Comparative analysis of docking motifs in MAP-kinases and nuclear receptors

Nuclear receptor (NR) agonists induce activation of mitogen-activated protein kinases (MAPK) through an yet unknown rapid non-genomic mechanism. Vice versa, NR are targets for phosphorylation by MAPK. By multiple alignment of the amino acid sequences and comparative analysis of the secondary and tertiary structures we identified four peptides in MAPK with similarity to bona fide protein-protein-interaction motifs in NR. In both molecule species, these motifs mediate selective docking to dimerization partners, coregulators or phosphoacceptors. We therefore propose that similar motifs may direct the site-specific association of NR with MAPK. Based on mutual allosteric interactions within a kinase-receptor complex, we discuss a novel principle how NR-agonists may regulate kinase activity and thus expression of hormone-dependent genes.     

FR3D: finding local and composite recurrent structural motifs in RNA 3D structures

New methods are described for finding recurrent three-dimensional (3D) motifs in RNA atomic-resolution structures. Recurrent RNA 3D motifs are sets of RNA nucleotides with similar spatial arrangements. They can be local or composite. Local motifs comprise nucleotides that occur in the same hairpin or internal loop. Composite motifs comprise nucleotides belonging to three or more different RNA strand segments or molecules. We use a base-centered approach to construct efficient, yet exhaustive search procedures using geometric, symbolic, or mixed representations of RNA structure that we implement in a suite of MATLAB programs, “Find RNA 3D” (FR3D). The first modules of FR3D preprocess structure files to classify base-pair and -stacking interactions. Each base is represented geometrically by the position of its glycosidic nitrogen in 3D space and by the rotation matrix that describes its orientation with respect to a common frame. Base-pairing and base-stacking interactions are calculated from the base geometries and are represented symbolically according to the Leontis/Westhof basepairing classification, extended to include base-stacking. These data are stored and used to organize motif searches. For geometric searches, the user supplies the 3D structure of a query motif which FR3D uses to find and score geometrically similar candidate motifs, without regard to the sequential position of their nucleotides in the RNA chain or the identity of their bases. To score and rank candidate motifs, FR3D calculates a geometric discrepancy by rigidly rotating candidates to align optimally with the query motif and then comparing the relative orientations of the corresponding bases in the query and candidate motifs. Given the growing size of the RNA structure database, it is impossible to explicitly compute the discrepancy for all conceivable candidate motifs, even for motifs with less than ten nucleotides. The screening algorithm that we describe finds all candidate motifs whose geometric discrepancy with respect to the query motif falls below a user-specified cutoff discrepancy. This technique can be applied to RMSD searches. Candidate motifs identified geometrically may be further screened symbolically to identify those that contain particular basepair types or base-stacking arrangements or that conform to sequence continuity or nucleotide identity constraints. Purely symbolic searches for motifs containing user-defined sequence, continuity and interaction constraints have also been implemented. We demonstrate that FR3D finds all occurrences, both local and composite and with nucleotide substitutions, of sarcin/ricin and kink-turn motifs in the 23S and 5S ribosomal RNA 3D structures of the H. marismortui 50S ribosomal subunit and assigns the lowest discrepancy scores to bona fide examples of these motifs. The search algorithms have been optimized for speed to allow users to search the non-redundant RNA 3D structure database on a personal computer in a matter of minutes.     

Nicotinamide riboside,an NAD+ precursor,attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

ObjectiveLiver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis.MethodsMale C57BL/6 J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR) for 20 weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro.ResultsNR supplementation significantly reduced body weight of mice only 7 weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of β-oxidation in skeletal muscle and brown adipose tissue.ConclusionNR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.     

