not DSIP itself!) in rabbits and rats revealed by our early studies; and (iv) significant SWS-promoting activity of a naturally occurring dermorphin-decapeptide that is structurally similar to DSIP (in five of the nine positions) and the sleep-suppressing effect of its optical isomer, as revealed in rabbits. Potential future studies are outlined, including natural synthesis and release of this DSIP-like peptide and its role in neuroendocrine regulation.

[Effect of delta-sleep-inducing peptide on trypsin and pronase E activities in a model experiment]. / Vliianie del'ta-son indutsiruiushchego peptida na aktivnost' tripsina i pronazy E v model'nykh opytakh.

A delta-sleep-inducing peptide in various concentrations inhibits the pharmacological activity of trypsin and pronase E under experimental conditions.

[Hypnogenic properties of DSIP peptide analogs: structural-functional relationship]. / Gipnogennye svoistva analogov peptida DSIP: strukturno-funktsional'nye vzaimootnosheniia.

The sleep-inducing activity of Delta Sleep-Inducing Peptide (DSIP) and its 13 synthetic analogs has been studied on rabbits with preliminary implanted electrodes. The peptides were injected into the lateral ventricle of cerebrum. Polygraphic computer monitoring of sleep-wake states was carried out at daytime for 7-12 h. DSIP and most analogs had no statistically significant effect on sleep compared to the control administration of saline to the same animals. [NMeAla2]DSIP and [Pro2]DSIP had a pronounced sleep-inducing effect and reliably increased the proportion of slow-wave sleep by 10-15% on average compared to the control. Several other analogs had a week sleep-inducing effect, increasing the proportion of slow-wave sleep during specific recording time only. [beta-Ala2]DSIP significantly suppressed sleep. In addition, this analog, as well as parent DSIP and four proline-containing nonapeptides, slightly increased the body temperature. The revealed differences may be due to both conformation properties and proteolytic resistance of the studied molecules, and it may reflect their indirect involvement in the control sleep-wake hormonal processes.

Conformational changes of neuromedin B and delta sleep-inducing peptide induced by their interaction with lipid membranes as revealed by spectroscopic techniques and molecular dynamics simulation.

Static and dynamic spectroscopic properties of the tryptophanil emission in conjunction with circular dichroism (CD) spectroscopy and molecular dynamics are used to investigate the interactions of the neuropeptide neuromedin B (NMB) and the membrane-permeable delta sleep-inducing peptide (DSIP) with the membrane lipid phase. Our data indicate that in solution both peptides exist in energetically equivalent conformations, whereas in the presence of the membrane specific conformational states are stabilized. By changing from the aqueous to the lipid phase, the static and the dynamic fluorescence properties of the NMB's tryptophan residue are clearly affected: the fluorescence steady-state spectrum as well as the resolved fluorescence decay-associated spectra (DAS) are shifted to the blue with a significant increase of the fluorescence intensity of the second lifetime component (tau 2-DAS). On the other hand, in the lipid environment the same parameters of DSIP are negligibly affected as compared to the aqueous buffer. The CD and molecular dynamics analyses are consistent with these results and indicate that, while NMB assumes a helix-like conformation with the tryptophan residue in the apolar surface, DSIP adopts a globule-like structure with the indole ring that is surface-exposed. As previously found for neuromedin C (Polverini, E., Neyroz, P., Fariselli, P., Casadio, R., and Masotti, L., Biochem. Biophys. Res. Commun. 214, 663-668, 1995), for NMB the stabilized "lipophilic" structure also may favor the correct peptide-receptor contact and recognition. For DSIP, the lipid-stabilized conformation does not support an amphiphilic structure-driven peptide-membrane interaction and suggests a hydrophobicity-driven diffusion across the bilayer.

Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product.

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

hDIP--a potential transcriptional regulator related to murine TSC-22 and Drosophila shortsighted (shs)--is expressed in a large number of human tissues.

We have cloned a 420 bp cDNA from a human fetal brain cDNA library in lambda encoding the human homologue of a DSIP-immunoreactive leucine zipper protein (DIP) isolated from porcine brain. The derived human protein (hDIP) shares a significant sequence identity with parts of the murine TSC-22 and Drosophila shs, both proteins which are discussed as functioning as transcriptional regulators. A similar role of hDIP is partially confirmed by the results of an RT-PCR analysis, demonstrating the widespread distribution of the protein among different human tissues.

Cyclo(-GLY-DSIP), A cyclic analog of the delta-sleep-inducing peptide: computer simulation of spatial structure involving NMR data.

The spatial structure of cyclo(-Gly-DSIP-), a physiologically active analog of the delta sleep-inducing peptide, was determined by computer modelling using 1H NMR data. An interesting feature of the spatial structure in DMSO was detected. One side of almost planar resulting conformation is formed by the side chains of the Asp5, Ser7 and Glu9 residues, the side chain of the Trp1 residue forming the other part of the outer surface. This feature may be associated with the functional properties of the peptide.

Delta sleep-inducing peptide in normal humans and in patients with sleep apnea and narcolepsy.

