An increase in the immunogenicity of bacterial antigens under the influence of one of the derivatives of muramyl dipeptide]

As revealed in animal experiments, glucosaminylmuramyl dipeptide (GMDP), the synthetic analog of muramyl dipeptide, when introduced intraperitoneally in a single injection or orally, exhibits adjuvant activity with respect to Citrobacter 0-antigens, Shigella flexneri and enhances the protective properties of dysentery and pertussis vaccines. The stimulating properties of GMDP depend on its dose, the route of its administration, the time elapsed after its administration, its ratio to the concomitant doses of bacterial antigens and to the dose of the virulent culture used for challenge.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

Effect of a synthetic adjuvant on the induction of primary immune responses in T cell-depleted spleen cultures.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) stimulates in vitro primary immune responses to SRBC in T cell-depleted (nude) spleen cultures. The stimulation of immune responses by muramyl peptide was antigen dependent. A microculture system was used to compare the T cell-replacing activities of several structural analogues of muramyl dipeptide and to compare the activity of muramyl dipeptide to helper T cells. In a limiting dilution analysis with excess helper T cells or muramyl dipeptide, the frequency of B cell precursors that respond to SRBC was similar, ranging from 1.5 to 5 X 10(-5). Decreasing the cell density in microcultures did not affect the efficiency of B cell precursor responses in the presence of muramyl dipeptide. Muramyl dipeptide was examined for mitogenic activity in spleen cell cultures. In serum-free medium, muramyl dipeptide stimulates slight (3-fold) increases in DNA synthetic activity. In medium supplemented with 5 to 20% fetal calf serum, muramyl dipeptide showed no significant mitogenic activity. There are a number of possible explanations for the T cell-replacing activity of muramyl dipeptide. The most likely is that muramyl dipeptide interacts directly with B cells to mimic the helper T cell signal in the inductive stimulus.     

hPepT1 selectively transports muramyl dipeptide but not Nod1-activating muramyl peptides

Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, 2 members of the newly characterized nod-like receptor (NLR) family of pattern recognition molecules. In the absence of bacterial invasion into the host cytosolic compartment, it remains unclear whether muramyl peptides can cross the plasma membrane and localize into the cytosol. We have recently demonstrated that the plasma membrane transporter, hPepT1, was able to efficiently translocate muramyl dipeptide (MDP), a specific Nod2-activating molecule, into host cells. We aimed to characterize the transport properties of hPepT1 towards a spectrum of muramyl peptides, including Nod1-activating molecules. To do so, we designed an original procedure based on the ectopic expression of hPepT1 in oocytes from Xenopus laevis. Our results demonstrated that hPepT1 transports MDP but no other Nod2-activating molecule. Moreover, we observed that Nod1-stimulating muramyl peptides were not transported by hPepT1. Since hPepT1 expression is strongly associated with intestinal epithelial cells, where Nod1 and Nod2 have been shown to play a key role, these observations suggest a distinct contribution of Nod1 and Nod2 in mucosal homeostasis following the cellular uptake of muramyl peptides by hPepT1.     

Immunological activities of muramyl peptides

Muramyl peptides are endowed with numerous modulatory effects on the immune and nervous systems. Studies with synthetic muramyl dipeptide (MDP), the smallest unit of bacterial cell walls that can replace Mycobacteria in Freund's complete adjuvant, revealed that this glycopeptide can regulate several functions of cells involved in the immune response. The adjuvanticity of MDP and the MDP-induced activation of macrophages against tumors were found to be potentiated in vitro and in vivo with monoclonal anti-MDP antibodies. When used on immunoadsorbent columns, the anti-MDP antibodies removed the somnogenic and pyrogenic activities contained in supernatants of stimulated rabbit peritoneal macrophages. Based on these data a hypothesis is put forward to explain the immuno- and neuro-modulatory effects of muramyl peptides.     

Recognition and classification of histones using support vector machine.

Histones are DNA-binding proteins found in the chromatin of all eukaryotic cells. They are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3, and H4. Two copies of H2A, H2B, H3, and H4 bind to about 160 base pairs of DNA forming the core of the nucleosome (the repeating structure of chromatin) and H1/H5 bind to its DNA linker sequence. Overall, histones have a high arginine/lysine content that is optimal for interaction with DNA. This sequence bias can make the classification of histones difficult using standard sequence similarity approaches. Therefore, in this paper, we applied support vector machine (SVM) to recognize and classify histones on the basis of their amino acid and dipeptide composition. On evaluation through a five-fold cross-validation, the SVM-based method was able to distinguish histones from nonhistones (nuclear proteins) with an accuracy around 98%. Similarly, we obtained an overall >95% accuracy in discriminating the five classes of histones through the application of 1-versus-rest (1-v-r) SVM. Finally, we have applied this SVM-based method to the detection of histones from whole proteomes and found a comparable sensitivity to that accomplished by hidden Markov motifs (HMM) profiles.     

