The KND olygopeptide as a putative endogenous prototype of Delta Sleep Inducing Peptide (DSIP). comparative study of biological properties

Biological properties of the known Delta Sleep-Inducing Peptide (DSIP, WAGGDASGE) were studied in vivo in comparison with those of a new DSIP-homologous peptide (WKGGNASGE — ([K ², N ⁵]DSIP, KND). This new peptide was recently discovered as the 324–332 fragment of the human lysine-specific histone demethylase 3B (EC 1.14.11, Swiss-Prot: Q7LBC6.1, 1761 a.a.) in the course of a computer search in available databases of proteins and nucleic acids. This demethylase belongs to the JmjC-domain-containing family of histone demethylases which are encoded by the JMJD1B gene and present in tissues of various mammals. These studies confirmed our preliminary conclusions on the functional similarity between the biological activities of DSIP and KND. The examined antioxidative, anticonvulsive, and behavioral effects of KND proved to be more pronounced than those of DSIP. The obtained results additionally sup-ported our hypothesis about KND being an endogenous prototype of a “real” DSIP.     

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.     

Organization of the human gene encoding the epididymis-specific EP2 protein variants and its relationship to defensin genes

The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to beta-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral beta-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for beta-defensin-2. While the EP2 gene transcribes beta-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.     

Delta Sleep-Inducing Peptide Modulates the Stimulation of Rat Pineal N-Acetyltransferase Activity by Involving the α1 -Adrenergic Receptor

Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of alpha 1-adrenergic receptors potentiates the "basic" effect of beta-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10(-6) M norepinephrine in vitro, and a similar effect was observed with 2nMP-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the alpha 1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.     

Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.     

MassSQUIRM

In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.     

MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity

In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.Key words: LSD1, lysine demethylase, mass spectrometry, reductive methylation, monoamine oxidase (MAO) inhibitors     

The hydroxylation activity of Jmjd6 is required for its homo-oligomerization

Jumonji C-terminal (JmjC) domain-containing proteins are protein hydroxylases and histone demethylases that control gene expression. Jumonji domain-containing protein 6 (Jmjd6) is indispensable for embryonic development and has both histone arginine demethylase and lysyl-hydroxylase activities. The protein undergoes post-translational homo-oligomerization, but the underlying mechanism remains unknown. In this study, we examined the enzymatic activity of Jmjd6 and uncovered the mechanism underlying its homo-oligomerization. An in vitro enzymatic assay monitored by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry indicates that Jmjd6 is unable to remove the methyl group from histone arginine residues but can hydroxylate the histone H4 tail at lysine residues in a 2-oxoglutarate (2-OG)- and Fe (II)-dependent manner. A mutational analysis reveals that the homo-oligomerization of Jmjd6 requires its enzymatic activity and the N- and C-termini. Using an in vitro enzymatic assay, we further demonstrate that Jmjd6 can hydroxylate its N-terminus but not its C-terminus. In summary, we did not detect arginine demethylase activity for Jmjd6, but we did confirm that it could catalyze the lysyl-hydroxylation of histone peptides. In addition, we demonstrated that the homo-oligomerization of Jmjd6 requires its own enzymatic activity and the N- and C-termini. We propose that Jmjd6 forms intermolecular covalent bonds between its N- and C-termini via autohydroxylation.     

Identification and gene structure of an azurin-like protein with a lipoprotein signal peptide in Neisseria gonorrhoeae

We have determined the DNA sequence of a cloned gonococcal gene and found that the predicted protein sequence is highly homologous to the class of blue copper-containing proteins known as azurins. However, the 127 amino acid sequence homologous to azurin is preceded by two unusual structural features. The gene possesses a typical 17 residue lipoprotein signal peptide and the N-terminal 39 amino acids are very rich in proline and alanine. The azurin gene of Pseudomonas aeruginosa has recently been characterized and possesses an ordinary signal peptide susceptible to signal peptidase I, causing export of a soluble protein to the periplasm. In gonococcus it would appear that the homologous product becomes an outer membrane protein.     

Synthesis and Biological Properties of a Number of Analogs of DSIP and Its KND Endogenous Prototype

Russian Journal of Bioorganic Chemistry - We performed a computer search for the DSIP homologous structures in the protein data bases and found the KND peptide (WKGGDNASGE), which was closely...     