We measured morning plasma concentrations of delta sleep-inducing-peptide-like-immunoreactivity (DSIP-LI) in 9 sleep apnea patients, 10 narcolepsy patients, and 11 normal controls. Comparisons between the three groups showed no significant differences, although there was a trend toward association with low levels of DSIP-LI in the narcoleptic group, particularly in patients not using medications. No differences were found in the morning or evening plasma DSIP-LI levels in a second group of 11 normal controls and 8 sleep apneics. Our findings do not appear to support a biological marker role of disease activity for single measures of plasma DSIP in sleep apnea.

Delta-sleep-inducing peptide: solution conformational studies of a membrane-permeable peptide.

Peptides and peptide-like molecules as a class have very poor permeability through biological membranes, which severely compromises their potential effectiveness as therapeutic agents. In order to gain insight into the problem of delivering peptide and protein drugs and to establish a model in which the effects of systematic structural variations on transport can be explored, an investigation of the solution conformation of a membrane-permeable peptide was undertaken. Delta-sleep-inducing peptide (DSIP, MW 849) was used in this investigation. DSIP is a charged, hydrophilic peptide that possesses the unusual ability to diffuse passively across the blood-brain barrier (BBB) in vivo [Kastin, A. J., Banks, W. A., Castellanos, P. F., Nissen, C., & Coy, D. H. (1982) Pharmacol. Biochem. Behav. 17, 1187-1191] and across monolayers of brain microvessel endothelial cells in vitro, a model of the BBB [Raeissi, S., & Audus, K. L. (1989) J. Pharm. Pharmacol. 41, 848-852]. This nonapeptide was studied in solution using one- and two-dimensional nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopies in conjunction with molecular modeling. Our spectroscopic findings suggest that DSIP exists in a dynamic equilibrium between unordered and folded structures. Residues 2-5 and 6-9 tend to form type I beta-turns in aqueous solution and a similar, but more ordered, helix-like structure inducible in 40% trifluoroethanol (TFE). NMR, FT-IR, and CD studies in aqueous solution support the dynamic equilibrium hypothesis with the IR data, suggesting that the beta-turn population is approximately 40%.(ABSTRACT TRUNCATED AT 250 WORDS)

Implication of tryptophan in the stimulatory effect of delta-sleep-inducing peptide on indole secretion from perifused rat pineal glands.

We have recently demonstrated that delta-sleep-inducing peptide (DSIP) stimulates indolamine secretion from rat pineal glands. In the present study, we show that tryptophan (TRP), as well as DSIP, stimulate melatonin (MEL) and 5-methoxy-tryptophol (5-ML) secretion in a dose-dependent manner between 5 x 10(-6) and 10(-4) M. The kinetic characteristics of the MEL and 5-ML secretion and the response induced by the two substances were similar. The increase in MEL secretion in response to 10(-4) M DSIP was completely inhibited by pretreatment of the pineals with 10(-5) M phenanthroline (amino-peptidase inhibitor), suggesting that stimulatory effect of DSIP was due to TRP liberated by peptide degradation. This mechanism occurring in the pineal was confirmed using 10(-4) M para-chlorophenylalamine (TRP hydroxylase inhibitor), which reduced the pineal response to 10(-4) and 10(-5) M DSIP by 50 and 100%, respectively.

[Demonstration of delta sleep inducing peptide in a strain of human small cell lung cancer by immunocytology]. / Mise en évidence par immunocytologie du delta sleep inducing peptide dans une lignée de cancer pulmonaire à petites cellules humain.

The "delta sleep inducing peptide" (DSIP) is a regulatory peptide localized in the brain, the hypophysis and some endocrine cells of the gut. The present immunological study, performed with a monoclonal antibody to DSIP, provides evidence for the presence of DSIP-like immunoreactivity (DSIP-LI) in a strain of small cell carcinoma. The specificity of the immunoreaction was assessed by the tests using heterologous antigen known to be secreted by these cells. The DSIP could play a role in the course of this disease.

Phosphorylation of delta sleep-inducing peptide (DSIP) by casein kinase II in vitro.

A phosphorylated analogue of DSIP at Ser7 has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent Km and Vmax values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.

Extraction and immunochemical characterization of delta sleep-inducing peptide-like material from the porcine pituitary and adrenal gland.

The naturally occurring forms of delta sleep-inducing peptide (DSIP) are not fully identified. In the present study, porcine pituitaries and adrenal glands were extracted in water, saline or acid under various conditions and immunoreactive DSIP (IR-DSIP) quantified by radioimmunoassay. The highest concentrations were measured in anterior pituitary extracts (40.8 +/- 2.6 ng/g tissue weight) recovered using water with aprotinin. However, high performance liquid chromatography (HPLC) indicated degradation of hydrophobic forms of IR-DSIP in water extracts. Extraction in acetic acid including C18 Sep-Pak purification resulted in an elution profile of IR-DSIP in adrenal extracts with a major peak coeluting with synthetic DSIP [DSIP(1-9)], whereas anterior pituitary extract showed material of higher hydrophobicity. Approximately 30% of IR-DSIP in anterior pituitary as well as in adrenal gland extracts seemed to be glucosylated, as based on concanavalin A chromatography. One of the DSIP-immunoreactive components by immunoblotting (molecular mass 25 kDa) was identified in both pituitary and adrenal gland extracts. In conclusion, several chromatographically distinct forms of IR-DSIP are present in the porcine pituitary and adrenal gland. IR material eluting as DSIP(1-9) is present in adrenal gland extract. The procedure and solution used for tissue extraction seem to be essential in order to obtain reliable elution positions on HPLC.