Protective effects of Da-cheng-qi decoction in rats with intracerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH), the most fatal subtype of stroke, has no disease-modifying treatment. Da-cheng-qi decoction (DCQ), composed of rhubarb, is one of the most commonly used Chinese traditional decoctions in ICH treatment. But the mechanism is not clear. Emodin is an active compound found in rhubarb.PurposeTo study the protective effects of DCQ on ICH and its possible mechanisms of action.MethodsThe ICH model was reproduced by injecting collagenase-VII into the left caudate putamen (CPu) of rats. DCQ and emodin were used to treat the ICH rats for 7 days. Behavior tests, proteomic analysis, morphological studies, and western blotting were performed.ResultsThe neurological deficits in the ICH rats recovered with DCQ and emodin on the 14^th day after ICH. The proteomics data revealed that DCQ significantly corrected the pathological signals in the CPu and hippocampus after ICH. The numbers of amoebic microglia in the CPu and M2 microglia in both CPu and hippocampus were significantly increased after DCQ and emodin treatment. The increase in GluN2B-containing NMDA receptor (NR2B) and postsynaptic density protein-95, activation of mitogen-activated protein kinase (MAPK) signals in the CPu, and secondary neurodegeneration (SND) in the hippocampus were significantly recovered in DCQ-treated rats. Inhibition of MAPK p38 (p38) in the hippocampus was observed after DCQ and emodin treatment.ConclusionThe protective effects of DCQ on ICH were confirmed in this study, and its mechanism may be related to the inhibition of MAPK and activation of M2 microglia. These results are beneficial to the development of ICH therapeutic targets.     

Molecular cloning and characterization of mitogen-activated protein kinase 2 in Toxoplasma gondii

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.     

Clinical and Biochemical Function of Polymorphic NR0B1 GGAA-Microsatellites in Ewing Sarcoma: A Report from the Children's Oncology Group

Background

The genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.

Objectives

Here we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target) in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.

Results

A complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20–26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21–25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.

Conclusions

These data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.     

Prenatal exposure to neurotoxic metals is associated with increased placental glucocorticoid receptor DNA methylation

Epigenetic alterations related to prenatal neurotoxic metals exposure may be key in understanding the origins of cognitive and neurobehavioral problems in children. Placental glucocorticoid receptor (NR3C1) methylation has been linked to neurobehavioral risk in early life, but has not been examined in response to neurotoxic metals exposure despite parallel lines of research showing metals exposure and NR3C1 methylation each contribute to a similar set of neurobehavioral phenotypes. Thus, we conducted a study of prenatal neurotoxic metals exposure and placental NR3C1 methylation in a cohort of healthy term singleton pregnancies from Rhode Island, USA (n = 222). Concentrations of arsenic (As; median 0.02 ug/g), cadmium (Cd; median 0.03 μg/g), lead (Pb; median 0.40 μg/g), manganese (Mn; median 0.56 μg/g), mercury (Hg; median 0.02 μg/g), and zinc (Zn; 145.18 μg/g) measured in infant toenails were categorized as tertiles. Multivariable linear regression models tested the independent associations for each metal with NR3C1 methylation, as well as the cumulative risk of exposure to multiple metals simultaneously. Compared to the lowest exposure tertiles, higher levels of As, Cd, Pb, Mn, and Hg were each associated with increased placental NR3C1 methylation (all P<0.02). Coefficients for these associations corresponded with a 0.71–1.41 percent increase in NR3C1 methylation per tertile increase in metals concentrations. For Zn, the lowest exposure tertile compared with the highest tertile was associated with 1.26 percent increase in NR3C1 methylation (P=0.01). Higher cumulative metal risk scores were marginally associated with greater NR3C1 methylation. Taken together, these results indicate that prenatal exposure to neurotoxic metals may affect the offspring's NR3C1 activity, which may help explain cognitive and neurodevelopmental risk later in life.     

A NR2E1‐interacting peptide of LSD1 inhibits the proliferation of brain tumour initiating cells

ObjectivesElimination of brain tumour initiating cells (BTICs) is important for the good prognosis of malignant brain tumour treatment. To develop a novel strategy targeting BTICs, we studied NR2E1(TLX) involved self‐renewal mechanism of BTICs and explored the intervention means.Materials and MethodsNR2E1 and its interacting protein‐LSD1 in BTICs were studied by gene interference combined with cell growth, tumour sphere formation, co‐immunoprecipitation and chromatin immunoprecipitation assays. NR2E1 interacting peptide of LSD1 was identified by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX‐MS) and analysed by in vitro functional assays. The in vivo function of the peptide was examined with intracranial mouse model by transplanting patient‐derived BTICs.ResultsWe found NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono‐ and di‐methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoter and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX‐MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides, LSD1‐197‐211 that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation.ConclusionPeptide LSD1‐197‐211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.