组蛋白去甲基化酶与肿瘤发生

组蛋白甲基化修饰是一个可逆的动态的调节过程。甲基化和/或去甲基化状态与表观遗传、转录调控和维持基因组完整性等密切相关。组蛋白甲基化状态异常会直接或间接影响各种生理和病理过程。已知组蛋白去甲基化酶包括赖氨酸特异性去甲基化酶（LSD）家族和含JmjC结构域的JMJD家族。研究发现,两者与肿瘤的发生均有着密切的关系。本文总结了组蛋白去甲基化酶在组蛋白甲基化修饰及肿瘤研究方面的最新进展,为组蛋白修饰的功能及肿瘤诊断、治疗、预后监测等研究提供新思路。在胃癌、乳腺癌、结肠癌等常见肿瘤中,组蛋白去甲基化酶可改变组蛋白的甲基化水平或者直接作用于癌基因,也可调节microRNA或转录因子等,促进或抑制肿瘤的发生发展与影响肿瘤的预后。     

Metabolic control of histone demethylase activity involved in plant response to high temperature

Jumonji C (JmjC) domain proteins are histone lysine demethylases that require ferrous iron and alpha-ketoglutarate (or α-KG) as cofactors in the oxidative demethylation reaction. In plants, α-KG is produced by isocitrate dehydrogenases (ICDHs) in different metabolic pathways. It remains unclear whether fluctuation of α-KG levels affects JmjC demethylase activity and epigenetic regulation of plant gene expression. In this work, we studied the impact of loss of function of the cytosolic ICDH (cICDH) gene on the function of histone demethylases in Arabidopsis thaliana. Loss of cICDH resulted in increases of overall histone H3 lysine 4 trimethylation (H3K4me3) and enhanced mutation defects of the H3K4me3 demethylase gene JMJ14. Genetic analysis suggested that the cICDH mutation may affect the activity of other demethylases, including JMJ15 and JMJ18 that function redundantly with JMJ14 in the plant thermosensory response. Furthermore, we show that mutation of JMJ14 affected both the gene activation and repression programs of the plant thermosensory response and that JMJ14 and JMJ15 repressed a set of genes that are likely to play negative roles in the process. The results provide evidence that histone H3K4 demethylases are involved in the plant response to elevated ambient temperature.

Histone H3K4me3 demethylases JMJ14, JMJ15, and JMJ18 function redundantly in the plant thermosensory response, which is affected by mutation of the cytosolic isocitrate dehydrogenase gene.     

A Novel Mammalian Flavin-dependent Histone Demethylase

Methylation of Lys residues on histone proteins is a well known and extensively characterized epigenetic mark. The recent discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled. Among the histone demethylases so far identified, LSD1 has the unique feature of functioning through a flavin-dependent amine oxidation reaction. Data base analysis reveals that mammalian genomes contain a gene (AOF1, for amine-oxidase flavin-containing domain 1) that is homologous to the LSD1-coding gene. Here, we demonstrate that the protein encoded by AOF1 represents a second mammalian flavin-dependent histone demethylase, named LSD2. The new demethylase is strictly specific for mono- and dimethylated Lys⁴ of histone H3, recognizes a long stretch of the H3 N-terminal tail, senses the presence of additional epigenetic marks on the histone substrate, and is covalently inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a biochemically stable complex with the C-terminal domain of the corepressor protein CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites that are not present in LSD1. We conclude that mammalian LSD2 represents a new flavin-dependent H3-Lys⁴ demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of chromatin-remodeling complexes that are distinct from those involving LSD1.     

Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

Delta-sleep inducing peptide (DSIP) and its fragments as the modulators of neural transmission in the central nervous system

The present study is a continuation of our previous experiments on DSIP activity which have revealed that nonapeptide DSIP inhibits hippocampal electrical activity of the 4-7 c/s frequency band. The aim of the present study was to find which of the known DSIP fragments is responsible for its activity, i.e. to find the active site of the molecule. The experiments were carried out with the entire DSIP molecule and its three different fragments. The method of threshold continuous arousal pattern (TCAP) monitoring was used as the indicator of DSIP activity. It was found that the entire DSIP molecule increased TCAP, while its 1-5 fragment decreased it 1-4 and 5-9 fragments had no noticeable effect.     

Oxidative stress alters global histone modification and DNA methylation

The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3 h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H₂O₂) for 3 h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′,7′-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H₂O₂ inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.