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.     

Identification of the core-histone-binding domains of HMG1 and HMG2

High mobility group (HMG) nonhistone chromosomal proteins are a group of abundant, conservative and highly charged nuclear proteins whose physiological role in chromatin is still unknown. To gain insight into the interactions of HMG1 and HMG2 with the fundamental components of chromatin we have introduced the methodology of photochemical crosslinking. This technique has allowed us to study the interaction of HMG1 and HMG2 with the core histones, in the form of an H2A X H2B dimer and an (H3 X H4)2 tetramer, for an effective time of crosslinking of less than 1 ms and under very mild conditions. This is achieved by using flash photolysis. With this procedure we found that both HMG1 and HMG2 interact with H2A X H2B and also with (H3 X H4)2. In the second case, they seem to do this through histone H3. To obtain more information about the interactions, we split HMG1 and HMG2 into their peptides using staphylococcal proteinase. The peptides obtained, which reflect the domain distribution of these proteins, were then used along with the histone oligomers to elucidate their interactions by means of photochemical crosslinking. Results obtained indicate that the domain of HMG1 and HMG2 involved in the interaction with H2A X H2B histones is the highly acidic C-terminal, whereas the N-terminal is involved in the interactions with (H3 X H4)2 histones. In all cases, the interactions found appear appreciably strong. Along with other data published in the literature, these proteins appear to have at least one binding site per domain for the chromatin components.     

Carboxyl-terminal peptides from histone H1 variants: DNA binding characteristics and solution conformation

The carboxyl-terminal domains of the histone H1 proteins bind to DNA and are important in condensation of DNA. Little is known about the details of the interactions between H1 histones and DNA, and in particular, there is little known about differences among variant H1 histones in their interactions with DNA. Questions concerning H1 histone-DNA affinity and H1 conformation were investigated using peptide fragments from the carboxyl terminal domains of four nonallelic histone H1 variant proteins (mouse H1-1, H1-4 and H1^°, and rat H1T). Three of the four peptides showed a slight preference for binding to a GC-rich region of a 214-base-pair DNA fragment, rather than to an AT-rich region. The fourth peptide, H1t, appeared to bind preferentially to the AT-rich region of the 214-base-pair fragment. The results show that these small peptides bind preferentially to a subset of DNA sequences; such sequence preference might be exhibited by the intact H1 histones themselves. CD spectra of the peptides, which are from regions of the proteins that are not compactly folded, showed that the α-helical content of the peptides was minimal if the peptides were in 10 mM phosphate buffer, but increased if the peptides were in 1 M NaClO₄ and 50% trifluoroethanol, conditions that are postulated to approximate certain aspects of binding to DNA. H1-4 peptide, which was predicted to be 70% α-helix, but was not α-helical in 10 mM phosphate buffer, appeared from difference CD spectra to be more α-helical when it was bound to DNA. The regions of the proteins from which these peptides are derived, which are extended in solution, may fold, forming α-helices, upon binding to DNA. © 1996 John Wiley & Sons, Inc.     

Dynamics of plant histone modifications in response to DNA damage

DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.     

Muramyl dipeptide and its synthetic analog as possible inducers of interleukin-2 production

Muramyl dipeptide and its synthetic derivative N-acetyl-glucosaminyl-N-acetyl-muramyl-alanyl - D-isoglutamine induce mitogenic factor production for Con A activated blasts (Con A-blasts) in splenocytes. The maximal factor production was revealed 24 h after stimulation with muramyl dipeptide or its derivative. Addition of the inducers to the culture of Con A-blasts led but to the negligible proliferative response. Therefore, the mitogenic action of muramyl dipeptide on target cells is mediated by the growth factor, evidently by interleukin-2. It has been also shown that muramyl dipeptide and its derivative with Con A exerted a synergic action in interleukin-2 induction.     

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.     