• NR2E1 and LSD1 complex directly represses Pten in BTICs by demethylating H3K4me and H3K4me2 at its promoter, and thus promotes the proliferation of BTICs.
• LSD1‐197‐211 peptide interferes the interaction between NR2E1 and LSD1 and restores Pten expression, which in turn inhibits the proliferation of BTICs.

     

Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High-Affinity Glycine Antagonist Binding Site

Abstract: The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophysiological and pharmacological properties characteristic of the N-methyl-d -aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)-4-(trans)-2-carboxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (K_D = 3.29 and 1.61 nM, respectively). B_max values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. K_i values for glycine site antagonists inhibiting [³H]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four- to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferring state. K_i values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands l -[³H]glutamate and d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP-39653) or the ion-channel radioligand [³H]dizocilpine ([³H]MK-801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiological role if present in vivo.     

Calorie restriction modulates hippocampal NMDA receptors in diet-induced obese rats

Calorie restriction (CR) has attracted increased interest since CR enhances lifespan and alters age-related decline in hippocampal-dependent cognitive functions. Obesity is associated with poor neurocognitive outcome including impaired hippocampal synaptic plasticity and cognitive abilities such as learning and memory. N-Methyl-d-aspartate receptors (NMDARs) are linked to hippocampal-dependent learning and memory, which may be stabilized by CR. In the present study, we aimed to establish the effects of CR on NMDARs in CA1 region of hippocampus in obese and non-obese rats. In addition, malondialdehyde (MDA) levels were determined as a marker for lipid peroxidation (LPO) in hippocampus. Four groups were constituted as control group (C, n?=?9), obese group (OB, n?=?10), obese calorie-restricted group (OCR, n?=?9), and non-obese calorie-restricted group (NCR, n?=?10). OCR and NCR were fed with a 60% CR diet for 10 weeks. After 10 weeks of CR, the MDA levels significantly decreased in the calorie-restricted groups. Obesity caused significant decreases in NR2A and NR2B subunit expressions in the hippocampus. The hippocampal NR2A and NR2B levels significantly increased in the OCR group compared with the OB group (P?<?0.05). In contrast, the hippocampal NR2A and NR2B levels significantly decreased in the NCR group compared with the C group (P?<?0.05). Oxidative stress can be prevented by CR, and these data may provide a molecular and cellular mechanism by which CR may regulate NMDAR-mediated response against obesity-induced changes in the hippocampus.     

Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits

The ionotropic N-methyl-d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares ~40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations.Figure The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-Å radius of the ligand (glutamate)     

RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells

Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf^V600E-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.     

Photooxidative damage to plastids affects the abundance of nitrate-reductase mRNA in mustard cotyledons

It was found previously that in the mustard (Sinapis alba L.) seedling (Schuster et al. 1989, Planta 177, 74–83) the action of nitrate and phytochrome on the appearance of cytosolic nitrate reductase (NR) is abolished if the plastids are damaged by photooxidation. In the present study this finding has been corroborated by the following results: (i) the appearance and disappearance of NR activity are strictly correlated with the appearance and disappearance of immunoresponsive NR protein; (ii) the appearance of NR correlates with the appearance of translatable NR mRNA; (iii) photodestruction of the plastids strongly reduces the level of NR mRNA. It is concluded that the dependence of the NR level on the state of the plastids can be detected at the level of its mRNA and is not attributable to an inactivation of the enzyme.Abbreviations NR nitrate reductase (EC 1.6.6.1) This research was supported by a grant from the Deutsche Forschungsgemeinschaft. We are greatly indebted to Dr. Ann Oaks (University of Guelph, Ontario, Canada) for the gift of antiserum.