Modulation of natural killer activity by muramyl peptides: relationship with adjuvant and anti-infectious properties

Natural killer (NK) activity of spleen cells was studied in DBA/2 mice, 24 and 72 h after intravenous injection of various muramyl peptides: muramyl dipeptide (MDP) and derivatives which are both adjuvant-active and able to increase resistance against Klebsiella pneumoniae; derivatives which are adjuvant-active but devoid of anti-infectious properties; derivatives which are anti-infectious but devoid of adjuvant activity, and derivatives which are devoid of both activities such as the stereoisomer MDP[D-Ala]1. An early increase in NK activity was observed 24 h after injection of all nonadjuvant derivatives, whatever their effect on infection. A stimulation of natural cytotoxicity was always induced 72 h after injection of MDP and derivatives able to protect mice against Klebsiella pneumoniae infection. So, even if the reverse was not true, there seems to exist some correlation between the anti-infectious effect of muramyl peptides and the late increase in NK activity. The modulation of NK activity by muramyl peptides appeared to be independent of interferon production. Moreover, inhibition of the stimulatory effect by a cell cycle-specific drug, hydroxyurea, observed 72 h after MDP suggests a requirement for proliferation.     

Structural basis of the chromodomain of Cbx3 bound to methylated peptides from histone h1 and G9a

Background

HP1 proteins are highly conserved heterochromatin proteins, which have been identified to be structural adapters assembling a variety of macromolecular complexes involved in regulation of gene expression, chromatin remodeling and heterochromatin formation. Much evidence shows that HP1 proteins interact with numerous proteins including methylated histones, histone methyltransferases and so on. Cbx3 is one of the paralogues of HP1 proteins, which has been reported to specifically recognize trimethylated histone H3K9 mark, and a consensus binding motif has been defined for the Cbx3 chromodomain.

Methodology/Principal Findings

Here, we found that the Cbx3 chromodomain can bind to H1K26me2 and G9aK185me3 with comparable binding affinities compared to H3K9me3. We also determined the crystal structures of the human Cbx3 chromodomain in complex with dimethylated histone H1K26 and trimethylated G9aK185 peptides, respectively. The complex structures unveil that the Cbx3 chromodomain specifically bind methylated histone H1K26 and G9aK185 through a conserved mechanism.

Conclusions/Significance

The Cbx3 chromodomain binds with comparable affinities to all of the methylated H3K9, H1K26 and G9aK185 peptides. It is suggested that Cbx3 may regulate gene expression via recognizing both histones and non-histone proteins.     

Muramyl dipeptide analogues as potentiators of the antitumor action of endotoxin

Summary The potentiation of endotoxin-induced necrosis and regression of solid syngeneic Meth A tumors in mice previously observed following administration of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was investigated further by use of various muramyl peptide analogues and two unrelated synthetic adjuvants, viz. the pluronic polyol L121 and dimethyldioctadecylammonium bromide (DDA) instead of MDP. All agents were administered in aqueous solution by the IV route. None of the muramyl peptide analogues nor L121 or DDA had any strong antitumor action of their own. Two 6-O-acylated muramyl peptides (L2-MDP and B30-MDP) and muramyl dipeptide stearoyllysine [MDP-Lys (L18)] clearly potentiated endotoxin-induced necrosis and regression. In contrast, MDP with L- instead of D-isoglutamine was completely inactive. Optimal activity of B30-MDP and MDP-Lys (L18) was only achieved by adding of suitable amounts of a nonionic surfactant. L121 and DDA could not replace muramyl peptides as potentiating agent. The combination of endotoxin, MDP, and L121 caused complete tumor regression in all mice, but was highly toxic.On the basis of the data in the literature on the biological response-modifying activities of the agents used it is concluded that the potentiating activity of muramyl peptides cannot yet be related to their immunoadjuvant action or their capacity to activate macrophages or to enhance nonspecific bacterial resistance.The work described in this paper was supported by grant UUKC 82-15 from the Koningin Wilhelmina Fonds, The Netherlands Cancer Organization     

Histones: a novel class of lipopolysaccharide-binding molecules

Unlike soluble and membrane forms of lipopolysaccharide (LPS)-binding proteins, intracellular LPS-binding molecules are poorly documented. We looked for such molecules in a murine lung epithelial cell line. Two proteins with LPS-binding activity were isolated and unambiguously identified as histones H2A.1 and H4 by mass spectrometry. Synthetic peptides representing partial structures indicated that the LPS binding site is located in the C-terminal moiety of the histones. Extending the study, we found that histones H1, H2A, H2B, H3, and H4 from calf thymus are all able to bind LPS. Bindings were specific, and affinities, determined by isothermal titration calorimetry, were (except for H4) higher than that of the LPS-binding antibiotic polymyxin B. In the presence of H2A the binding of LPS to the macrophage cell line RAW 264.7, and the LPS-induced production of TNF-alpha and nitric oxide by these cells, were markedly reduced. Histones may thus represent a new class of intracellular and extracellular LPS sensors.     

Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Presence of histones in Aspergillus nidulans

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea- starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.     

A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells

An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.